Carlos Casado

Asier González

Maria Platara

Amparo Ruiz

IBB Joaquín Ariño
email: joaquin.arino@uab.es

The role of the

Keywords: alkaline stress, gene expression, PKA protein kinase, Msn2 and Msn4 transcription factors, Saccharomyces cerevisiae

INTRODUCTION

The regulation of the activity of the cAMP-PKA pathway plays a major role in the control of metabolism and cell proliferation in yeast cells, mostly linked to the available carbon source. In S. cerevisiae, for instance, in response to a rapidly fermentable carbon source, such as glucose, the pathway activates the Cyr1 adenylate cyclase, which results in a transient increase in cAMP levels. PKA is a heterotetramer composed of two catalytic subunits and two regulatory subunits. The catalytic subunits can be encoded by three, largely redundant genes (TPK1, TPK2 and TPK3), whereas the regulatory subunits are encoded by a single gene (BCY1). Binding of the second messenger cAMP to the regulatory subunits, results in dissociation of the complex and activation of PKA. Restoration of cAMP levels is controlled by the low-and high-affinity phosphodiesterases, encoded by PDE1 and PDE2, respectively, which hydrolyze cAMP to AMP. Sequentially, PKA affects diverse downstream targets, often at the gene transcription level, including stimulation of cell growth and cell cycle progression, upregulation of glycolysis, downregulation of gluconeogenesis and mobilization of glycogen and trehalose [START_REF] Thevelein | Novel sensing mechanisms and targets for the cAMPprotein kinase A pathway in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Santangelo | Glucose signaling in Saccharomyces cerevisiae[END_REF][START_REF] Gancedo | The early steps of glucose signalling in yeast[END_REF][START_REF] Tamaki | Glucose-stimulated cAMP-protein kinase A pathway in yeast Saccharomyces cerevisiae[END_REF][START_REF] Smets | Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae[END_REF].

PKA can be activated in response to glucose by two parallel signalling pathways. The first one involves the Ras1 and Ras2 small GTPases, which are activated by glucose uptake and phosphorylation. The active (GTP-bound) Ras proteins increase the activity of the adenylate cyclase. In turn, the GDP/GTP exchange on the Ras proteins is controlled by the guanine nucleotide exchange factors (GEF) Cdc25 and Sdc25. The reverse process is accelerated by the Ira proteins (encoded by IRA1 and IRA2), which act as Ras GTPase activating proteins (GAP) and maintain Ras in the GDPbound, inactive state. The second involves Gpr1, a putative G protein-coupled receptor, and its Gα protein Gpa2. Both pathways converge to activate adenylate cyclase, resulting in the generation of cAMP [START_REF] Thevelein | Novel sensing mechanisms and targets for the cAMPprotein kinase A pathway in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Santangelo | Glucose signaling in Saccharomyces cerevisiae[END_REF][START_REF] Gancedo | The early steps of glucose signalling in yeast[END_REF].

Activation of PKA has a major impact on gene expression. Consequently, several transcription factors are among the known PKA targets. Two of those are Msn2 and Msn4, which mediate the transcription of the so-called stress response element (STRE)-controlled genes [START_REF] Estruch | Two homologous zinc finger genes identified by multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae[END_REF][START_REF] Martinez-Pastor | The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE)[END_REF][START_REF] Schmitt | Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae[END_REF]. STRE-regulated genes are involved in important processes, such as carbohydrate metabolism and growth regulation, as well as in adaption to diverse types of stress, including heat, DNA damage, oxidative and osmotic stresses [START_REF] Mai | Xbp1, a stress-induced transcriptional repressor of the Saccharomyces cerevisiae Swi4/Mbp1 family[END_REF][START_REF] Moskvina | A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements[END_REF][START_REF] Smith | Yeast PKA represses Msn2p/Msn4p-dependent gene expression to regulate growth, stress response and glycogen accumulation[END_REF][START_REF] Gasch | Genomic expression programs in the response of yeast cells to environmental changes[END_REF]. Under growth-promoting conditions (growth on glucose, absence of stress,..) Msn2 and Msn4 are phosphorylated and reside in the cytosol. Upon glucose exhaustion or other stress conditions [START_REF] Gorner | Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity[END_REF], they become hypophosphorylated and translocate to the nucleus, where they induce expression of the STRE-controlled genes. PKA plays a very important role inhibiting nuclear import of Msn2/4, either through direct phosphorylation of their nuclear localization signal [START_REF] Gorner | Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity[END_REF][START_REF] Gorner | Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor[END_REF][START_REF] Garreau | Hyperphosphorylation of Msn2p and Msn4p in response to heat shock and the diauxic shift is inhibited by cAMP in Saccharomyces cerevisiae[END_REF] or indirectly via the protein kinases Yak1 and Rim15.

S. cerevisiae grows far better at acidic than at neutral or alkaline pH and, consequently, even a modest alkalinization of the medium represents a stress situation that is able to trigger a compensatory multifactorial response (see [START_REF] Arino | Integrative responses to high pH stress in S. cerevisiae[END_REF] for review). Alkaline stress activates diverse signaling pathways, including the Rim101-Nrg1 [17;18], the calcium / calcineurin [START_REF] Serrano | The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling[END_REF][START_REF] Viladevall | Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae[END_REF][START_REF] Ruiz | Direct regulation of genes involved in glucose utilization by the calcium/calcineurin pathway[END_REF], and the Wsc1-Pkc1-Slt2 MAP kinase pathways [START_REF] Serrano | Signaling alkaline pH stress in the yeast Saccharomyces cerevisiae through the Wsc1 cell surface sensor and the Slt2 MAPK pathway[END_REF]. Alkalinisation of the environment also disturbs nutrient homeostasis, as deduced from its impact on iron/copper and phosphate uptake/utilization pathways [19;23]. Work in our laboratory in the past years has evidenced that alkaline pH stress has also a profound impact on the expression of genes encoding glucose uptake and metabolism-related proteins, in the sense that exposure to high pH would mimic a situation of glucose starvation [20;21]. Given the strong link between carbohydrate metabolism and the PKA pathway, we speculated that alkaline stress might involve changes in the activity of this pathway and, specifically, that the correct adaptation to high pH could entail its downregulation. Indeed, in this paper we show that alkaline pH stress causes a transient decrease in cAMP levels and that change in the activity of the PKA pathway alters tolerance to alkaline pH. Our data also indicates that the adaptive response to high pH involves PKA-regulated, Msn2/Msn4-mediated gene remodelling.

Experimental

Yeast strains and growth conditions Yeast cells (strains are listed in Table 1) were grown at 28°C in YPD medium (10 g/l yeast extract, 20 g/l peptone, and 20 g/l dextrose) or, when carrying plasmids, in synthetic complete medium [START_REF] Adams | Methods in Yeast Genetics[END_REF] containing 2% glucose and lacking the appropriate selection requirements. Single kanMX deletion mutants, in the BY4741 background, except MAR231, were generated in the context of the Saccharomyces Genome Deletion Project [START_REF] Winzeler | Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis[END_REF].

Strain MAR231 was made by transforming BY4741 with a bcy1::kanMX4 cassette obtained from the strain KKY385 [START_REF] Kafadar | Integration of stress responses: modulation of calcineurin signaling in Saccharomyces cerevisiae by protein kinase A[END_REF] by PCR amplification using primers 5´-bcy1_disr (GAGGAGCATACGACTTCGGC) and 3´-bcy1_disr (CTGTCTTGTAGATCCTTTGG). Strains CCV35 and CCV36 were constructed as follows. A 1.6 Kbp pde1::kanMX4 cassette was amplified from genomic DNA of the BY4741 pde1::kanMX4 strain with oligonucleotides 5'-pde1 (CAAGGATCGTTACCCGGTA) and 3'-pde1 (GACTTATGTTGGGATAGGGG). The purified DNA fragment was used to transform strains W303 or PM942 [START_REF] Ma | The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonistinduced cAMP signaling[END_REF], to yield CCV35 and CCV36, respectively. Strains CCV37 and CCV38 were obtained transforming W303-1A wild type and MCY5278 strains with a SNF1::LEU2 disruption cassette [START_REF] Ruiz | Direct regulation of genes involved in glucose utilization by the calcium/calcineurin pathway[END_REF] and strains CCV174 and CCV175 were generated by transforming W303-1A and MCY5278 strains with the 2.1 kb nrg1::nat1 cassette from plasmid pBS-nrg1::nat1 as described in [START_REF] Platara | The Transcriptional Response of the Yeast Na+-ATPase ENA1 Gene to Alkaline Stress Involves Three Main Signaling Pathways[END_REF]. Strain AGS66 contains an integrated STRE(7x)-lacZ reporter system at the URA3 locus [START_REF] Gonzalez | Normal function of the yeast TOR pathway requires the type 2C protein phosphatase Ptc1[END_REF].

Plasmids

The following plasmid constructs were used in this work. The reporter plasmid pHXK1-lacZ was generated as follows. The HXK1 upstream DNA region containing -624 and +39, relative to the starting ATG, was amplified by PCR with added BamHI/PstI restriction sites and cloned into the same sites of YEp357 [START_REF] Myers | Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions[END_REF]. Plasmid YCp50-RAS2 Ala18Val19 [START_REF] Xue | GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway[END_REF] expresses the RAS2 Ala18Val19 hyperactive Ras2 allele [START_REF] Toda | In yeast, RAS proteins are controlling elements of adenylate cyclase[END_REF] from the centromeric YCp50. Plasmid pG2CT-112.2 expresses the constitutively active GPA2 R273A allele from the episomal YEplac112 backbone [START_REF] Xue | GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway[END_REF]. Plasmid pAMS366 contains a tandem of four CDRE elements from the FKS2 gene fused to the lacZ reporter [33]. pPHO84-LacZ contains a PHO84-LacZ reporter fusion as described in [START_REF] Serrano | The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling[END_REF]. pKC201 contains the ENA1 promoter fused to LacZ ( [34;35].

Growth Tests

The sensitivity of different yeast strains to alkaline pH was assayed by drop test on YPD plates containing 50 mM TAPS adjusted with KOH at different pH values. Growth in liquid medium at high pH was performed in 5 ml cultures or in 96-well plates (250 µl), at 28 °C in YPD medium buffered with 50 mM TAPS and adjusted with KOH at the indicated pH values. Growth was monitored by measuring the A at 660 nm. BY4742 and DC90 cells, at an initial A 660 of 0.001, were grown for 17 and 24 h respectively. Strains prepared from the W303-1A genetic background were grown from an initial A 660 of 0.01 for 17 h (wild type and MCY5278 strains) and for 21h (CCV37 and CCV38 strains).

Measurements of cAMP levels

Measurements were made as described in [START_REF] Marquez | Multiple transduction pathways regulate the sodium-extrusion gene PMR2/ENA1 during salt stress in yeast[END_REF]. Briefly, BY4741 cells were grown to an OD 660 of approximately 1.0 in YPD at 28ºC, and then the culture was centrifuged and resuspended in YPD containing 50 mM TAPS pH 8. Aliquots of 10 ml were filtered at the indicated times and extracted in the cold with 1 ml 2 M perchloric acid. After neutralization with 1 ml of 1.8 M KOH and 0.4 M KHCO 3 and centrifugation, samples were purified with Amprep SAX minicolumns (Amersham, code RPN 1918), eluted with methanol-HCl and dried under vacuum. Assays were performed by a competitive binding method with the Amersham cAMP enzymeimmunoassay (EIA) system (code RPN 225). For calculation of yeast concentrations, one unit of absorbance at 660 nm was equivalent to 2.6 mg wet weight/ml. In other set of experiments, pH was raised to 8.2 by addition of KOH (35 mM, final concentration). Control cells received the same concentration of KCl. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

β-Galactosidase Activity Assay

Yeast cells were grown to saturation in the appropriate dropout media and then inoculated into YPD (or YP plus 4% glucose, when indicated) at pH 5.5. Growth was resumed until A 660 0.5-0.7, and cultures were centrifuged for 5 min at 1620 × g. Cells were resuspended in YPD or YP 4% glucose where indicated (no induction) or YPD (or YPD 4% glucose) plus 50 mM TAPS adjusted to pH 8.0 (alkaline stress), and growth was resumed for the indicated times. In all cases, β-galactosidase activity was measured as described previously [START_REF] Reynolds | Yeast Vectors and Assays for expression of cloned genes[END_REF].

Microscopy techniques

For Msn2 subcellular localization experiments, the indicated strains were transformed with plasmid pMSN2-GFP (a generous gift of F. Estruch, University of Valencia, Spain), a YCplac111-based vector that contains a C-terminal Msn2-green fluorescent protein fusion [START_REF] Gorner | Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity[END_REF]. Cells were grown in YPD or YP 2% ethanol until an A 660 of 0.8 to 1.0 was reached. Cultures (5 ml) were treated as follows: addition of 100 μl of 1 M KCl (control cells, pH 5.5) or addition of 100 μl of 1 M KOH (alkaline stress, pH 8.0). Samples (500 μl) were taken at the appropriate times and fixed for 5 min by adding 30 µl of 37% formaldehyde. Cells were harvested, washed three times with a phosphate-buffered saline solution (PBS), and concentrated 10-fold before visualization. In all cases the cells were visualized with a fluorescein filter using a Nikon Eclipse E800 fluorescence microscope (magnification, x1000). Digital images were captured with an ORCA-ER 4742-80 camera (Hamamatsu) using the Wasabi software. Intracellular distribution of Msn2-GFP was quantified by scoring at least in 200 cells per sample into one of three possible categories: cytoplasmic (fluorescence in the cytoplasm only), nuclearcytoplasmic (fluorescence in the cytoplasm and nucleus), and nuclear (fluorescence in the nucleus only).

RNA purification, cDNA synthesis, and DNA microarray experiments

For RNA purification, 50 ml of yeast culture (strains W303-1A and MCY5278) was grown at 28°C in YPD medium until an A 660 of 0.6 to 0.8, KOH or KCl was added from a concentrated stock solution (1 M) to reach a final concentration of 20 mM (pH 8.05 and pH 5.5 respectively). Yeast cells were harvested by filtration after 10 and 30 min and washed with cold water; dried cells were kept at -80 °C until RNA purification. Total RNA was purified using a RiboPure-Yeast kit (Ambion, Inc.) following the manufacturer's instructions. RNA quality was assessed by denaturing 0.8% agarose gel electrophoresis, and RNA quantification was performed by measuring absorbance at 260 nm in a BioPhotometer (Eppendorf). Transcriptional analyses were performed using DNA microarrays containing PCR-amplified fragments from 6014 S. cerevisiae open reading frames [20;38]. Fluorescent Cy3-and Cy5-labeled cDNA probes were prepared from 8 μg of purified total RNA by the indirect dUTP labelling method using a CyScribe post-labelling kit (Amersham Biosciences).

Pre-hybridization, hybridization, and washes were carried out as recommended by The Institute for Genomic Research with minor modifications. Briefly, prehybridizations of the DNA microarrays were carried out at 42 °C for 1 h in a solution containing 5× SSC, 0.1% SDS and 1% bovine serum albumin. For hybridization, dried Cy3-and Cy5-labeled probes were resuspended in 35 μl of hybridization solution (50% formamide, 5× SSC, 0.1% SDS) each and mixed. Five μg of salmon sperm DNA was added to the mix before denaturation for 3 min at 95 °C. DNA microarrays were hybridized in an ArrayBooster hybridization station (Sunergia Group) for 14 h at 42 °C. The scanner ScanArray 4000 (Packard Instrument Co.) was used to obtain the Cy3 and Cy5 images with a resolution of 10 μm. The fluorescent intensity of the spots was measured and processed using the GenePix Pro 6.0 software (Molecular Devices). Spots with either diameter smaller than 120 μm, or fluorescence intensity for Cy3 and Cy5 lower than 150 units were not considered for further analysis.

For each condition assayed, two independent experiments were performed, and dye swapping was carried out for each experiment. Microarray data was deposited at the Gene Expression Omnibus (GEO) and can be retrieved under accession number GSE27925. Data from different experiments were combined, and the mean was calculated. A given gene was considered to be induced or repressed when the mean of the ratios (alkaline stress versus no stress) was >2.0 or <0.50, respectively. Software from the GEPAS server (gepas.bioinfo.cipf.es/) was used to carry out clustering and other data analyses [START_REF] Herrero | GEPAS: A web-based resource for microarray gene expression data analysis[END_REF]. According to the expression of these genes in the msn2 msn4 strain, different levels of dependence on these transcription factors were defined. Thus, genes showing a mutant/wild type ratio 0.67 > X > 0.50 were considered "weakly dependent" (WD); those with a ratio 0.50 > X > 0.25 were ranked as "strongly dependent" (SD) and those with a ratio ≤ 0.25 were defined as "totally dependent" (TD). Likewise, genes induced more than 2.5-fold in wild type cells and considered not induced (i.e ratio alkaline stress/no stress) <1.3) in msn2 msn4 cells were also considered as totally dependent. This score is the same employed in our previous report on calcineurin dependence of high pH response [START_REF] Viladevall | Characterization of the calcium-mediated response to alkaline stress in Saccharomyces cerevisiae[END_REF].

RESULTS

Manipulation of upstream elements of the PKA pathway affects growth at alkaline pH.

Our starting hypothesis was that adaptation to high pH stress should entail a decrease in the PKA pathway activity. Therefore, we speculated that mutations increasing the activity of the pathway should result in decreased tolerance to alkaline pH. We first tested strains lacking components of the Gpr1/Gpa2 sensing pathway, such as gpr1, gpa2, rgs2 (lacking a GAP of Gpa2), gpb1 or gpb2 (lacking multistep regulator of cAMP-PKA signalling) (Figure 1A). However, none of these strains showed altered tolerance to alkaline pH (not shown). In contrast, deletion of ira1 or ira2, the GAPs of Ras1 and Ras2 results in increased sensitivity. This phenotype was enhanced in the double ira1 ira2 mutant (Figure 1B). Conversely, expression of the Cdc25 WΔN1 allele, which lacks the N-terminal domain of Cdc25 and elicits constitutive activation of Ras proteins, also results in poor growth at high pH (Figure 1B). All these manipulations lead to increased Ras activity and hyperactivation of the PKA pathway. In contrast, the effect on high pH tolerance of the expression of the Cdc25 WΔN2 allele, which does not lead to increase in PKA activity, was barely noticeable. These results were confirmed by expression of the hyperactive Ras allele RAS2 Ala18Val19 from a centromeric plasmid [START_REF] Xue | GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway[END_REF]. Among other phenotypes, expression of this allele decreases heat shock tolerance. As it can be observed (Figure 1C), expression of the RAS2 Ala18Val19 allele decreases alkaline pH tolerance. In contrast, expression of a constitutively active form of Gpa2 (pG2CT-112.2) constructed by replacing the arginine at position 273 with an alanine [START_REF] Xue | GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway[END_REF] did not alter high pH tolerance.

Alkaline pH stress transiently decreases the levels of cAMP.

The levels of cAMP determine the interaction between the regulatory subunit (Bcy1) and the catalytic subunits (Tpk) of PKA and are, therefore, crucial for the activity of the kinase. We have measured the levels of cAMP after shifting the cells from pH 5.5 to media buffered to pH 8.0 and found that this treatment drastically decreases the concentration of the second messenger in the first 5-15 minutes, followed by a recovery to the initial levels after 30 min of stress (Figure 2A). A similar result was obtained when KOH was directly added to the exponentially growing cultures to raise the pH. In this case, decrease in cAMP levels was apparent even only 2 min after stress (not shown). Raising pH of the liquid medium up to values of 8.2 did not result in detectable cell lysis, as determined by microscopic examination, viability counting, or release of alkaline phosphatase activity to the medium.

Our data was consistent with the possibility that a drop in cAMP levels, leading to downregulation of PKA activity, could be an adaptive strategy to confront high pH stress. Therefore, we considered that mutations directly resulting in increased cAMP levels should be deleterious for cells subjected to alkaline pH stress. The PDE1 and PDE2 genes encode the yeast cyclic AMP phosphodiesterase activity, which degrades cAMP to AMP. When the single mutants are grown on alkaline pH plates, a growth defect can be observed for the pde2 mutant (lacking the high affinity isoform) that is exacerbated in the double pde1 pde2 mutant strain (Figure 2B).

Lack of Bcy1, the regulatory subunit of PKA, leads to constitutive PKA activity, which results in a variety of phenotypes (impaired growth on different carbon sources, temperature sensitivity, etc., see [START_REF] Santangelo | Glucose signaling in Saccharomyces cerevisiae[END_REF] and references therein). Although the quantification of the effect is difficult, because of the relatively poor growth of the bcy1 mutant even under standard conditions, we can show that this strain exhibits a dramatic alkaline pH-sensitive phenotype (Figure 2C). The same effect was observed in the W303-1A background (data not shown). Similarly, we tested if a decrease in PKA activity could improve growth at high pH. Since complete lack of PKA activity is not compatible with survival, for A c c e p t e d M a n u s c r i p t this experiment we resorted to a strain lacking TPK1 and TPK3 and carrying an attenuated allele of TPK2 (tpk2 w3 ) as sole source of PKA activity. As it can be observed in Figure 2D, the relative growth of this strain in alkaline pH medium versus non-restrictive conditions is higher than the wild-type strain. In essence, the data presented so far shows that yeast cells respond to the alkalinisation of the medium with a sharp decrease in the intracellular level of cAMP. The observation that when PKA activity is attenuated cells grow better at high pH, whereas any situation resulting in increased PKA activity makes cells more sensitive to this condition, supports the notion that downregulating PKA activity contributes to adaptation to alkaline pH stress.

Alkaline pH stress promotes entry of Msn2 in the nucleus and STRE-dependent promoter activation.

Nuclear localization of Msn2, a transcription factor that, together with Msn4, regulates the general stress response of S. cerevisiae, is controlled by PKA activity [13;14;40]. We considered that alkaline pH-triggered downregulation of the PKA activity could result in the translocation of Msn2 to the nucleus and that this transition might have a relevant effect in the pH-induced adaptive response. To test this possibility, an Msn2-GFP fusion was introduced into wild type cells and the cultures subjected to alkaline treatment. As it can be observed in Figure 3A andB, exposure to high pH (8.0) triggers an almost immediate entry of Msn2 into the nucleus, which reached a maximum after 5 min (Figure 3B). Nuclear localization of Msn2 was transient and after 10-15 min the transcription factor was again mostly cytosolic. Interestingly, when the same experiment was carried out in a strain lacking the IRA2 gene (sensitive to alkaline pH, see Figure 1B), entry of Msn2 to the nucleus was somewhat delayed and nuclear localization was a less general effect than in the wild type strain. Nuclear translocation of Msn2 was also evaluated in cells subjected to alkaline stress (pH 8.0) growing in the presence of ethanol as carbon source (Figure 3B). In this case, Msn2 showed both cytoplasmic and nuclear/cytoplasmic distribution in unstressed cells. Interestingly, exposure of cells to alkalinisation resulted in only a modest increase in the percentage of cells showing nuclear/cytoplasmic distribution, but very few cells with only nuclear Msn2. Therefore, subcellular distribution of Msn2 in response to alkaline stress is affected by the carbon source in which cells are grown.

We then investigated if the Msn2 and Msn4 transcription factors are necessary for normal tolerance to high pH stress. As shown in Figure 4A, lack of the transcription factors reduces somewhat tolerance to high pH, suggesting that they are components of the adaptive response to alkalinisation. Remarkably, additional deletion of all three PKA catalytic subunits (which is feasible in the absence of the transcription factors) yields cells that are even more sensitive than the msn2 msn4 mutant. It should be noted that in this case the msn2 msn4 strain was transformed with a centromeric plasmid which contains a TRP1 marker. The introduction of this marker in the reference strain was necessary for accurate comparison because the tpk msn2,4 strain was constructed using the TRP1 gene as marker, and it has been reported [START_REF] Gonzalez | Use of the TRP1 auxotrophic marker for gene disruption and phenotypic analysis in yeast: a note of warning[END_REF] that the absence of this gene somewhat decreases tolerance to high pH. This results suggested that, besides its role on Msn2,4 function, regulation of PKA activity may exert additional effects on high pH tolerance. This notion was reinforced by the result of the experiment shown in figure 4B. In this case, we observed that expression of the hyperactive form of RAS2 further decreases high pH tolerance even in the absence of Msn2/Msn4 transcription factors, suggesting again that not all the effects relevant for alkaline tolerance mediated by PKA are necessarily based in the activation of Msn2/Msn4. On the other hand, cells lacking the Snf1 kinase are sensitive to alkaline pH and Snf1 has been reported to repress Msn2 function. Therefore, we considered whether the sensitive phenotype of snf1 cells could be due to deregulation of Msn2 function. As shown in Figure 4C, snf1 cells are more sensitive than the msn2 msn4 mutant to alkaline pH. In addition, deletion of msn2 msn4 further increases sensitivity to alkaline pH of the snf1 mutant. Therefore, deregulation of Msn2/Msn4 is not at the basis of the snf1 alkali-sensitive phenotype.

Because Msn2 is known to bind to STRE elements in response to glucose shortage and other forms of stress, we tested the activation of a synthetic promoter containing a tandem of seven STRE elements fused to the LacZ reporter (strain AGS66). As shown in Figure 5A, this promoter exhibited a very fast activation, with a peak of activity after 15 min of shifting cells to pH 8.0. This activation was much faster than that observed when the LacZ gene is expressed from a PHO84 promoter, which is considered a gene with a relatively late response to high pH stress [START_REF] Serrano | The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling[END_REF] and it was completely abolished in a msn2 msn4 strain (not shown). Remarkably, the response was also faster than that obtained from a synthetic promoter based in a cluster of four copies of the CDRE motif from the calcineurin-responsive FKS2 gene. It must be noted that calcineurin/Crz1 mediated response is considered to be an early response to alkaline pH stress [19;20]. We also tested the response to alkaline pH of HXK1, encoding hexokinase, a gene with predicted STRE elements in its promoter [START_REF] Moskvina | A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements[END_REF] and known to be induced by high temperature (37ºC) and oxidative stress in a msn2 msn4 dependent way [START_REF] Gasch | Genomic expression programs in the response of yeast cells to environmental changes[END_REF]. As it is shown in Figure 5B, expression from the HXK1 promoter induced by high pH stress was as fast as the one induced by exposure to 37ºC, although the level of β-galactosidase activity declines faster (likely due to yeast-promoted acidification of the medium). Remarkably, in the msn2 msn4 strain the basal activity of the promoter, as well as the response to both stress conditions, is dramatically reduced. However, a small increase can still be detected after 30 min of exposure to high pH stress, suggesting an Msn2/4-independent input to the promoter, which could be attributed to activation of calcineurin, as this gene was demonstrated to be induced by calcium as well as by high pH [START_REF] Ruiz | Direct regulation of genes involved in glucose utilization by the calcium/calcineurin pathway[END_REF].

Relevance of Msn2/Msn4 transcription factors in the transcriptional response to high pH stress.

Since these results indicated that Msn2/4 could be responsible for the response of certain genes to alkaline pH stress, we considered necessary to evaluate up to what extent this pathway is responsible for the remodelling of gene expression observed upon exposure to high pH. To this end, DNA microarray analysis was conducted using wild type and msn2 msn4 strains subjected to pH 8.0 for 10 and 30 min. After 10 min of exposure to high pH, 331 genes were induced at least 2-fold in wild type cells, whereas only 186 were found in the msn2 msn4 cells strain (from a total number of 3463 genes with valid data, Figure 6 and Supplemental Data).

Gene Ontology (GO) analysis of the genes induced in the wild type strain yielded, as expected, an excess of genes related to carbohydrate metabolism (p<7.74E-09), in particular to trehalose (p<5.05E-08) and glucose (p<1.42E-06) and glycogen (p<1.61E-03) metabolisms. Analysis of the msn2 msn4 dependence for induction revealed that 157 genes required the presence of the transcription factors for full induction (Figure 6). While dependence was limited in some cases (62 genes were rated as weakly dependent, WD), the majority of the short-term induced genes were strongly (57) or totally [START_REF] Alberola | A new set of DNA macrochips for the yeast Saccharomyces cerevisiae: features and uses[END_REF] dependent of the presence of Msn2 and Msn4. Gene Ontology analysis of the 95 genes defined as SD or TD revealed a strong excess of genes encoding proteins involved in trehalose (p<9.40E-09) and glycogen (p<1.46E-07) metabolism. A similar analysis of genes showing weak dependence provided no distinctive profile.

Exposure of cells to pH 8.0 for 30 min resulted in 241 genes induced in the wild type strain, whereas 296 genes increased expression at least 2-fold in the msn2 msn4 mutant (Figure 6). In this case, the level of dependence of the transcription factors was much lower (only 50 genes). In addition, the vast majority (40) was weakly dependent; nine were ranked as SD and only one as TD. Gene Ontology analysis of the set of SD plus TD genes did not produce any specific profile. Therefore, Msn2 and Msn4 transcription factors are responsible for the induction of a substantial subset of the early alkali-responsive genes. Analysis of repressed genes showed 146 and 279 genes repressed at least 0.5-fold in the wild type strain after 10 and 30 min of shift to high pH, respectively. The absence of msn2 msn4 did not result in activation of any of these genes.

The ENA1 gene encodes a Na + -ATPase that has been repeatedly reported to be strongly induced upon alkaline pH stimulation. Interestingly, our microarray data indicate that deletion of MSN2/4 does not affect induction of the ATPase gene at short-term (10 min) but results in higherthan-normal expression after 30 min of stress (Figure 7A). In an attempt to identify a possible cause for this behaviour we searched our microarray raw data for changes in expression of genes encoding known regulatory components of ENA1. We observed that after 10 min after alkalinisation of the medium, the expression of NRG1, a known repressor of ENA1 expression, was essentially identical in wild type and msn2 msn4 cells. However, after 30 min the expression of NRG1 was decreased by 37% in the msn2 msn4 strain compared to the wild type. To explore a possible influence of this change on the response of ENA1, we evaluated the expression from the ATPase gene promoter in wild type and msn2 msn4 cells deleted for NRG1 by means of a LacZ reporter. As it can be observed (Figure 7B), high-pH dependent expression from the ENA1 promoter is stronger in the msn2 msn4 mutant than in the wild type strain, thus confirming the microarray data. Moreover, the induction observed upon alkaline pH stimulation in the nrg1 strain is virtually identical to that observed in the msn2 msn4 nrg1 triple mutant, suggesting that changes in the Nrg1 levels could be at the basis of the abnormal response of ENA1 in the msn2 msn4 mutant.

DISCUSSION

Exposure to alkaline stress triggers a set of responses that are known to be mediated by different signaling pathways, such as the calcineurin/Crz1, the Rim101, and the Slt2 pathways [START_REF] Arino | Integrative responses to high pH stress in S. cerevisiae[END_REF]. PKA mediates a major signaling pathway that is crucial for linking stress responses and cell proliferation. Therefore, the major goal of this work was to evaluate the relevance of the PKA pathway in the adaptive response to high pH stress. We observe that mutations leading to activation of PKA result in decreased alkaline pH tolerance, whereas those that lead to inhibition of the pathway yield cells with increased tolerance. Our results also show that exposure to high pH provokes a sharp and transient decrease in the cAMP levels, which may lead to downregulation of PKA activity. In this regard, a previous work reported a transitory increase of cAMP levels as a result of sudden acidification of the cytosol [42]. It is worth noting that these authors noticed that Gpa2 was not required for the stimulation of cAMP accumulation in response to intracellular acidification. Remarkably, we observe that only alterations in the small G proteins Ras1 and Ras2 branch of the pathway do result in changes in tolerance to alkaline pH, whereas mutations in diverse genes involved in the Gpr1/Gpa2 glucose sensor system do not produce phenotypic effects. This suggests that the observed changes are due to alteration of intracellular glucose metabolism and not to defects in the extracellular glucose detection system and reinforce the notion that changes in cytosolic pH may result in fluctuations in cAMP levels and consequent regulation of PKA activity. In the case of sudden alkalinisation of the medium, the sharp decrease in cAMP levels would inhibit PKA activity and allow cells for adaptation to the stress.

Our results show that alkalinisation of the medium triggers a very fast entry of the Msn2 transcription factor to the nucleus (2-5 min). This time frame is very similar to that observed when cells are shifted to low glucose [13;14]. Interestingly, the fact that we observe only a very modest response in ethanol-grown cells suggests that nuclear transition of Msn2 is related to alkaline stressinduced alterations in glucose signalling. We also show that Msn2 and Msn4 are important for mediating a substantial part of the transcriptional response induced by alkalinisation of the medium. It is conceivable that this response constitutes one of the factors that allow normal tolerance to the stress, since the msn2 msn4 mutant is sensitive to high pH (Figure 4A). Moreover, our results suggest that regulation of Msn2 function in response to high pH is the result of inhibition of PKA. It is known that intracellular localization of Msn2 can be regulated not only by PKA but also by the Snf1 kinase [43;44] and the TOR pathway [44;45], thus raising the possibility that these two pathways could contribute to the alkaline pH-triggered entry of Msn2 into the nucleus. In fact, activation of the Snf1 pathway has been previously linked to high pH stress [21;28;46]. However, we observe that mutation of SNF1 yields cells even more sensitive to alkaline pH than those lacking msn2 msn4, and that deletion of both factors in a snf1 background further decreases tolerance. Furthermore, we have observed that, within the time-span shown in Figure 3A, entry into the nucleus of Msn2 is not affected by the lack of the protein kinase Snf1 after alkaline treatment (data not shown). Finally, comparison of the dependence level for Msn2,4 of the short-term transcriptional response to high pH described in this work with the dependence on Snf1, evaluated in a parallel project (Casamayor, A., Ruiz, A., Serrano, R., Platara, M., Ferrer-Dalmau, J. & Ariño, J. unpublished work), indicates low overlap (only 34 of the 157 genes reported as Msn2,4-dependent in this report are found to exhibit some degree of dependence for the Snf1 kinase). As an example, most of the genes encoding enzymes involved in trehalose metabolism whose expression is increased by alkalinisation are Msn2,4-dependent, but not Snf1dependent. Therefore, our results do not support the notion that activation of Msn2/4 by high pH is mediated by the Snf1 kinase.

The PKA and TOR pathways illustrate diverse ways of functional interaction in the regulation of cell growth, showing some functional overlap [START_REF] Chen | Coordinate regulation of multiple and distinct biosynthetic pathways by TOR and PKA kinases in S. cerevisiae[END_REF]. However, a role of the TOR pathway in regulation of Msn2 function in response to high pH is questionable, because with the only exception of the ure2 strain (a very pleiotropic strain), mutants in genes of the pathway do not show altered tolerance to high pH nor alkalinisation triggers increased expression of well-established readouts for inhibition of the TOR pathway, such as GAP1, GLN1 or GDH1 (our own data, not shown). Two protein kinases, Yak1 and Rim15, have been reported to modulate Msn2 function in response to PKA activation (see [START_REF] Smets | Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae[END_REF] for review). However, yak1 and rim15 mutant strains are not sensitive to high pH (data not shown) and, furthermore, it has been proposed that Yak1 regulates Msn2 function by an unknown mechanism that does not implicate the control of Msn2 subcellular localization. Therefore, the most likely scenario is that Msn2 entry into the nucleus after high pH stress is caused by inhibition of PKA activity.

Comparison of the time sequence of the events described in our work is rather coherent, with a very fast decrease in cAMP levels and subsequent entry of Msn2 in the nucleus, followed by a massive, fast (10 min), and transient activation of Msn2/4 dependent genes, as deduced by DNA microarray transcriptomic analysis. It is remarkable that, after 30 min of high pH stress, the transcriptional response becomes largely msn2 msn4-independent. In fact, our data (Figure 5A) suggest that Msn2/Msn4 may be responsible for the first chronological set of transcriptional responses to alkaline stress, even a faster response than that mediated by the calcineurin/Crz1 pathway, which were currently defined as early responses [19;20].

It has been widely documented that expression of the ENA1 Na + -ATPase is potently induced by alkaline pH (see [START_REF] Ruiz | Function and regulation of the Saccharomyces cerevisiae ENA sodium ATPase system[END_REF] and references therein). An interesting observation derived from our microarray data is that the increase in ENA1 expression is virtually identical in wild type and msn2 msn4 cells at short times (10 min), but is further enhanced by the lack of Msn2/4 at 30 min. Interestingly, we observed long time ago a similar effect by using a LacZ translational fusion of the ENA1 promoter and a different msn2 msn4 mutant strain [START_REF] Serrano | The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling[END_REF], although at that moment it was not further characterized. Our observations suggest that lack of Msn2/4 results in either activation of a positive regulatory element for ENA1 expression or removal of a negative regulator. It is well known that Nrg1 acts as a repressor of ENA1 expression by directly binding to the gene promoter, thus participating in alkaline pH signalling [18;49;50]. We observe here that NRG1 levels decrease after 30 min of high pH stress and that alkaline pH induction of ENA1 is not further increased by mutation of msn2 msn4 in a nrg1 deletion background. This suggests that the enhanced expression of the ATPase gene in the msn2 msn4 mutant can be due to a decrease in the amount of the Nrg1 repressor. In this regard, it must be noted that the remarkable effect of lack of RIM101 in the expression of the ATPase gene [18;19;28], is fully mediated by a rather small change (2.8-fold) in NRG1 mRNA levels [START_REF] Lamb | The transcription factor Rim101p governs ion tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and SMP1 in Saccharomyces cerevisiae[END_REF].

Our data also suggest involvement of the PKA pathway in high pH tolerance in way that would be independent of the Msn2/4 transcription factors. This is based in the combination of tpk and msn2,4 mutations and in the observation that hyperactivation of RAS2 in the msn2 msn4 mutant still increases high pH sensitivity (Figure 4B). A possible explanation could be based in a cooperative role of PKA and calcineurin pathways in the adaptive response to high pH stress. In this regard, it was reported that PKA can phosphorylate Crz1, promoting its exit from the nucleus and thus opposing the action of calcineurin [START_REF] Kafadar | Integration of stress responses: modulation of calcineurin signaling in Saccharomyces cerevisiae by protein kinase A[END_REF]. Therefore, inhibition of PKA may result in further enhancement of calcineurin/Crz1-mediated responses. A further step of complexity may exist in the interaction between the calcineurin and the PKA pathways, as it was recently reported that Crz1 may exert a destabilization effect on Msn2 levels upon calcium treatment [START_REF] Takatsume | Calcineurin/Crz1 destabilizes Msn2 and Msn4 in the nucleus in response to Ca(2+) in Saccharomyces cerevisiae[END_REF]. In this context, it is conceivable that entry of Crz1 in the nucleus, following activation of calcineurin by high pH may serve, in addition to activate calcineurin/Crz1-responsive promoters, as a negative feed-back system to avoid persistent activation of Msn2/4-responsive genes. This regulatory system may contribute to the transient Msn2/4-dependent transcriptional effect observed in our work. Supported by grants BFU2008-04188-C03-01, GEN2006-27748-C2-1-E/SYS (SysMo) and EUI2009-04147 (SysMo2) to JA (Ministry of Science and Innovation, Spain and FEDER). J.A. was recipient of an "Ajut 2009SGR-1091" (Generalitat de Catalunya). C. Casado was supported by a predoctoral fellowship from the Spanish Ministry of Science and Technology.

Legends

Figure 1. Effect of mutations in the upstream components of the PKA pathway on alkaline pH tolerance. A) A simplified schematic depiction of the PKA signaling pathway. See Introduction for additional information. B) Upper panel. Three dilutions of cultures of wild type BY4741 cells and the isogenic kanMX disruption derivatives ras1, ras2, sds25 and ira2 mutants were spotted on YPD plates adjusted at the indicated pHs. Middle panel. Wild type strains W303-1A and its ira1::LEU2 (strain SC7), ira2::URA3 (SC8) and ira1::LEU2 ira2::URA3 (PM903) derivatives were spotted as indicated above. Lower panel. Wild type strains W303-1A and isogenic cells expressing the W ∆N1 or W∆N2 alleles of CDC25 were spotted. All plates were incubated for 3 days. C) Wild type strain W303-1A was transformed with the indicated plasmids. Positive clones grown overnight in synthetic selective medium and spotted on YPD plates adjusted at the indicated pH and growth was monitored after 2 days. YCp50-RAS2* expresses a hyperactive allele (Ras2 Ala18Val19 ) of the Ras protein. YEp-GPA2* generates a constitutively active Gpa2 R273A version of the protein. Wild type W303-1A alone (WT) was plated along with strain MCY5278, a W303-1A derived strain lacking both MSN2 and MSN4 genes. Lower panel, strain MCY5278 was transformed with centromeric plasmid YCp22 (which carries a TRP1 gene marker, see main text for explanation) and plated with the equivalent strain but lacking all three TPK genes [START_REF] Gorner | Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor[END_REF]. Dilutions of the cultures were grown for 2 days at the indicated pH. B) Strain W303-1A (WT) and the msn2 msn4 derivative transformed with the empty plasmid YCp50 or the same plasmid carrying a hyperactive RAS2 Ala18Val19 allele (RAS2*). Dilutions of the cultures were spotted on YPD plates at the indicated pHs and growth monitored after 2 days. C) Strain W303-1A (open bars) and its msn2 msn4 derivative (closed bars) carrying a wild type allele of SNF1 (+) or a snf1 deletion (-) were grown at the indicated pHs. Growth is denoted as the percentage compared to the same strains cultured at pH 5.5. Data are mean ± SEM from two independent experiments performed by triplicate.

Figure 5. Alkaline pH stress induces STRE-mediated transcriptional activation.

A) The wild type strain DBY746 was transformed with episomal plasmids pPHO84-LacZ (pPHO84) and pAMS366 (pCDRE). These strains, together with strain AGS66 (carrying a STRE(7x)-LacZ reporter system integrated at the URA3 locus) were grown and subjected to high pH stress (pH 8.0). β-galactosidase activity was measured as described and it is represented as percentage over the maximum value for each strain. B) Wild type strain W303-1A (continuous lines) and its msn2 msn4 derivative (discontinuous lines) were transformed with the HXK1-LacZ reporter. Cultures grown on YP (plus 4% glucose) were subjected to high pH (8.0, triangles) or high temperature (37 ºC, squares) and samples taken at different times for β-galactosidase activity measurements. Non-induced cultures are denoted by circles. In all cases, data correspond to means ± SEM from 6 independent experiments. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607

  Biochemical Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607

Biochemical

  Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 2 .

 2 Figure 2. Changes in PKA pathway activity influence high pH tolerance. A) Wild type (WT) BY4741 cells were subjected to alkaline pH stress as described in Materials & Methods and cultures processed for cAMP determination. Data correspond to the means ± S.E.M of at least four independent experiments. B) Wild type W303-1A cells and its derivatives pde1 (strain CCV35), pde2 (PM942) and pde1 pde2 (CCV36) were spotted on YPD plates adjusted at the indicated pHs. Growth was monitored after 2 days. C) Strain BY4741 (WT) and its bcy1 derivative (strain MAR231) were grown on YPD plates adjusted at the indicated pH for 3 days. D) Wild type strain BY4742 (open bars) and its derivative DC90 (tpk1 tpk2 w3 tpk3, closed bars) were grown on liquid YPD media as described in Materials and Methods. Growth is represented as percentage over the same strain at initial pH 5.5. Data are the average ± SEM from two independent experiments.

Figure 3 .

 3 Figure 3. Alkaline pH triggers nuclear entry of the Msn2 transcription factor. Cultures of wild type BY4741 (WT) and its ira2::kanMX derivative (right panels) were transformed with plasmid pMsn2-GFP and subjected to pH stress (8.0) on YPD medium as described in the text. Samples collected at the specified times and fixed for fluorescence microscopy. B) BY4741 cells carrying the pMsn2-GFP construct were grown to exponential phase (A 660 0.8) on YPD or YP containing ethanol (2%) as carbon source, shifted to pH 8.0. The subcellular distribution of the Msn2-GFP fusion was monitored for at least 200 cells per time point. Open bars denotes fully cytoplasmic, closed bars, nuclear/cytoplasmic, and crossed bars, fully nuclear localization. Data shown correspond to a representative experiment. Three independent experiments were performed with similar results.

Figure 4 .

 4 Figure 4. Effect of lack of Msn2 and Msn4 transcription factors on high pH tolerance. A) Upper panel.Wild type W303-1A alone (WT) was plated along with strain MCY5278, a W303-1A derived strain lacking both MSN2 and MSN4 genes. Lower panel, strain MCY5278 was transformed with centromeric plasmid YCp22 (which carries a TRP1 gene marker, see main text for explanation) and plated with the equivalent strain but lacking all three TPK genes[START_REF] Gorner | Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor[END_REF]. Dilutions of the cultures were grown for 2 days at the indicated pH. B) Strain W303-1A (WT) and the msn2 msn4 derivative transformed with the empty plasmid YCp50 or the same plasmid carrying a hyperactive RAS2 Ala18Val19 allele (RAS2*). Dilutions of the cultures were spotted on YPD plates at the indicated pHs and growth monitored after 2 days. C) Strain W303-1A (open bars) and its msn2 msn4 derivative (closed bars) carrying a wild type allele of SNF1 (+) or a snf1 deletion (-) were grown at the indicated pHs. Growth is denoted as the percentage compared to the same strains cultured at pH 5.5. Data are mean ± SEM from two independent experiments performed by triplicate.

Figure 6 .

 6 Figure 6. Msn2/Msn4 dependence of the transcriptional response to alkaline stress. Upper panel, number of genes induced in the wild type and the msn2 msn4 strain after 10 or 30 min of alkaline stress, deduced from the DNA microarray analysis. Lower panel, evaluation of the Msn2/Msn4 level of dependence of the transcriptional response to high pH after 10 min (open bars) and 30 min (closed bars). Ind, Independent; WD, weakly dependent; SD, strongly dependent; TD, totally dependent. See main text for definitions.

Figure 7 .

 7 Figure 7. The dependence of ENA1 expression on the presence of Msn2/Msn4. A) Fold-change of ENA1 expression deduced from microarray data for W303-1A wild type cells (WT, empty bars) and

  MCY5278 (msn2 msn4, filled bars) exposed to pH 8.0 for the indicated times. Data are mean ± SEM from 4 microarray experiments. B) Strains with the indicated genotype (+, wild type allele; -, deletion mutant) were transformed with plasmid pKC201, which bears a LacZ fusion of the ENA1 promoter. Exponential cultures were resuspended in medium buffered at pH 5.5 (empty bars) or pH 8.0 (filled bars) for one hour and β-galactosidase activity measured. Data are mean ± SEM from 6 to 9 independent experiments. Biochemical Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1

 1 Figure 1 Casado et al.

Figure 2 Figure 3

 23 Figure 2 Casado et al.

7 Figure 4 Figure 5 Figure 6

 7456 Figure 4 Casado et al.

Figure 7

 7 Figure 7 Casado et al.

  Biochemical Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607 A c c e p t e d M a n u s c r i p t

TABLE 1 .

 1 Yeast strains used in this work. Except otherwise indicated, single BY4741derived kanMX disruptans not listed here correspond to the systematic gene disruption project[START_REF] Winzeler | Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis[END_REF].

		Biochemical Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607
		Name	Relevant genotype	Source / reference
		BY4741	MATa his3Δ1 leu2Δ met15Δ ura3Δ	[25]
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607	MAR231 W303-1A SC7 SC8 PM903 WΔN1 WΔN2 CCV35 PM942 CCV36 MCY5278 CCV174 CCV175 tpk123 msn2 msn4 CCV37 CCV38 BY4742 DC90 DBY746 AGS66 A c c e p t e d M a n u s c r i p t BY4741 bcy1::kanMX4 This work MATa ade2-1, can1-100, his3-112, leu2-3, trp1-1, ura3-1 [52] W303-1A ira1::LEU2 [42] W303-1A ira2::URA3 [42] W303-1A ira1::LEU2 ira2::URA3 [42] W303-1A cdc25::CDC25 aa907-1589/URA3 [53] W303-1A cdc25::CDC25 aa1147-1589/URA3 [53] W303-1A pde1::kanMX4 This work W303-1A pde2::URA3 [27] W303-1A pde1::kanMX4 pde2::URA3 This work W303-1A msn2::kanMX4 msn4::hphMX4 Gift from M. Carlson W303-1A nrg1::nat1 This work W303-1A msn2::kanMX4 msn4::hphMX4 nrg1::nat1 This work W303-1A tpk1::URA3 tpk2::HIS3 tpk3::TRP1 msn2::HIS3 msn4::TRP1 [14] W303-1A snf1::LEU2 This work W303-1A msn2::kanMX4 msn4::hphMX4 snf1::LEU2 This work MATα his3Δ1 leu2Δ met15Δ ura3Δ [25] BY4742 tpk1::kanMX::HIS3 tpk2 w3 tpk3::LEU2 J.M. Thevelein MATa ura3-52 leu2-3112 his3-1 trp1-239 D. Botstein DBY746 URA3-STRE(7x)-lacZ [29]

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Biochemical Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements

We thank Raquel Serrano and Antonio Casamayor for support. The excellent technical assistance of Montserrat Robledo and Anna Vilalta is acknowledged. We thank the generous help of Francisco Estruch, Martha Cyert, Jeanne Hirsch, Johan Thevelein, Christoph Schüller, Marian Carlson, Renata Tisi and Enzo Martegani for constructs, strains and advice.

Funding

Biochemical Journal Immediate Publication. Published on 13 Jul 2011 as manuscript BJ20110607 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110607

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


Alejandra Collazos

Nicholas Michael

Richard Dh Whelan

Gavin Kelly

Harry Mellor

Leon C H Pang

Nick Totty

Peter J Parker
email: peter.parker@cancer.org.uk

Site recognition and substrate screens for PKN family proteins

Keywords: Protein Kinase N, substrates, phosphorylation consensus motif AA, arachidonic acid, PKC, protein kinase C, PKN, protein kinase N, EGFR epithelial growth factor receptor, PI3K, phosphoinositide3-kinase

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The PKC-related kinase family are a subfamily of the serine/threonine kinases with a Cterminal kinase domain closely related to those of PKC while their amino termini are distinct comprising a conserved repeated domain (HR1a,b,c) followed by a C2-related domain. PKNs are activated by fatty acids [START_REF] Mukai | Activation of PKN, a novel 120-kDa protein kinase with leucine zipper-like sequences, by unsaturated fatty acids and by limited proteolysis[END_REF] and phospholipids in vitro [START_REF] Yu | Isolation and characterization of a structural homologue of human PRK2 from rat liver. Distinguishing substrate and lipid activator specificities[END_REF], although the in vivo significance of this activation remains unclear [START_REF] Palmer | Activation of PRK1 by phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. A comparison with protein kinase C isotypes[END_REF].

The PKN family play an important role in diverse functions including regulation of cell cycle [START_REF] Schmidt | Rho GTPases regulate PRK2/PKN2 to control entry into mitosis and exit from cytokinesis[END_REF], receptor traffic [START_REF] Gampel | Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB[END_REF], vesicle transport [START_REF] Torbett | Hyperosmotic-induced protein kinase N 1 activation in a vesicular compartment is dependent upon Rac1 and 3phosphoinositide-dependent kinase 1[END_REF] and apoptosis [START_REF] Takagi | Activation of PKN mediates survival of cardiac myocytes in the heart during ischemia/reperfusion[END_REF]. The PKNs are implicated in signal transduction as effectors of Rho, Rac and PI3Kinase [START_REF] Bourguignon | Hyaluronan-CD44 interaction stimulates Rac1 signaling and PKN gamma kinase activation leading to cytoskeleton function and cell migration in astrocytes[END_REF][START_REF] Leenders | PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase[END_REF][START_REF] Zhu | Signaling via a novel integral plasma membrane pool of a serine/threonine protein kinase PRK1 in mammalian cells[END_REF]. In particular, PKN1 has been linked to stress-induced pathways implicated upstream of c-Jun transcription via p38 activation induced on hyperosmotic stress [11] with associated activation of PKN1 [START_REF] Torbett | Hyperosmotic-induced protein kinase N 1 activation in a vesicular compartment is dependent upon Rac1 and 3phosphoinositide-dependent kinase 1[END_REF]. PKN is also linked to the ligand-dependent transcriptional activation of the androgen receptor [START_REF] Metzger | A novel inducible transactivation domain in the androgen receptor: implications for PRK in prostate cancer[END_REF][START_REF] Metzger | Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation[END_REF]. PKN1 has been implicated as well in milk secretion by regulating the tight junction in the mammary gland [START_REF] Fischer | Impaired tight junction sealing and precocious involution in mammary glands of PKN1 transgenic mice[END_REF]. PKN2 plays a role alongside fyn in controlling cell-cell adhesion in keratinocytes [15] and the maturation of apical juctions [START_REF] Wallace | The Rho target PRK2 regulates apical junction formation in human bronchial epithelial cells[END_REF]. PKN2 can modulate as well migration in astrocytes by up-regulating cortactin phosphorylation [START_REF] Bourguignon | Hyaluronan-CD44 interaction stimulates Rac1 signaling and PKN gamma kinase activation leading to cytoskeleton function and cell migration in astrocytes[END_REF]. PKN3 has been identified as an effector required for malignant cell growth downstream of activated PI3K [START_REF] Leenders | PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase[END_REF]. More recently, it has been shown that knock-down of PKN3 can decrease the growth of prostate and pancreatic tumours and prevents lung metastasis in mouse models [17,18].

More than 20 proteins and several peptides have been shown to be phosphorylated by PKN1 and PKN2 including α-actinin, adducin, Cdc25C, vimentin and TRAF1 [START_REF] Mukai | Activation of PKN, a novel 120-kDa protein kinase with leucine zipper-like sequences, by unsaturated fatty acids and by limited proteolysis[END_REF][START_REF] Yu | Isolation and characterization of a structural homologue of human PRK2 from rat liver. Distinguishing substrate and lipid activator specificities[END_REF][START_REF] Metzger | Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation[END_REF][19][START_REF] Fukata | Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility[END_REF][START_REF] Harrison | Protein kinase C-related kinase targets nuclear localization signals in a subset of class IIa histone deacetylases[END_REF][START_REF] Kato | Negative regulation of constitutive NF-kappaB and JNK signaling by PKN1-mediated phosphorylation of TRAF1[END_REF][START_REF] Kim | Protein kinase Crelated kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation[END_REF][24][START_REF] Lee | Protein kinase Crelated kinase 2 phosphorylates the protein synthesis initiation factor eIF4E in starfish oocytes[END_REF][26][START_REF] Misaki | PKN delays mitotic timing by inhibition of Cdc25C: possible involvement of PKN in the regulation of cell division[END_REF][28][START_REF] Takahashi | Regulation of a mitogen-activated protein kinase kinase kinase, MLTK by PKN[END_REF][30]. In contrast, there are no known substrates for PKN3. The phosphorylation consensus motif of a protein helps to determine which substrates are phosphorylated by a kinase. Concerning PKN phosphorylation consensus motifs, Zhu et al. had evaluated the importance of Arg in 7 Nterminal and 3 C-terminal positions of serine directed peptides for several kinases, including PKN1. [START_REF] Zhu | A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families[END_REF]. Very recently, Shiga et al confirmed these results using a combinatorial peptide library method. [START_REF] Shiga | Development of an intracellularly acting inhibitory peptide selective for PKN[END_REF]. These observations provide a theoretical prediction for PKN phosphorylation motifs.

The developing of new technologies such as peptide and protein arrays has made it practical to investigate the optimal phosphorylation consensus motif and identify new substrates and partners, not only for one kinase, but for several proteins at the same time [START_REF] Zhu | A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families[END_REF][START_REF] Schnack | Identification of novel substrates for Cdk5 and new targets for Cdk5 inhibitors using high-density protein microarrays[END_REF][START_REF] Vlad | A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substrates[END_REF]]. Here we have investigated kinase substrate specificity of PKN1 and PKN3 re-evaluated using a peptide array and assessed utility using newly reported substrates for the kinase. We confirm that PKN1 follows the phosphorylation consensus motif by the phosphorylation in vitro of a newly identified PKN substrate, CLIP-170. Furthermore, novel substrates for PKN1 were identified using a protein array. These results were validated by identifying the site of EGFR phosphorylation by PKN1 as Thr-654; this site conforms to the consensus defined by peptide array and is shown to be modulated in vivo through PKN.

EXPERIMENTAL

Reagents

Arachidonic acid was purchased from Sigma. P]-ATP was purchased from GE Healthcare. EGF was from Calbiochem. The peptide arrays (Pepchip kinomics) were bought from Pepscan Presto BV, Lelystad, The Netherlands. The protoarrays were from Invitrogen. CLIP-170 was a kind gift from Frank Perez [START_REF] Perez | CLIP-170 highlights growing microtubule ends in vivo[END_REF].

DNA cloning and Protein production

The full length cDNAs for human PKN1 and PKN3 were subcloned into a modified pFastbac-1 vector (Invitrogen) that encodes an N-terminal glutathione S-transferase (GST) tag and a thrombin cleavage site. Recombinant baculoviruses were generated using the Bacto-Bac Baculovirus Expression System (Invitrogen). Routine baculovirus production of human GST-PKN1 and GST-PKN3 in Spodoptera frugiperda cells (Sf9 cells) was achieved.

The first 650 amino acids of CLIP-170 or the whole protein was fused to GST into the pGEX-2TK vector. To subclone the first 650 amino acids and the whole CLIP-170, one common forward primer was used: 5'ggatccatgagtatgctaaagccaagtggg3'. A reverse primer 5'gaattctcacctcaaggcttccatctcttt3' was used to subclone the first 650 amino acids and the primer 5'gaattctcagaaggtttcgtcgtcattgca3' was used to subclone the whole protein. The recombinant proteins were produced and purified from E. coli. The GST fused to CLIP-170 was removed by incubating the purified protein with thrombin at 4 o C overnight.

Peptide Array

A peptide array (Pepchip kinomics) with specific phosphorylation sites was used to study PKNs phosphorylation consensus motif. Each array has 960 peptides of 11 amino acids long derived from known phosphorylation sites from human protein sequences. In addition, each peptide comes annotated with reported upstream kinases. Moreover, the peptides that belong to a substrate have a physiological relevance in stress, growth, differentiation and other responses. The glass slides contain 976 peptides (including 16 control peptides) spotted in triplicate.

For array kinase assays, the glass slides containing the peptides were incubated with each PKN in the following buffer: 20 mM Tris pH.7.5, 40 μM Arachidonic acid, 4 mM MgCl 2 , 10% Glycerol, 0.01 mg/ml BSA, 0.01% V/V Brij-35, 10 μM ATP and 300 μCi/ml [γ-33 P]-ATP. PKN1 and PKN3 were diluted to 26 μg/ml and 13 μg/ml respectively in a buffer containing 20 mM Tris pH 7.5, 1 mM EDTA pH 8, 0.2% Triton X-100 and 1 mM DTT. PKN1, PKN3 or no kinase were incubated on the PepChip kinomics arrays for 2 h in a humidified incubator at 37°C. Subsequently, the arrays were washed in 2 M NaCl, 1% Triton X-100, PBS, 0.1% Tween 20 and water, after which slides were dried and exposed to an Xray film screen for 2h or 24h for PKN1 and 3 respectively, in order to visualise in the linear range the reaction. Imaging of the incorporated [γ-33 P]-ATP was performed using a conventional scanner (Epson Expression 1680). Data analysis was performed using Microsoft Excel and the R Statistical Package (http://www.R-project.org). After image acquisition and quantification using ImageQuantTL software (Amersharm, Biosciences), the data of each triplicate was normalized with a median-centring method. Signals with high intensity in the PKN slide but low intensity in the control (no PKN) slide were considered as peptide substrates recognised by PKN1 or PKN3. After performing an ANOVA F-Test, the peptides that had a p-value higher than 9.7 x 10 -4 were eliminated. The peptides were sorted by spot intensity and the top 20 peptides based were chosen to analyse the phosphorylation consensus motif of each kinase using the WebLogo v3 application (http://weblogo.threeplusone.com/) [START_REF] Crooks | WebLogo: a sequence logo generator[END_REF].

Protein arrays

Glass slides containing 4700 human proteins from Invitrogen were used to identify PKN1 substrates. The slides were incubated with PKN1 following the manufacturer's instructions P]-ATP. PKN1-GST was diluted in a buffer containing 20 mM Tris pH 7.5, 1 mM EDTA pH 8, 0.2% Triton X-100 and 1 mM DTT (the final concentrations in the protein array assay were: 168 μM EDTA, 0.033% Triton X-100 and 168 μM DTT). The final PKN1-GST concentration in the assay was 52 nM. The slides were incubated with or without (control) kinase for 1h at 37 o C. The slides were exposed to an X-ray film for different times, scanned and the spots were analysed using the software provided by Invitrogen in order to identify the hits.

Kinase Assay

Three μg of CLIP-170 or 0. Western blot SDS-PAGE was performed using the NuPAGE system (Invitrogen) with 4-12% Bis-Tris gels. Proteins were transferred to a PVDF membrane and probed as indicated in the Figure legends. Levels of PKN1, 2 and 3 in A431 were quantified against recombinants PKN1, 2 and 3 by westerns blot. Quantitation was derived using an ImageQuant LAS 4000 mini instrument to obtain a series of exposures of the ECL readout; ImageJ was used to determine the levels of expression relative to recombinant PKN proteins.

Mass spectrometry

In vitro phosphorylated CLIP-170 was reduced with 10 mM dithiothreitol (Sigma) followed by alkylation with 50 mM iodoacetamide (Sigma) prior to digestion in-gel with trypsin (Promega) overnight at 37 o C. Extracted peptides were analysed by on-line nanoLC-MS/MS using a linear ion trap instrument (Applied Biosystems Q-TRAP 4000).

siRNA Transfection A431 cells were maintained in DMEM/10% FBS (Gibco) + penicillin/streptomycin at 37 o C, in a humified atmosphere containing 10% CO 2 . On the first day, the cells were transfected with 25 nM siRNA Luciferase (AATCGAAGTATTCCGCGTACG) or PKNs (8.33 nM each PKNs: PKN1 (AAGGGCACGGGAACTGGAGTT), PKN2 (AAGCATGGCATGTGTCTCTATT) and PKN3 (GAGAGCCTGTACTGCGAGAAG; as described elsewhere [START_REF] Leenders | PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase[END_REF]). siRNA transfections were carried out with Lullaby (OZ-Biosciences). 24 h later, the medium was replaced by fresh medium. 96 h after the siRNA transfection, the media was replaced by CO 2 independent media/0.5% FBS (Gibco) for 5h. The cells were collected on Nupage LDS sample buffer 2x (Invitrogen) + 2 mM EDTA.

RESULTS

Biochemical

PKN phosphorylation consensus motif

We used a peptide library in order to study the influence of the residues up to 5 amino acids away (C-terminal and N-terminal) from candidate phosphorylation sites for PKN1 and PKN3. Based on the top 20 phosphorylated peptides (Fig. 1A), PKN1 and PKN3 were shown to have a preference for the phosphorylation of Serines, which have an Arg in position -3 (Fig. 1B and Fig. S1). In fact PKN1 and 3 show an 80 and 85% preference for Arg in position -3, respectively. In contrast, PKN1 and 3 do not tolerate Arg in position +1 and -1 respectively. PKN1 shows a 50% and 40% of preference for Arg in positions +2 and -2 respectively. In addition, PKN3 shows a 70% preference for hydrophobic amino acids (Ile, Met, Leu, Val, Phe, Trp, Tyr) in position +1 against a 55% preference for PKN1. These PKNs show a strong preference (at least 70%) for neutral amino acids (Ser, Gly, His, Thr, Ala, Pro) in position -5 for PKN1 and -1 and +5 for PKN3. Finally, PKN1 shows a 70% preference of hydrophilic amino acids (Arg, Lys, Asp, Glu, Asn, Glu) in position -2. If we take a 70% preference cut-off, the PKN1 phosphorylation consensus motif is Ne-x-Arg-Hi-x-Ser-Z-x-x-x-x and the PKN3 is x-x-Arg-x-Ne/Z-Ser-Ho-x-x-x-Ne. (where x denotes a position with no strict amino acid requirements, Z a position strictly not tolerating Arg, Ne a neutral amino acids, Hi a hydrophilic amino acid and Ho a hydrophobic amino acid). Even though, PKN1 and 3 phosphorylated peptides were not identical, the kinases share peptide substrates in the array. Of the 20 top hits for each kinase, 5 peptides were hits for both PKN1 and 3; of the top 30, 12 peptides were hits for PKN1 and 3. The results indicate that PKN1 and PKN3 share recognition, but retain some specificity.

PKN phosphorylates CLIP-170

The identification of a PKN phosphorylation consensus motif was assessed using the hitherto uninvestigated PKN substrate CLIP-170 (H. Mellor and P. Parker unpublished data). We confirmed that CLIP-170 is phosphorylated by PKN1 and 3 in vitro. Figure 1C shows that PKN1 phosphorylates equally the N-terminal half of CLIP170 and intact CLIP-170. PKN3 phosphorylates as well the N-terminal half of CLIP-170 as shown in Fig. 1D. By mass spectrometry we identified S312 as a target for PKN, this is a site identified previously as being occupied in CLIP-170 (http://www.phosphosite.org/). Sequence surrounding S312 follows the Arg-x-Ne-S-Z-x-x-x-x-Ne pattern, consistent with the specificity analysis derived from the peptide array (Fig. 1E).

New PKN substrates and partners

To avoid any bias from the known substrate approach, we performed a protein array in order to identify PKN1 substrates. We found 22 substrates (Fig. 2 and Table 1), of which 20 were previously unidentified (the two known substrates validated the screening approach: CDC25C and Adducin 2 [START_REF] Fukata | Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility[END_REF][START_REF] Misaki | PKN delays mitotic timing by inhibition of Cdc25C: possible involvement of PKN in the regulation of cell division[END_REF]). For the identified substrates, 21 of 22 retain at least one motif that follows the phosphorylation consensus motif Arg-x-x-[Ser/Thr]-x.

PKNs phosphorylate EGFR at a consensus site, Thr-654

In order to validate the protein array results, we analysed EGFR. To confirm this phosphorylation event, we performed a kinase assay in solution with purified proteins. We compared the sequences of different EGFR constructs spotted on the Invitrogen protein array and we observed 2 different EGFR sequences, only one of which was phosphorylated by PKN1 (Fig. 3A). Sequence analysis of these 2 different versions of EGFR indicates 2 possible sites of phosphorylation that follow the PKN consensus. One site corresponds to Ser 1071. However, the peptide that contains this Ser (Leu-Gln-Arg-Tyr-Ser-Ser-Asp-Pro-Thr-Gly-Ala) was ranked number 235 in the PKN1 peptide array, so it was unlikely to be the PKN1 phosphorylation site. The second site was Thr-654 and it although it had previously been shown that PKN can phosphorylate in vitro a peptide corresponding to this site (Val- Arg-Lys-Arg-Thr-p-Leu-Arg-Arg-Leu) [START_REF] Yu | Isolation and characterization of a structural homologue of human PRK2 from rat liver. Distinguishing substrate and lipid activator specificities[END_REF][START_REF] Morrice | A cardiolipinactivated protein kinase from rat liver structurally distinct from the protein kinases C[END_REF] it was unknown if the protein was targeted by PKNs at this site. In order to determine if PKNs could phosphorylate EGFR at Thr-654, we used a site-specific antibody. Figure 3B and Figure S4 shows that Thr-654 is phosphorylated by PKN1 and PKN3 in vitro.

PKNs modulate phosphorylation of EGFR on Thr-654 in cells

Having demonstrated the phosphorylation by PKN in vitro, we evaluated if PKNs contributed to EGFR Thr-654 phosphorylation in vivo. Using siRNAs targeting all three PKN isoforms (all three PKN proteins are expressed in these cells), we observed in A431 cells that the constitutive phosphorylation of Thr-654 in EGFR was decreased in comparison with the control condition (Fig. 3C and3D). In response to EGF, PKN1/2/3 knock-down did not block the increase in Thr-654 phosphorylation, although there was a reduced total phosphorylation reflective of the basal effect. This indicates that the PKN effect on Thr-654 under basal conditions is independent of EGF and is reflective of a transmodulation pathway.

In order to determine which PKN family member(s) is responsible for this effect, we deconvoluted the PKN siRNAs. PKN1 knock-down was found to have a very similar effect on EGFR Thr 654 phosphorylation as that seen for the knock-down of all PKNs (Fig. 3E,F). PKN2 had a minor effect and PKN3 none at all, although we note that in contrast to PKN1 and PKN2, the knock-down of PKN3 is not efficient in A431 cells. The evidence suggests that the PKN responsible for controlling basal EGFR Thr654 phosphorylation in A431 cells is principally PKN1. This result may reflect the fact that the PKN1 is the major PKN isoform expressed in these cells; quantitative analysis of expression in A431 cells shows that PKN1 is expressed at 0.44 μg/mg of cell protein, PKN2 0.13 μg/mg of cell protein and PKN3 0.03 μg/mg of cell protein.

DISCUSSION

We have analysed the phosphorylation consensus motifs of PKN1 and 3 using a peptide array. PKN1 and 3 selected for similar phosphorylation consensus motifs based on residues both in the N-terminal and C-terminal sides of the phosphorylation site. The phosphorylation consensus motifs are consistent with a previously unmapped site we identify here in the PKN substrate CLIP-170. To further assess the predictive capacity of the motif identified, we used an array approach to identify protein substrates for PKN1. This screen identified a number of in vitro substrates amongst which was the EGFR. The arrayed proteins (two different length forms of EGFR) and the determined sequence specificity of PKN predicted that Thr-654 was targeted by PKN on the EGFR. In vitro we show that EGFR is phosphorylated by PKN1 on Thr-654, consistent with this specificity. Finally, we show that PKN depletion by siRNA decreased the constitutive EGFR phosphorylation on Thr-654 in A431 cells and that this is primarily a function of PKN1. We conclude that PKN specificity has predictive capacity as applied to both a predetermined substrate (CLIP170) and a newly identified protein substrate (EGFR). In the case of the latter, the phosphorylation at Thr-654 has hitherto been ascribed to PKC; the evidence here is that this is more accurately defined as a PKC superfamily target site.

Phosphorylation consensus motif

Previous groups have studied PKN phosphorylation motifs. Zhu et al. evaluated only the importance of Arg in positions near the phosphorylated serine in peptides phosphorylated by PKN1 and they did not analyse the importance of other amino acids as the study does here. Zhu et al. showed that Arg was important in position -3, -2 and +2 and Arg was not allowed in position +1 and +3 [START_REF] Zhu | A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families[END_REF]. We confirm these findings, except that we did not find a nonallowance of Arg in position +3: PKN1 and 3 have a preference for Arg in position -3 (80 and 85% respectively, Fig. 1 and Fig. S1) and a preference in position -2 (40 and 30% respectively). More recently, Shiga et al. using a combinatorial peptide library method, confirmed the results of Zhu et al. and showed that both PKN1 and PKN3 phosphorylate preferentially serines with an Arg in position -3. Shiga et al. also showed that these kinases have a preference for hydrophobic amino acids in position +1 and basic amino acids downstream of the phosphorylation site [START_REF] Shiga | Development of an intracellularly acting inhibitory peptide selective for PKN[END_REF], concluding that the best phosphorylation consensus motif for PKN1 and PKN3 is Lys-Arg-Arg-Lys-Pro-Ser-Phe-Arg-Asn-Pro. Shiga et al. found a preference for lysine in position -5 and -2. In general, we see a very similar pattern of recognition here (albeit basic residues rather than any lysine specific preferences). However Shiga et al. noted a preference for proline in position -1, asparagine in position +3 and proline in position +5; we did not find these preferences. These differences between the three studies likely reflects the nature of the arrays: Zhu et al. used degenerate peptides where only Arg is changed, Shiga el al. used degenerate peptides were one amino acid is keep constant and our study used a large array of synthetic peptides based on known target sites. The key feature is however consistent between all three studies and that is the very strong preference for Arg at the -3 position.

Since PKN belongs to the AGC family we expected that the PKN phosphorylation consensus motif would follow a pattern similar to that of other AGC family members. If we compare with other kinases that belong to distinct branches of kinases, it is evident that the pattern is very different. For example, CDC2 phosphorylates peptides with the pattern x-Ser-Pro-x-Lys while CK2 targets the consensus [Ser/Thr]-[Asp/Glu]-x-[Asp/Glu] [38, 39]. These motifs are very different from the PKN motif Arg-x-x-[Ser/Thr]-x-x-x. The presence of Arg in position -3 is similar to other AGC family (basophilic) kinases. (PKC: [Lys/Arg]-x-x-[Ser/Thr]-x-[Lys/Arg]; PKA: Arg-Arg-x-[Ser/Thr]-Phe) [START_REF] Ubersax | Mechanisms of specificity in protein phosphorylation[END_REF]. It is possible that the presence of peptides phosphorylated by members of the AGC family could influence the PKN results, however if we analyse the whole collection of peptides spotted in the slide (960 peptides), only 20% present Arg in position -3 (Fig. S2). Therefore it is not the case that the preference of Arg in position -3 by PKN1 and 3 only reflects a bias in the array composition since peptides substrates identified were enriched to ~80% probability of Arg at the -3 site for PKN1.

PKN and PKC are close phylogenetic neighbours. As expected, they share some similarities in the phosphorylation consensus motif (Fig. S3). The preference of Arg in position -3 and the hydrophobic amino acid in position +1 are significant similarities. If we analyse more closely, PKN1 and 3 have a preference for Arg in position -2 (40 and 30% respectively, Fig. 1 and Fig. S1) and many PKC substrate peptides have Lys or Arg in this position as well. In addition, PKN1 presents a preference for Arg (50%) in position +2 as do PKCα, and η; PKCβ and γ prefer a Lys residue here. Concerning PKN3, there is a preference for Arg in position -5 (>30%), as for PKCα, γ, δ and η. So, there is a substantial overlap in specificity indicative of shared targets in vivo where phosphorylation is triggered under different circumstances by regulatory inputs particular to individual members this family of proteins [40], controlling the same downstream events. Evidence for such properties have come from the use of a dPKN-dPKC chimera to rescue dPKN loss [START_REF] Betson | A rho-binding protein kinase C-like activity is required for the function of protein kinase N in Drosophila development[END_REF]42].

It was proposed that PKN has a pseudosubstrate in the ACC1 region, overlapping the Rho binding domain (Fig. S3). This region corresponds to amino acids 39 to 53. The substitution of Ile-46 with a Ser converts a peptide based on this sequence into a potent substrate for PKN, while the authentic Ile counterpart inhibits activity of PKN [43]. However, the deletion A c c e p t e d M a n u s c r i p t of this region does not activate PKN [START_REF] Yoshinaga | Mutational analysis of the regulatory mechanism of PKN: the regulatory region of PKN contains an arachidonic acid-sensitive autoinhibitory domain[END_REF]. If we compare this region with the phosphorylation consensus motif, it has an Arg for PKN1, and PKN2 and a Lys for PKN3 in position -3. However, there are some discrepancies between the sequences. For example, the PKN1 region has an Arg in position +1, while the phosphorylation consensus motif does not permit this amino acid in this position. This could explain why this region (amino acid 39 to 53) does not operate as a pseudosubstrate site. However, as for other proteins, the presence of a consensus phosphorylation site in a protein, does not guarantee that the protein is a substrate in vivo and authentic phosphorylation sites do not always conform to the consensus, [START_REF] Ubersax | Targets of the cyclin-dependent kinase Cdk1[END_REF]. Evidently this is the case as well for PKN. Some peptides that have been shown to be phosphorylated by PKN1 do not follow the phosphorylation consensus pattern (Fig. S3).

PKN substrates and EGFR

We found 22 susbstrates in vitro of which 15 are kinases. This is a high proportion, reflective of their representation in the protein array. However these are not false positives lighting up because they are protein kinases, since the software is designed to eliminate the kinases that autophosphorylate; in order to define a protein as a hit the mean signal of the replicates (in the PKN1 slide) needs to be at least 1.5 times that of the mean signals from the corresponding proteins on the negative control (no kinase). So, we are confident that these kinases are PKN substrates in vitro.

We have shown that PKN1 phosphorylates EGFR on Thr-654 in vitro and in vivo. The Thr-654 site is a known phosphorylation site for PKC in vivo [START_REF] Davis | Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor[END_REF]47]. Thr-654 is located on the cytoplasmic side of the plasma membrane nine residues distant from the transmembrane domain. It has been shown that the phosphorylation of EGFR at Thr-654 by PKC reduces the Tyrosine kinase activity of the receptor [47], inhibits lysosomal degradation to promote recycling back to the cell surface [48] and positively mediates drug inhibition of prostate tumour growth in vivo [49].

We found that the PKN effect on Thr-654 phosphorylation was only observed under basal conditions and had little influence on the response to EGF. This suggests that the effect of PKN in Thr-654 phosphorylation under basal conditions is EGF-independent. By contrast the EGFR phosphorylation on Thr-654 by PKC can be the product of EGF binding [50]. That indicates that PKC and PKN can control the same substrate however the input that regulates this phosphorylation event is different. EGFR can be transmodulated by other growth factors [51] and it is anticipated that PKN lies on one of these pathways reflected in its requirement for the basal Thr-654 phosphorylation in A431 cells.

In summary, the peptide and protein library approach has provided information about substrate specificity for PKN1 and PKN3 and new substrates for PKN1. In addition, we have defined CLIP-170 and EGFR as substrates for PKN1 and PKN3 and determined the sites involved. The predictive nature of the specificities defined and the protein substrates identified in vitro should serve to help determine the signalling pathways for these kinases. 3D and with de-convoluted PKNs siRNA. After 96h, the cells were depleted of serum for 5 h in a CO 2 independent medium containing 0.5% FBS. The cells were treated or not with EGF 100ng/ml for 30 min. The cells were collected and probed for EGFR Thr-654, total EGFR, PKN1, PKN2 and tubulin-α as a loading control. (F) Quantification of 2 different experiments.

AUTHOR CONTRIBUTION

Table and figure legends

Supplementary Figures

  Site recognition and substrate screens for PKN family proteins. Alejandra Collazos * , Nicholas Michael †, Richard, D. H. Whelan * , Gavin Kelly ‡, Harry Mellor §, Leon C. H. Pang ¶, Nick Totty † and Peter J. Parker * , ** , 1

  Journal Immediate Publication. Published on 12 Jul 2011 as manuscript BJ20110521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110521 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 12 Jul 2011 as manuscript BJ20110521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110521 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2011 as manuscript BJ20110521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110521 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 12 Jul 2011 as manuscript BJ20110521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110521

  Alejandra Collazos made the Clip-170 constructs and the experiments presented in the figures of the paper. Nicholas Michael and Nick Totty performed the mass spectrometry experiments. Richard, D. H. Whelan helped with the preliminary EGFR experiments. Gavin Kelly analysed the peptide array data. Harry Mellor identified Clip-170 as a PKN susbstrate. Leon C. H. Pang made the PKN1 and PKN3 recombinant baculoviruses constructs. Alejandra Biochemical Journal Immediate Publication. Published on 12 Jul 2011 as manuscript BJ20110521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110521 A c c e p t e d M a n u s c r i p t Collazos and Peter J. Parker planned the experiments, analysed the data and wrote the manuscript. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .

 1 Figure 1. PKN phosphorylation consensus motif. (A) Representative image of pepchip array (Pepscan) of one of three arrays phosphorylated by PKN1. The open headed arrows show the top 20 phosphorylated peptides. The 25 th and 50 th phosphorylated peptides are also shown for reference. (B) Phosphorylation consensus motif using WebLog version 3.0 based on the top 20 phosphorylated peptides. The x axis shows the positions in relation to the Ser/Thr (position 0). The y axis in the upper panel shows the bits. The graphs show the amino acid preference near the Ser/Thr. The height of the letter is proportional of the frequency in the top 20 peptides. The lower graph shows the grouped amino acid probability: S groups neutral amino acid (Ser, Gly, His, Thr, Ala, Pro); R groups hydrophilic amino acids (Arg, Lys, Glu, Asp, Asn, Gln); I groups hydrophobic amino acids (Ile, Met, Leu, Val, Phe, Trp, Tyr). The red line shows 70 % probability. (C) Phosphorylation of CLIP-170 by PKN1 in an in vitro kinase assay with CLIP-170 as substrate. The first 650amino acids or the whole CLIP-170 (1393 amino acids) were tested. The samples were incubated for 0-60 min, resolved by SDS-PAGE, stained using Coomassie, and dried. γ[-32 P]-incorporation was analysed by exposing the gel to a X-film. The data is representative of 3 different experiments. (D) Phosphorylation of CLIP 170 (first 650 amino acids) by PKN1 and PKN3 was carried out as in 1C. The autoradiographs are shown. E, Identification of phosphorylation

Figure 2 .

 2 Figure 2. PKN1 protein array. An image of substrate protein array is shown. Comparison of the control slide (no kinase) and the slide incubated in presence of PKN1-GST. The positive controls (proteins that autophosphorylate) are present in the negative control slide and the slide incubated with PKN1; these are important for alignment and normalisation. The 4 th and 21 st hits are shown in the magnified region.

Figure 3 .

 3 Figure 3. Phosphorylation of EGFR by PKN1. (A) EGFR sequences present in the susbstrate array; only the second sequence was phosphorylated by PKN1.In the first region of this EGFR substrate, there is a Thr that has an Arg in position -3, Thr-654. X denotes a position with no strict amino acid requirements, Z a position strictly not tolerating Arg, Ne a neutral amino acids (Ser, Gly, His, Thr, Ala, Pro), Hi a hydrophilic amino acids (Arg, Lys, Glu, Asp, Asn, Gln) and Ho a hydrophobic amino acids (Ile, Met, Leu, Val, Phe, Trp, Tyr). (B) In vitro kinase assay using the kinase domain of EGFR L861Q as a substrate. The samples were incubated for 15 min, resolved by SDS-PAGE, western blotted and probed with the EGFR Thr-654 antibody and total EGFR antibody as indicated. (C) A431 cells were transfected with 20 nM Luciferase or 20 nM PKNs siRNA. After 96h, the cells were depleted of serum for 5 h in a CO 2 independent medium containing 0.5% FBS. The cells were treated or not with EGF 100ng/ml for 30 min. The cells were collected and probed for EGFR Thr-654, total EGFR, PKN1, PKN2, PKN3, and tubulin-α as a loading control. This blot is representative of at least 4 different experiments. (* p<0.05) (D) Quantification of 4 different experiments. (E) A431 cells were treated as in Fig.3Dand with de-convoluted PKNs siRNA. After 96h, the cells were depleted of serum for 5 h in a CO 2 independent medium containing 0.5% FBS. The cells were treated or not with EGF 100ng/ml for 30 min. The cells were collected and probed for EGFR Thr-654, total EGFR, PKN1, PKN2 and tubulin-α as a loading control. (F) Quantification of 2 different experiments.

  Figure S1. PKN3 phosphorylation consensus motif. A phosphorylation motif illustrated using Weblog version 3.0 based on the top 20 phosphorylated peptides phosphorylated by PKN3. The x axis shows the positions in relation to the Ser/Thr (position 0). The y axis in the upper graph show the bits. The graphs show the amino acid preference near the Ser/Thr. The height of the letter is proportional of the frequency in the top 20 peptides. The lower graph shows the grouped amino acid probability: S groups neutral amino acids (Ser, Gly, His, Thr, Ala, Pro); R groups hydrophilic amino acids (Arg, Lys, Glu, Asp, Asn, Gln); I groups hydrophobic amino acid (Ile, Met, Leu, Val, Phe, Trp, Tyr). The red line shows 70 % probability.

Figure S2 .

 S2 Figure S2. Phosphorylation motif in the peptide library. (A) The phosphorylation motif of peptides phosphorylated by proteins members of the AGC family. 204 peptides printed on the array were analysed using the WebLogo version 3.0. The left and right graph show the bits and the probability respectively, as Fig 1B. (B) Phosphorylation motif of all peptides present on the Pepchip kinomics. 960 peptides printed on the peptide slide were analysed using WebLogo version 3.0.

Figure S4 .Biochemical

 S4 Figure S4. Phosphorylation of EGFR by PKN1 and PKN3. In vitro kinase assay using the kinase domain of EGFR L861Q as a substrate. The samples were incubated for 15 min, resolved by SDS-PAGE, western blotted and probed with the EGFR Thr-654 antibody and total EGFR antibody as indicated.

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  with the following modifications. Briefly, the slides were incubated with the kinase in 20 mM Tris pH 7,5, 40 μM Arachidonic Acid, 4 mM MgCl 2 , 1% NP40, 5 mg/ml BSA, 1 μM ATP, and 33 nM [γ-

		Biochemical Journal Immediate Publication. Published on 12 Jul 2011 as manuscript BJ20110521
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110521	A c c e p t e d M a n u s c r i p t

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Table 1 PKN1

 1

	Database

Table 1 .

 1 Substrates identified for PKN1. The proteins identified as PKN1 substrates in the substrate screen are classified by function. The protein full name and the database ID are also shown.

  3, Thr-654. X denotes a position with no strict amino acid requirements, Z a position strictly not tolerating Arg, Ne a neutral amino acids (Ser, Gly, His, Thr, Ala, Pro), Hi a hydrophilic amino acids (Arg, Lys, Glu, Asp, Asn, Gln) and Ho a hydrophobic amino acids (Ile, Met, Leu, Val, Phe, Trp, Tyr). (B) In vitro kinase assay using the kinase domain of EGFR L861Q as a substrate. The samples were incubated for 15 min, resolved by SDS-PAGE, western blotted and probed with the EGFR Thr-654 antibody and total EGFR antibody as indicated. (C) A431 cells were transfected with 20 nM Luciferase or 20 nM PKNs siRNA. After 96h, the cells were depleted of serum for 5 h in a CO 2 independent medium containing 0.5% FBS. The cells were treated or not with EGF 100ng/ml for 30 min. The cells were collected and probed for EGFR Thr-654, total EGFR, PKN1, PKN2, PKN3, and tubulin-α as a loading control. This blot is representative of at least 4 different experiments. (* p<0.05) (D) Quantification of 4 different experiments. (E) A431 cells were treated as in Fig.

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

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ACKNOWLEDGEMENTS

We should also like to acknowledge the support of CRT who provided the protein arrays. A.C. was funded by the FRM (Foundation de la Recherché Medical, France) and CR-UK.

FUNDING

This work was supported in part by a grant from the European Commission Research [project no. 503467] and by Cancer Research UK and Cancer Research Technology. Biochemical Journal Immediate Publication. Published on 12 Jul 2011 as manuscript BJ20110521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110521 A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.