Annie Rodolosse

Maria-Luisa Campos

Ilse Rooman

Mathieu Lichtenstein

Francisco X Real
email: preal@cnio.es

R Macdonald

R Wagener

P/CAF MODULATES THE ACTIVITY OF THE TRANSCRIPTION FACTOR p48/Ptf1a INVOLVED IN PANCREATIC ACINAR DIFFERENTIATION

Keywords: pancreas, p48/PTF1a, p

p48, also called Ptf1a, is a tissue-restricted basic helix-loop-helix transcription factor critical for pancreatic commitment during development and for the activation/maintenance of the acinar differentiation program in exocrine pancreas. High-level expression of exocrine digestive enzymes, a hallmark of mature acinar cells, depends largely on the trimeric complex PTF1 (pancreas transcription factor 1), formed by p48, RBP-L and a class A bHLH protein. In addition, p48 induces cell cycle exit by controlling G1-S progression. However, the mechanisms that mediate the PTF1-dependent gene activation are poorly understood. Here, we report that p48 increases transcription through two activation domains located in its N-terminal region by recruiting transcriptional coactivators. The histone acetyltransferase cofactor p/CAF interacts in vivo with p48 in acinar cells and is associated with the promoter region of acinar genes targeted by the PTF1 complex. P/CAF potentiates the PTF1 transcriptional activity by selectively enhancing the p48 transactivation activity. P/CAF promotes the nuclear accumulation of p48 and its in vivo acetylation in Lys residue K200. The K200R mutation abolishes the transcriptional activity of p48 as well as its capacity to functionally cooperate with RBP-L to ensure an effective PTF1-driven transcription, indicating that p/CAF-mediated acetylation of p48 is required for PTF1 full transcriptional activity. By contrast, p/CAF did not cooperate with p48 in its growth regulatory effects. These results support a critical and selective role of p/CAF in the PTF1-dependent gene activation during acinar differentiation.

INTRODUCTION

The basic-helix-loop (bHLH) transcription factor p48 (also designated Ptf1a or Ptf1α) exerts critical functions during both early and late pancreatogenesis: it is required for acquisition of pancreatic fate by undifferentiated foregut endoderm [START_REF] Kawaguchi | The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors[END_REF] and is also necessary for activation/maintenance of the acinar differentiation program in exocrine pancreas. In murine pancreatic buds, p48 expression is detected starting at E9.5 [START_REF] Krapp | The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas[END_REF][START_REF] Obata | p48 subunit of mouse PTF1 binds to RBP-J kappa/CBF-1, the intracellular mediator of Notch signalling, and is expressed in the neural tube of early stage embryos[END_REF] in a progenitor population that gives rise to all acinar and ductal cells, as well as the majority of insulin-and glucagonproducing cells, and becomes restricted to the acinar compartment from E14 onward [START_REF] Kawaguchi | The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors[END_REF][START_REF] Krapp | The p48 DNA-binding subunit of transcription factor PTF1 is a new exocrine pancreasspecific basic helix-loop-helix protein[END_REF]. In the absence of p48, progenitors from the dorsal pancreatic bud give rise to the four differentiated intestinal cell types [START_REF] Kawaguchi | The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors[END_REF]. Inactivation of p48 by homologous recombination in the mouse also results in pancreatic and cerebellar agenesis as well as in defective specification of GABAergic neurons in the spinal cord and horizontal/amacrine cells in developing retina [START_REF] Kawaguchi | The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors[END_REF][START_REF] Krapp | The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas[END_REF][START_REF] Hoshino | Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum[END_REF][START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF][START_REF] Pascual | Cerebellar GABAergic progenitors adopt an external granule cell-like phenotype in the absence of Ptf1a transcription factor expression[END_REF]. A similar pancreatic and cerebellar phenotype is observed in patients with permanent neonatal diabetes mellitus harbouring mutations leading to the expression of a truncated form of p48 lacking the C-terminal 32 amino acids [START_REF] Sellick | Mutations in PTF1A cause pancreatic and cerebellar agenesis[END_REF]. These observations highlight a crucial role of the C-terminal domain of p48 in pancreatic and neuronal specification. Additionally, the C-terminal region of p48 has been shown to be responsible for the antiproliferative activity of the protein [START_REF] Rodolosse | PTF1alpha/p48 transcription factor couples proliferation and differentiation in the exocrine pancreas (corrected)[END_REF] and to interact with the mammalian Suppressor of Hairless (RBP-Jkappa) and its paralogue RBP-L [START_REF] Beres | PTF1 is an organ-specific and notch-independent basic helix-loop-helix complex containing the mammalian suppressor of hairless (RBP-J) or its paralogue[END_REF][START_REF] Obata | p48 subunit of mouse PTF1 binds to RBP-J kappa/CBF-1, the intracellular mediator of Notch signalling, and is expressed in the neural tube of early stage embryos[END_REF].

The establishment of the acinar differentiation program relies on the formation of transcriptional complexes thought to be responsible for the activation of expression of all acinar digestive enzymes. Thus, the tissue-specific transcription of the acinar genes at high levels is largely controlled by the pancreas-specific transcription complex PTF1 [START_REF] Cockell | Identification of A Cell-Specific Dna-Binding Activity That Interacts with A Transcriptional Activator of Genes Expressed in the Acinar Páncreas[END_REF][START_REF] Rose | The role of PTF1-P48 in pancreatic acinar gene expresión[END_REF]. PTF1 is a hetero-oligomeric complex composed of the ubiquitously expressed class A bHLH proteins E2A or HEB, RBP-Jkappa or its paralogue RBP-L [START_REF] Beres | PTF1 is an organ-specific and notch-independent basic helix-loop-helix complex containing the mammalian suppressor of hairless (RBP-J) or its paralogue[END_REF][START_REF] Masui | Early pancreatic development requires the vertebrate Suppressor of Hairless (RBPJ) in the PTF1 bHLH complex[END_REF], and p48 [START_REF] Roux | The Cell-Specific Transcription Factor Ptf1 Contains 2 Different Subunits That Interact with the DNA[END_REF]. In the adult, RBP-L and Ptf1a are mainly expressed in acinar cells in the pancreas [4;10]. The PTF1 binding site is a bipartite sequence with an E-box and a TC box (TTTCCCA) present in the promoter of all the acinar digestive enzymes genes [START_REF] Cockell | Identification of A Cell-Specific Dna-Binding Activity That Interacts with A Transcriptional Activator of Genes Expressed in the Acinar Páncreas[END_REF]. An antisense RNA-mediated reduction of p48 synthesis in cultured exocrine pancreatic cells led to inhibition of the exocrine transcription program [START_REF] Krapp | The p48 DNA-binding subunit of transcription factor PTF1 is a new exocrine pancreasspecific basic helix-loop-helix protein[END_REF] and in vitro acinar-to-ductal transdifferentiation of normal exocrine pancreas is associated with a loss of PTF1 activity and the selective down-regulation of p48 and RBP-L expression ( [START_REF] Adell | Role of the basic helix-loop-helix transcription factor p48 in the differentiation phenotype of exocrine pancreas cancer cells[END_REF], unpublished data). These findings indicate that p48 and RBP-L are essential for maintaining the terminally differentiated state in acinar cells. However, the mechanisms supporting PTF1mediated gene activation are still unknown. P/CAF [START_REF] Yang | A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A[END_REF] and the closely related GCN5 (also called p/CAF-B) [START_REF] Smith | Cloning of Drosophila GCN5: conserved features among metazoan GCN5 family members[END_REF] are transcriptional co-activators with intrinsic histone acetyltransferase (HAT) activity which participate in transcriptional regulation by modifying chromatin and transcription factors. Histone acetylation on Lys residues is predominantly associated with gene activation but the precise mechanism by which this post-translational modification augments transcription is not fully understood. It has been proposed that acetylation would weaken the nucleosomal structure and disrupt contacts between nucleosomes, thereby facilitating the access of transcription factors and the progression of RNA polymerase II onto chromatin [START_REF] Bannister | Regulation of gene expression by transcription factor acetylation[END_REF]. In human cells, p/CAF is part of a large multisubunit complex of more than twenty polypeptides including TATA box-binding protein associated factors [START_REF] Ogryzko | Histone-like TAFs within the PCAF histone acetylase complex[END_REF] and may associate with the HATs CBP/p300 [START_REF] Yang | A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A[END_REF]. It is thought that the combination of HATs ensuring the optimal transcription of a given promoter may be determined at least in part by their interaction with specific transcription factors bound to this promoter. In addition to histones, a growing number of transcription factors have been identified as substrates for p/CAF. They include p53 [START_REF] Gu | Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain[END_REF], pRb [START_REF] Nguyen | Acetylation regulates the differentiation-specific functions of the retinoblastoma protein[END_REF] and the bHLH transcription factors MyoD [START_REF] Dilworth | In vitro transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation[END_REF][START_REF] Sartorelli | Acetylation of MyoD directed by PCAF is necessary for the execution of the muscle program[END_REF], Tal1 [START_REF] Huang | P/CAF-mediated acetylation regulates the function of the basic helix-loop-helix transcription factor TAL1/SCL[END_REF], E2A [START_REF] Bradney | Regulation of E2A activities by histone acetyltransferases in B lymphocyte development[END_REF], and Beta-2 [START_REF] Qiu | Acetylation of the BETA2 transcription factor by p300-associated factor is important in insulin gene expresión[END_REF]. P/CAF-mediated acetylation of these transcription factors is required for their function, illustrating the participation of p/CAF in proliferation, apoptosis or differentiation processes.

Here, we report on the mechanisms through which p48 contributes to transcriptional activation. We demonstrate that the N-terminus of p48 contains activation domains and we show that p/CAF, through its interaction with p48, contributes to the transcriptional activity of 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.

vectors or the corresponding empty vectors were transfected with increasing amounts of pCX-Flag-p/CAF or pCX-Flag-p/CAF(HAT-) and 0.1 μg of 6XA 26 -luc.Transcriptional activity assays of wild-type and Lys-mutated forms (p48K-R) of p48 were performed by transfecting 0.2 μg of p48 or p48K-R point mutant expression plasmids, 0.2 μg of RBP-L expression vector and 0.1 μg of 6XA 26 -luc.

Immunoprecipitation and western-blotting. HEK293T cells were transfected with 3 μg of pIRESp48 expression vector and 3 μg of Flag-p/CAF or Flag-GCN5 plasmids. After 48 h, cells were lysed in 1 ml of immunoprecipitation (IP) buffer (50 mM Tris pH8, 120 mM NaCl, 0.5% NP40) supplemented with protease inhibitors and lysates were incubated overnight. Endogenous p/CAF was immunoprecipitated from AR42J lysates with anti-p/CAF and protein G-agarose beads (Roche, Basel, Switzerland). Flag-tagged p/CAF and GCN5 were immunoprecipitated with anti-Flag M2 affinity gel (Sigma). P48 and p48 K200R interaction with RBP-L or E47 was analysed in HEK293T cells transfected with 3 μg of pFlag-p48 or pFlag-p48 K200R and 3 μg of pcDNA 3 RBP-L or pCDNA 3 E47 expression vectors. Flag-tagged p48 or p48 K200R were immunoprecipited with anti-flag M2 affinity gel as described above. The immune complexes were pelleted by gentle centrifugation, washed 3 times with IP buffer, resuspended in loading buffer, and fractionated by SDS-PAGE. The following antibodies were used for western blotting: affinity-purified rabbit p48 (1:800), E2A/E12 rabbit polyclonal antibody (1:500), affinity-purified rabbit anti-RBP-L (1:300) and anti-Flag M2 mAb (1:10000).

In vitro acetylation assays. Flag-tagged p48 and N+bHLH proteins were immunoprecipitated from HEK293T transfected cell extracts with anti-Flag M2 affinity gel. After immunoprecipitation, the beads were washed with IP buffer, divided into aliquots and stored at -80 °C. For acetylation assays, immunoprecipitated proteins were incubated with 300 ng of recombinant GST-p/CAFHAT domain (Upstate, Temecula, CA) or GST alone and 10 nmol of acetyl-CoA (Sigma) in HAT buffer (25 mM Tris pH8; 2.5% glycerol; 0.5 mM EDTA; 25 mM KCl) at 30 ºC for 1 h. Reactions were stopped by adding sample buffer. Proteins in the input samples were visualised by Coomassie blue staining and acetylated proteins were detected by immunoblotting using an anti-acetyl-Lys antibody.

In vivo acetylation assays. HEK293T cells were transfected with 3 μg of Flag-p48 expression vector and 3 μg of pCX-Flag-p/CAF or pCX-Flag-p/CAF(HAT-) plasmids. Acetylation of wildtype and K200R mutated forms of p48 N+bHLH fragment was assessed by transfecting 3 μg of the corresponding expression plasmids and 3 μg of pCX-Flag-p/CAF. Lysates were prepared as described above and immunoprecipitation was performed with 2.5 μg of anti-acetyl-Lys antibody and 20 μl of protein G-agarose beads. For western-blotting, the anti-Flag M2 mAb (1:10000) was used. For reciprocal assay, immunoprecipitation was performed with anti-Flag affinity gel. Acetylated proteins were detected with anti-acetyl-Lys (1:1000). To detect the immunoprecipitated proteins, membranes were stripped and re-probed with anti-Flag mAb.

Indirect immunofluorescence. Cos-7 cells were seeded on sterile coverslips and transfected with 0.2 μg of p48GFP expression vector and 0.2 μg of pCX-Flag-p/CAF or pCX-Flagp/CAF(HAT-) plasmids. Cells were fixed for 10 min with 4% paraformaldehyde and washed. After blocking with 1% BSA, 0.1% saponin, 0.1% Triton X-100 in PBS (blocking buffer) for 30 min, coverslips were incubated with anti-Flag mAb (1:1000), washed, and incubated with rhodamine-conjugated anti-mouse Ig (1:200) (Dako, Glostrup, Denmark). Samples were visualized using a Leica TCS SP2 confocal microscope.

Chromatin immunoprecipitation. 266-6 cells were transfected with lipofectamine with either 3 μg Flag-p/CAF or with 3 μg Flag-tagged p48. Fourty-eight h later, cells were cross-linked with 1% formaldehyde. Nuclear extracts were prepared by lysis in 50 mM Tris (pH 8), 2 mM EDTA, 0.1% NP40, 10% glycerol with protease inhibitors. After centrifugation, the pellet was resuspended in SDS lysis solution (50 mM Tris (pH8), 10 mM EDTA, 1% SDS) with protease A c c e p t e d M a n u s c r i p t inhibitors. Lysates were sonicated and precleared with protein G-agarose beads for 3 h at 4 °C. Half of the chromatin was immunoprecipitated overnight at 4 °C with pre-blocked agarose beads coupled to anti-Flag M2 mAb, the other half with pre-blocked agarose coupled to anti-HA mAb (Sigma, St-Louis, MO). After washing, bound chromatin was eluted in 100 mM Na 2 CO 3 with 1% SDS and proteinase K (1mg/ml) (Sigma). Cross-linking was reversed at 65 °C. After treatment with RNase A (0.04mg/ml) (Roche, Mannheim, Germany) and proteinase K, the immunoprecipitated DNA was purified by the GFX PCR DNA purification kit (Amersham, Buckinghamshire, UK). Quantitative SYBR Green PCR (Applied Biosystems, Foster City, CA) was performed comparing the Ct values for anti-HA versus anti-Flag precipitates. Results were expressed as fold increase of Flag-immunoprecipitated DNA over HA-immunoprecipitated DNA. Two independent experiments were performed and all samples were analyzed in triplicate. Primer sequences were as follows: chymotrypsinogen B1 promoter: 5'-GCTGGCCACTACCAATGTTC-3' and 5'-CTGAGGCTCTTTTATGTCCC-3'; carboxypeptidase A1 promoter: 5'-CCATGGTCAAGGGTGAAAGC-3' and 5'-TCTGGGGCCTTTTTAAACAC-3'. Transfection efficiency of 266-6 cells was <10%, as determined using a p48GFP plasmid and counting of GFP positive cells.

Quantitative RT-PCR analysis of elastase, carboxypeptidase and chymotrypsinogen. AR42J cells were transfected using lipofectamine with 6 μg of pIRES-GFP-p48, pIRES-GFP-p48 K200R or pIRES-GFP empty vector and 20 h later GFP positive cells sorted by FACSvantage SE (Becton Dickinson, San Jose, CA) were isolated. To examine the effects of glucocorticoids, AR42J cells were treated with 100 nM dexamethasone for 72 h. RNA was extracted and treated with DNAse I using the DNA free kit (Ambion, Austin, TX). cDNA generated by Taqman reverse transcription was analysed by quantitative PCR (Applied Biosystems). The relative Ct values were calculated as previously described [START_REF] Sellick | Mutations in PTF1A cause pancreatic and cerebellar agenesis[END_REF]. Primer sequences used were as follows: HPRT: 5'-GTTCTTTGCTGACCTGCTGGA-3' and 5'-TTATGTCCCCCGTTGACTGGT-3'; p48 or p48 K200R : 5'-GCTCCTGGAGCATTTTCCCG-3' and 5'-CTGAGGAACTCTACCTCCGC-3'; elastase: 5'-TCCGTGAAGACCAACATGGTG-3' and 5'-CCAAAGCTCACAATACCGTGC-3'; carboxypeptidase A1: 5'TCCAGATCGGCAACACCTTT-3' and 5'TCCCTAGAATGGATGCCAGTG-3'; chymotrypsinogen B: 5'-GCAAGACCAAATACAATGCCC-3' and 5'-TGCGCAGATCATCACATCG-3'. HPRT mRNA was used as internal control and total p48 mRNA was used as reference to normalize for transfection efficiency. For comparison of RNA levels in cells expressing transfected p48 or p48 K200R , Mann-Whitney test was used.

RESULTS

The N-terminal region of p48 functions as a transactivation domain

To identify the p48 transcriptional activation domain, a series of deletion derivatives of p48 fused to the yeast Gal4-DBD was expressed in AR42J cells. Their transcriptional activity on a Gal4-driven promoter was measured using the Gal4-TKluc reporter (Figure 1). Full-length p48 (residues 1-326) and its N-terminal region (residues 1-168) exhibited an 87-and 55-fold activation of luciferase activity, respectively, whereas the p48 N-terminal region including the bHLH domain (residues 1-217) and the p48 C-terminal region (residues 211-326) did not transactivate the Gal4 promoter (Figure 1A). These results indicate that p48 activates transcription through its N-terminal region and that the transactivation of the N-terminal region is negatively modulated by the bHLH domain. The comparison between the activities of the full-length p48 and its N-terminal region suggests that the C-terminus, inactive by itself, may potentiate the transactivation of the N-terminal region.

To further define the domains responsible for transcriptional activation, fine deletion analysis of the p48 N-terminal region was performed (Figure 1B). The products of a construct coding for residues 1-43 of p48 and, to a lesser extent, a construct coding for residues 38-108 efficiently activated the Gal4-promoter. By contrast, residues 104-138 exhibited minimal activity and the construct coding for residues 133-168 was inactive. From these results we A c c e p t e d M a n u s c r i p t conclude that p48 activates transcription through two activation domains located in its Nterminal region between residues 1-43 and 43-138.

p48 interacts in vivo with p/CAF and GCN5

The primary sequence of the most N-terminal region of p48 is particularly rich in acidic amino acids, a feature previously reported for several transcriptional activating domains that recruit coactivators possessing an intrinsic HAT activity. We therefore tested whether p48 could interact in vivo with coactivators of the p/CAF family using co-immunoprecipitation experiments. Protein extracts from HEK293T cells cotransfected with pIRESp48 vector in combination with Flag-p/CAF or Flag-GCN5 were immunoprecipitated with the anti-Flag antibody. The presence of p48 in the immunoprecipitates was assessed by western-blotting with anti-p48 antibodies. As shown in Figure 2A, an efficient in vivo interaction of p48 was observed with p/CAF (left panel) and with its homologue GCN5 (right panel). A similar experiment performed with a CBP expression vector failed to reveal any interaction between p48 and CBP (data not shown). To determine whether endogenous p48 and p/CAF did interact in acinar cells, protein extracts from AR42J cells were immunoprecipitated with an anti-p/CAF antibody and p48 was assayed in the immunocomplexes by western-blot. As shown in Figure 2B, p48 and p/CAF interact in vivo in acinar cells.

p/CAF potentiates the transcriptional activity of p48

In acinar cells, p48 is part of the PTF1 complex which also includes RBP-L. Both proteins have been shown to physically interact and functionally cooperate to ensure the high transcriptional activity of PTF1 on a reporter driven by tandem repeats of a PTF1 binding sites (6XA 26 luc) [START_REF] Beres | PTF1 is an organ-specific and notch-independent basic helix-loop-helix complex containing the mammalian suppressor of hairless (RBP-J) or its paralogue[END_REF]. To determine the effect of p/CAF on the transcriptional activity of p48 and RBP-L, AR42J cells were cotransfected with increasing amounts of p/CAF (Figure 3, left panel), p/CAF(HAT-) plasmid (Figure 3, right panel), or the corresponding empty vector, in combination with expression vectors for p48, RBP-L, and the 6XA 26 luc construct as a PTF1responsive reporter. As expected for a cofactor, p/CAF alone did not activate the PTF1responsive promoter (not shown) whereas p48-mediated activation increased in a dosedependent manner from 3.5 to 16.4-fold upon p/CAF overexpression (Figure 3, left panel). In contrast, p/CAF did not enhance RBP-L mediated activation. Transfection with the p/CAF expression plasmid induced an 8-fold increase in the activity of the PTF1-responsive promoter upon co-transfection of p48 and RBP-L cDNAs. P/CAF coactivation was selective for p48/RBP-L as it was not observed when RBP-J kappa, the RBP-L paralogue, was expressed in the same conditions (data not shown). In addition, the plasmid coding for a p/CAF mutant lacking HAT activity failed to cooperate with p48 (Figure 3, right panel). These results show that p/CAF functions as a coactivator of p48 on a PTF1-responsive promoter and suggest that p/CAF coactivation of p48/RBP-L transcriptional activity is mediated by p48 as RBP-L activity is not affected by p/CAF.

Glucocorticoids have been shown to increase the mRNA levels of digestive enzymes [START_REF] Logsdon | Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells[END_REF]. We analyzed whether these effects were accompanied by an induction of Ptf1a, p/CAF, or Gcn5 using quantitative RT-PCR: treatment with dexamethasone for 72 h induced a 3-fold increase in amylase mRNA levels (p=0.013) but had no significant effects in the expression of Ptf1a, PCAF, or Gcn5 transcripts.

p/CAF enhances nuclear accumulation of p48

To determine whether p/CAF could affect the subcellular distribution of p48, Cos-7 cells were co-transfected with a p48GFP fusion protein plasmid and Flag-p/CAF or Flagp/CAF(HAT-) plasmids. p48GFP distribution was quantified in cells expressing, or not, exogenous p/CAF or p/CAF(HAT-) and the latter proteins were detected using rhodaminelabeled secondary antibodies. In the absence of exogenous p/CAF, p48GFP was distributed in either the nucleus or both the nucleus and the cytoplasm in the vast majority of the cells (Figure 4A). As expected, p/CAF and p/CAF(HAT-) were predominantly found in the nucleus. Importantly, p/CAF co-localized with nuclear p48GFP. As shown in Figure 4B A c c e p t e d M a n u s c r i p t distribution of p48GFP (from 36.7% to 81%) and concomitantly reduced the proportion of the cells with cytoplasmic p48GFP. HAT deficient p/CAF mutant, which retains the capacity to interact with p48 (not shown), also increased the nuclear distribution of p48GFP although to a lesser extent. Thus, p/CAF promotes nuclear accumulation of p48 through a mechanism that is partially dependent of its acetyltransferase activity.

p48 is acetylated by p/CAF in vitro and in vivo.

We next investigated whether p48 is a target of p/CAF acetyltransferase activity. Immunoprecipitated Flag-tagged p48 protein was incubated with either GST or recombinant GST-p/CAF HAT domain in HAT buffer containing acetyl-CoA. After SDS-PAGE, proteins were stained by Coomassie blue and acetylated proteins were detected with anti-acetyl-Lys antibodies. Figure 5A shows that addition of p/CAF HAT domain, but not GST, results in the detection of a band corresponding to p48 by anti-acetyl-Lys antibodies, indicating that p48 is acetylated by p/CAF in vitro. For in vivo acetylation assay, a plasmid coding for Flag-tagged p48 was expressed in HEK293T cells alone or in combination with plasmids coding for p/CAF-Flag or p/CAF(HAT-)-Flag proteins. Lysates from transfected cells were immunoprecipitated with anti-acetyl-Lys antibodies and Flag-tagged acetylated proteins were detected by westernblot with anti-Flag antibodies (Figure 5B). P48-Flag was not immunoprecipitated with antiacetyl-Lys antibodies in the absence of co-transfected p/CAF, indicating that p48 is not acetylated or that p48 acetylation level is too weak to be detected in this assay in HEK293T cells. Upon expression of p/CAF-Flag, both p48 and p/CAF were detected in the immunoprecipitates using anti-acetyl-Lys antibodies. Auto-acetylation of p/CAF has been previously reported [START_REF] Santos-Rosa | Mechanisms of P/CAF auto-acetylation[END_REF] and confirmed the functionality of its HAT domain in this assay. By contrast, acetylated p48 was not detected when p48 was co-expressed with the HAT-deficient p/CAF demonstrating the critical role of its enzymatic activity. These results were confirmed by the reciprocal assay where, as shown in Figure 5C, the two Flag-tagged proteins p48 and p/CAF were exclusively detected by anti-acetyl-Lys antibodies upon p/CAF expression.

The K200 residue of p48 is essential for PTF1 transcriptional activity and is a target of p/CAF acetylation.

To identify the p48 residues targeted by p/CAF acetylation and study the functional relevance of p48 acetylation, we generated K-R mutants at each Lys residue of p48. The eight Lys residues of p48 are conserved through species. The first two Lys are in the bHLH domain while the six other Lys residues are all located in the C-terminal region of the protein (Figure 6A). After confirming by western-blot the correct expression of the K-R mutant forms of p48 in HEK293T transfected cells, we tested their transcriptional activity and their capacity to functionally cooperate with RBP-L on the PTF1-responsive promoter. As shown in Figure 6B, all single Lys-mutated forms of p48, with the exception of the p48 K200R mutant, exhibited a transcriptional activity similar to that of the wild-type protein. In addition, they were able to cooperate with RBP-L, although to variable extents, for an efficient activation of the PTF1responsive promoter. By contrast, The K200R mutation completely abolished the transcriptional activity of p48 as well as its cooperation with RBP-L. Moreover, this mutant exhibited a normal capacity to interact with p/CAF (Figure 6C), RBP-L (Figure 6D, upper part) and E47 (Figure 6D, lower part) indicating that loss of p48/RBP-L transcriptional activity due to K200R mutation does not apparently result from an alteration of p48 interaction with RBP-L, E47 or p/CAF.

To determine whether the K200 residue of the bHLH domain is a target of p/CAFmediated acetylation, we performed in vitro and in vivo acetylation assays using the wild-type or the K200R mutated form of the N+bHLH region of p48. The N+bHLH K200R fragment is neither detected nor immunoprecipitated by anti-acetyl-Lys antibodies in both in vitro and in vivo acetylation assays (Figure 7 A and 7B, left panel). By contrast, the N+bHLH wild-type fragment is acetylated in vitro by p/CAF HAT domain (Figure 7A). Acetylation of the wild-type p48 fragment is also observed in vivo when it is coexpressed with exogenous p/CAF (right part of Figure 7B), thus demonstrating that the K200 residue is a target of p/CAF acetylation. Taken together, these data indicate that the K200 residue of p48 is essential for its transcriptional activity and strongly suggest that its acetylation by p/CAF is required for full transcriptional activity of p48 and PTF1 complex.

The K200 mutation alters the expression of the endogenous PTF1 target genes.

The importance of K200 residue acetylation for p48 function was assessed by comparing the expression of the three PTF1 target genes carboxypeptidase A1 (Cpa1), chymotrypsynogen B (Ctrb) and elastase in cells overexpressing the wild-type or the mutated form of p48. Protein extracts and RNA were prepared from AR42J cells overexpressing p48 or p48 K200R and expression of the three PTF1 target genes was measured by quantitative RT-PCR. Both wild-type and mutated forms of exogenous p48 were similarly expressed as assessed by western-blot (data not shown). Figure 8 shows that p48 K200R expression results in a decrease of 42.2%, 47.35% and 44.2% of the RNA levels of Cpa1, Ctrb and elastase, respectively. Thus, substitution of K200 for a residue that cannot be acetylated severely compromises the p48 capacity to sustain a high level expression of the three PTF1 target genes.

p/CAF is associated with the promoter of PTF1 target genes

To determine whether p/CAF is associated with the promoter region of acinar genes targeted by the PTF1 complex, ChIP experiments were performed using acinar 266-6 cells transfected with Flag-p/CAF or Flag-p48; for these experiments transfected p48 was used as control. Chromatin immunoprecipitation with anti-Flag from p48 and p/CAF transfected cells lysates showed an enrichment of the promoter region of chymotrypsinogen B1 of 4.3±0.9-fold and 2.6±0.6-fold, respectively. Similar enrichment levels were demonstrated at the promoter of carboxypeptidase A1: 3.5±1.3-fold (p48-Flag) and 1.9±0.1-fold (p/CAF-Flag), indicating that p/CAF and p48 are associated with the same region of the chymotrypsinogen B1 or the carboxypeptidase A1 promoter.

DISCUSSION

Cell differentiation relies on coordinated transcriptional regulation as well as on changes in cellular organization leading to functional maturation. Tissue-specific gene expression depends on the expression of tissue-specific transcription factors and on changes in chromatin structure. Histone acetylation has been predominantly correlated with gene activation [18;32] and the activity of HAT appears to be critical for gene expression. Association of HAT cofactors with site-specific DNA binding factors allows to direct acetylation-dependent regulation to specific loci. In addition to core histones, HATs may also acetylate Lys residues of architectural DNA binding factors, basal transcription factors and tissue-specific DNA binding factors [START_REF] Bannister | Regulation of gene expression by transcription factor acetylation[END_REF].

In pancreatic acinar cells, expression of genes coding for digestive enzymes is largely controlled by PTF1 [START_REF] Beres | PTF1 is an organ-specific and notch-independent basic helix-loop-helix complex containing the mammalian suppressor of hairless (RBP-J) or its paralogue[END_REF][START_REF] Cockell | Identification of A Cell-Specific Dna-Binding Activity That Interacts with A Transcriptional Activator of Genes Expressed in the Acinar Páncreas[END_REF][START_REF] Rose | The role of PTF1-P48 in pancreatic acinar gene expresión[END_REF]. However, the mechanisms responsible for the activation of gene expression by PTF1 remain to be deciphered. Here, we show that p/CAF interacts in vivo with p48 in acinar cells and potentiates PTF1 transcriptional activity through p48 acetylation at K200. P48 interacts with Class A bHLH proteins through its bHLH domain [START_REF] Roux | The Cell-Specific Transcription Factor Ptf1 Contains 2 Different Subunits That Interact with the DNA[END_REF] and with RBP-J kappa and RBP-L through its carboxy-terminal region [START_REF] Beres | PTF1 is an organ-specific and notch-independent basic helix-loop-helix complex containing the mammalian suppressor of hairless (RBP-J) or its paralogue[END_REF][START_REF] Obata | p48 subunit of mouse PTF1 binds to RBP-J kappa/CBF-1, the intracellular mediator of Notch signalling, and is expressed in the neural tube of early stage embryos[END_REF]. Recently, a new model of the PTF1 complex has been proposed according to which recruitment of RBP-L, in place of RBP-Jkappa, by a DNA binding dimer formed by p48/Class A bHLH protein provides high transcriptional activity in the adult pancreas [START_REF] Beres | PTF1 is an organ-specific and notch-independent basic helix-loop-helix complex containing the mammalian suppressor of hairless (RBP-J) or its paralogue[END_REF][START_REF] Masui | Early pancreatic development requires the vertebrate Suppressor of Hairless (RBPJ) in the PTF1 bHLH complex[END_REF]. The recruitment of RBP-L would result in ensuring the formation of a PTF1 complex with highest transcriptional activity by excluding RBP-Jkappa [START_REF] Beres | PTF1 is an organ-specific and notch-independent basic helix-loop-helix complex containing the mammalian suppressor of hairless (RBP-J) or its paralogue[END_REF], leading to full acinar cell differentiation. Here, we show that mutation of the p/CAF-acetylated residue K200 results in the loss of p48 transcriptional activity and of its functional cooperation with RBP-L without affecting its capacity to interact with RBP-L and the class A bHLH protein E47 (Fig. 6C), or p/CAF (Fig. 6D). We propose that p/CAF-mediated acetylation of p48 on K200 residue could, by favouring the selective cooperation between RBP-L and p48, help to confer to the PTF1 complex the high and specific activity required for full differentiation of acinar cells. 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. P/CAF has been reported to interact with, and to regulate the function of, several bHLH transcription factors required for cell fate specification or terminal differentiation. For example, MyoD interacts with, and is acetylated by, p/CAF and both effects are necessary for its transcriptional activity on chromatin-associated templates and for triggering terminal myogenic differentiation [START_REF] Dilworth | In vitro transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation[END_REF][START_REF] Puri | Differential roles of p300 and PCAF acetyltransferases in muscle differentiation[END_REF]. BETA2/NeuroD, essential for the differentiation of pancreatic islet and neural cells, is an in vivo target of p/CAF acetylation in pancreatic β cells. Acetylation of BETA2 affected its transcriptional activity on the insulin gene promoter supporting the idea that gene activation by BETA2 is regulated by acetylation [START_REF] Qiu | Acetylation of the BETA2 transcription factor by p300-associated factor is important in insulin gene expresión[END_REF]. The close relation between p/CAFmediated bHLH transcriptional regulation and cell differentiation is further illustrated by TAL1, a critical bHLH regulator of hematopoietic and vascular development. Inhibition of p/CAFmediated TAL1 acetylation in erythroleukemia cells resulted in an inhibition of TAL1 transcriptional activity and compromised differentiation [START_REF] Huang | P/CAF-mediated acetylation regulates the function of the basic helix-loop-helix transcription factor TAL1/SCL[END_REF]. As reported for BETA2 and TAL1, p48 is acetylated in vivo by p/CAF on a Lys residue located in the bHLH domain. Several Lys residues are found at conserved positions in the helix 2 region of bHLH factors and, in a few cases, have been demonstrated to be acetylated by p/CAF and essential for transcriptional activation (underlined):

p48 KRLSKVDTLRLA TAL1 KKLSKNEILRLA BETA2/NeuroD QKLSKIETLRLA Math1 KKLSKYETLQMA MASH2 KKLSKVETLRSA Mist1 KKLSKIETLTLA Neurogenin 3 AKLTKVETLRFA
A comparison of these sequences indicates that p/CAF acetylation takes place in a highly conserved region of the bHLH proteins. It is conceivable that the activity of other bHLH factors required for neuronal and/or pancreatic differentiation such as Mash2 [34], Mist1 [START_REF] Lemercier | Mist1: A novel basic helix-loop-helix transcription factor exhibits a developmentally regulated expression pattern[END_REF][START_REF] Pin | The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity[END_REF], or the Neurogenin-3 [START_REF] Apelqvist | Notch signalling controls pancreatic cell differentiation[END_REF], could be similarly regulated by p/CAF acetylation. Whether HAT-mediated acetylation of p48 is required for its function in pancreas and cerebellar organogenesis remains to be determined. Despite the fact that the K200R mutation impairs the acetylation of the N-terminal region of p48, the p48 K200R mutant is immunoprecipitated by anti-acetyl-Lys antibodies upon p/CAF co-transfection (data not shown), supporting the notion that additional Lys residues are targeted by p/CAF in the full-length protein. Interestingly, five out of the eight potentially acetylated Lys residues are in the most C-terminal part of p48, close to the interaction domains with RBP-Jkappa and RBP-L [START_REF] Beres | PTF1 is an organ-specific and notch-independent basic helix-loop-helix complex containing the mammalian suppressor of hairless (RBP-J) or its paralogue[END_REF], within a region shown to be crucial for pancreatic and cerebellar organogenesis [START_REF] Sellick | Mutations in PTF1A cause pancreatic and cerebellar agenesis[END_REF] and for p48 antiproliferative activity [START_REF] Rodolosse | PTF1alpha/p48 transcription factor couples proliferation and differentiation in the exocrine pancreas (corrected)[END_REF]. We have previously described that p48-mediated inhibition of proliferation is associated with the up-regulation of p21 mRNA, the down-regulation of Cyclin D2 mRNA levels, and increased p27 protein levels. Up-regulation of p21 mRNA levels by MyoD has been shown to depend on MyoD/p/CAF interaction and p/CAF-mediated acetylation in differentiated muscle cells [START_REF] Mal | A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program[END_REF][START_REF] Puri | P300 is required for MyoD-dependent cell cycle arrest and musclespecific gene transcription[END_REF]. Upregulation of p21 mRNA levels by the Krüppel-like Factor 6 (KLF6) also required KLF6 acetylation by p/CAF and CBP cofactors [START_REF] Li | Regulation of Kruppel-like factor 6 tumor suppressor activity by acetylation[END_REF]. To determine the contribution of p/CAF to the effects of p48 on cell proliferation, we used cell cycle analysis, p21 promoter reporter assays, and p21 qRT-PCR. p/CAF did not cooperate with p48 in any of these assays (see Supplementary data). We cannot rule out a contribution of endogenous p/CAF; however, these findings indicate a distinct role of p/CAF in enhancing transcriptional activation of acinar genes and other p48-mediated cellular effects. Whether p/CAF plays a role in a p48-dependent recovery from injury upon induction of experimental acute pancreatitis also merits additional study [START_REF] Molero | Pancreas transcription factor 1 alpha expression is regulated in pancreatitis[END_REF].

Regarding the mechanisms through which p/CAF contributes to enhanced transcriptional activity, we show that -in Cos-7 cells -p/CAF promotes the nuclear accumulation of p48, an effect that has been reported for other transactivators [START_REF] Spilianakis | Acetylation by PCAF enhances CIITA nuclear accumulation and transactivation of major histocompatibility complex class II genes[END_REF]. Should HAT activity be required for nuclear targeting or retention of p48, expression of the p/CAF(HAT-) A c c e p t e d M a n u s c r i p t mutant would not modify the subcellular distribution of p48 or might compete with endogenous p/CAF resulting in an increase of cytoplasmic p48. However, we observed that p/CAF(HAT-) also leads, though to a lesser extent, to nuclear p48 accumulation indicating that HAT activity is not fully required for this effect. We therefore propose that the p/CAF-mediated effect on the subcellular distribution of p48 relies mainly on its interaction with p48 rather than on its capacity to acetylate it. However, we cannot exclude that modulation of p48 distribution results from an indirect effect of p/CAF. It is thought that E2A could, by interacting with p48, be responsible for its nuclear import [START_REF] Sommer | Nuclear Targeting of the Transcription Factor Ptf1 Is Mediated by A Protein Subunit That Does Not Bind to the Ptf1 Cognate Sequence[END_REF]. Obata et al. reported that, in Cos-7 cells, overexpression of E47 (produced from E2A gene) shifted the predominant localization of a myc-tagged p48 from the cytoplasm to the nucleus [START_REF] Obata | p48 subunit of mouse PTF1 binds to RBP-J kappa/CBF-1, the intracellular mediator of Notch signalling, and is expressed in the neural tube of early stage embryos[END_REF]. However, using a different tag, we have found that the distribution of the GFP-p48 in Cos-7 cells is predominantly nuclear in 80% of the cells. Additionally, overexpression of E47 or HEB in acinar and non-acinar cultured cells had minor effects on p48 subcellular localization (M. Lichtenstein, unpublished data). The importance of analyzing the mechanisms responsible for the nuclear targeting of p48 is underlined by the study of acinar tumors. We have reported that in some acinar pancreatic tumours, endogenous p48 was found exclusively in the cytoplasm and was associated with low expression of amylase [START_REF] Adell | Role of the basic helix-loop-helix transcription factor p48 in the differentiation phenotype of exocrine pancreas cancer cells[END_REF]. p48 is also partially redistributed to the cytoplasm in ElasCCK2 transgenic mice in association with defects in acinar cell differentiation [START_REF] Bierkamp | Expression of cholecystokinin-2/gastrin receptor in the murine pancreas modulates cell adhesion and cell differentiation in vivo[END_REF]. These findings suggest that altered subcellular distribution of p48 may contribute to reduced expression of acinar genes and compromised exocrine function. Additional experiments need to be performed, using p/CAF null mice [START_REF] Yamauchi | Distinct but overlapping roles of histone acetylase PCAF and of the closely related PCAF-B/GCN5 in mouse embryogenesis[END_REF], to determine the p/CAF contribution to p48 nuclear localization and transcriptional activity.

To further assess the role of P/CAF in cell differentiation, knock-down RNA interference experiments were conducted in cultured acinar cells. However, we could not detect any effect, possibly because of redundancy with GCN5, a HAT protein, as has been suggested to be the case in p/CAF null mice [START_REF] Yamauchi | Distinct but overlapping roles of histone acetylase PCAF and of the closely related PCAF-B/GCN5 in mouse embryogenesis[END_REF]. GCN5 displays a very high amino acid identity with p/CAF and is also expressed in the pancreas (unpublished data). Therefore, it is possible that the results reported for P/CAF may be extensive to GCN5.

Altogether, the results presented here provide new insights into the mechanisms through which PTF1 complex mediates acinar gene expression during exocrine differentiation. Deciphering the mechanisms by which p/CAF and/or other HAT cofactors may regulate p48 functions is an important step towards the understanding of acinar differentiation and fate determination in the pancreas and in the nervous system. In addition, it may contribute to a better understanding of the role of acinar transcriptional regulators in disease processes such as chronic pancreatitis and pancreatic cancer [START_REF] Hernández-Muñoz | Pancreatic ductal adenocarcinoma: cellular origin, signalling pathways and stroma contribution[END_REF].

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080293 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080293

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080293 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080293

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080293 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080293

  , exogenous p/CAF expression strongly enhanced the proportion of cells with exclusively nuclear Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080293 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080293

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080293 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080293

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080293 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080293

BiochemicalFigure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. p48 interacts in vivo with p/CAF and GCN5. (A) Whole-cell extracts from HEK293T cells expressing p48 and Flag-p/CAF (left panel) or p48 and Flag-GCN5 (right panel) were immunoprecipitated with anti-Flag or mouse IgG antibodies. Immune complexes were analysed by western blotting using anti-p48 (top) or anti-Flag (bottom) antibodies. (B) AR42J cell lysates were immunoprecipitated with rabbit preimmune (PI) or anti-p/CAF antiserum. Endogenous p48 and p/CAF were detected by western blotting with affinity purified anti-p48 (top) and antip/CAF (bottom). Input corresponds to 10% of the amount of extracts used for immunoprecipitation.

Figure 3 .

 3 Figure 3. p/CAF enhances p48-mediated transcription. AR42J cells were transiently transfected with increasing amounts (0, 50, and 100 ng) of pCX-Flag-p/CAF (left panel) or pCX-Flagp/CAF(HAT-) (right panel) together with 0.1 μg of pIRESp48 (white bars), 0.1 μg of pcDNA 3 RBP-L (light-grey bars), or 0.1 μg of both (dark-grey bars) and 0.1 μg of 6XA 26 -luc reporter plasmid. pRL-TK was included as an internal control for normalization. The results are displayed as -fold activation over the transfection of reporter with equivalent amounts of empty vectors and represent an average of two experiments performed in triplicate.

Figure 4 .

 4 Figure 4. p/CAF promotes nuclear accumulation of p48. Cos-7 cells were transfected with p48GFP plasmid and pCX-Flag-p/CAF, pCX-Flag-p/CAF(HAT-) or empty vector. Twenty hours after transfection, cells were stained with an anti-Flag antibody followed by rhodamineconjugated secondary antibody and analysed for either GFP fluorescence or rhodamine staining. (A) Subcellular distribution of p48GFP upon p/CAF or p/CAF(HAT-) co-expression. Representative fields are shown. (B) Quantitative analysis of the p48 distribution. The percentage of cells with GFP fluorescence exclusively nuclear (N), predominantly nuclear (N>C), or homogeneously distributed in both nucleus and cytoplasm (N=C), in the presence of empty vector or the indicated p/CAF expression plasmids, is shown. Results are the average percentages with their standard deviation derived from three independent experiments. For each experiment, more than 300 cells were scored.

Figure 5 .

 5 Figure 5. p48 is acetylated by p/CAF. (A)In vitro acetylation assay of p48. Immunoprecipitated p48-Flag was incubated with GST or with the recombinant GST-p/CAFHAT domain. Acetylated proteins were analysed by SDS-PAGE and western blotting with anti-acetyl-Lys antibody. Coomassie blue staining indicates the input protein bands. (B) and (C) In vivo acetylation assays. Total extracts from HEK293T cells transfected with 3 μg of pCMVFlag-p48 and 3 μg of pCx-Flag-p/CAF or pCx-Flag-p/CAF(HAT-) were immunoprecipitated with antiacetyl-Lys or control rabbit IgG. Immune complexes were analysed by western blotting using anti-Flag antibody (panel B). In reciprocal experiment, extracts from HEK293T transfected cells were immunoprecipitated with anti-Flag or mouse IgG and immune complexes were analysed by western blotting using anti-acetyl-Lys (C, upper panel). The anti-acetyl-Lys blot was stripped and re-probed with anti-Flag (C, lower panel) to confirm that the bands corresponding to p48 and p/CAF-Flagged proteins overlap with the acetylated proteins shown in the upper panel.

Figure 6 .

 6 Figure 6. The K200 residue of p48 is essential for transcriptional activity. (A) Comparison of the amino acid sequences of mouse, rat, human, Xenopus and zebrafish p48. The bHLH domain is represented in bold and Lys residues are marked with asterisks. (B) Transcriptional activity of wild-type and K-R p48 mutants on a PTF1-responsive promoter. AR42J cells were transiently transfected with pIRESp48 or pIRESp48 K-R , in the presence (black bars) or in the absence (white bars) of pcDNA 3 RBP-L, 6XA 26 -Luc reporter construct, and pRL-TK. pRL-TK vector was included as an internal control for normalization. The results are displayed as -fold activation over the transfection of reporter with equivalent amounts of empty vectors and represent an average of three experiments performed in triplicate. (C) p48 K200R mutant interacts with p/CAF. Whole-cell extracts from HEK293T cells expressing p48 K200R mutant and Flagp/CAF were immunoprecipitated with anti-Flag or mouse IgG antibodies. The immunoprecipitates were assessed by western-blot using anti-p48 antibodies. (D) p48 and p48 K200R interact with RBP-L and E47. Total extracts from HEK293T cells expressing Flag-p48 or the Flag-p48 K200R mutant in association with RBP-L or E47 were immunoprecipitated with either anti-Flag antibody or mouse IgG. Immunoprecipitates were tested for RBP-L (upper panel) or E47 (lower panel) by western blotting using anti-E2A.E12 and anti-RBP-L, respectively.

Figure 7 .

 7 Figure 7. The K200 residue of p48 is acetylated by p/CAF. (A) In vitro acetylation of N+bHLH. Wild-type N+bHLH or N+bHLH K200R fragments of p48 were incubated with GST or recombinant p/CAFHAT domain in HAT buffer containing acetyl-CoA. After incubation, acetylated proteins were analysed by SDS-PAGE and western-blotting with anti-acetyl-Lys antibody. Coomassie staining of the gel indicates the input protein bands. (B) In vivo acetylation of N+bHLH. Total extracts from HEK293T cells expressing flagged wild-type N+bHLH or N+bHLH K200R p48 fragments in association with Flag-p/CAF were immunoprecipitated with rabbit anti-acetyl-Lys antibody or rabbit IgG. Immune complexes were analysed by western blotting using anti-Flag. Results of acetylation assays with N+bHLH K200R and N+bHLH fragments are shown in the left and right panels, respectively.

Figure 8 .

 8 Figure 8. Expression of endogenous PTF1 target genes is reduced by the K200 mutation of p48. RNA from AR42J cells overexpressing p48 (white columns) or p48 K200R (black columns) was extracted and analysed by quantitative RT-PCR for carboxypeptidase A1 (CPA1), chymotrypsinogen B (CTRB) and elastase expression. RNA levels were calculated using HPRT as internal control and normalized by dividing by the total p48 RNA value. Each value represents the mean of three independent experiments performed in triplicate. Statistical analysis: *P<0.001 (Mann-Whitney test).

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

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. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

We thank the investigators mentioned in the text for providing reagents, R. MacDonald for discussion of unpublished data, S. Peiro and F. Mateo for valuable advice for ChIP experiment and acetylation assays. Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20080293 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080293 A c c e p t e d M a n u s c r i p t

Santos-Rosa (The Wellcome Trustand p48 K200R PCR products into pIRES-hrGFP-2a (Stratagene, La Jolla CA). pFlagCMV2-GCN5 was obtained from T. Honjo This work was supported, in part, by grants GEN2001-4748-C01 and SAF2004-01137 (Plan Nacional de I+D, Ministerio de Ciencia y Tecnología, Madrid, Spain), C03/010 from Instituto de Salud Carlos III, Ministerio de Sanidad, Madrid, and SGR-00410, CIRIT-Generalitat de Catalunya. M.L. Campos was supported by Fundaçao para a Ciência e Tecnologia, Portugal (SFRH / BD / 17661 / 2004; POCI2010) and I. Rooman was recipient of the Marie Curie Intra-European fellowship.


Mario A Pagano

Jenny Bain

Zygmunt Kazimierczuk
email: zygmunt_kazimierczuk@sggw.pl

Stefania Sarno

Maria Ruzzene

Giovanni Di Maira

Matthew Elliott

Andrzej Orzeszko

Giorgio Cozza

Flavio Meggio

Lorenzo A Pinna
email: lorenzo.pinna@unipd.it.

The selectivity of inhibitors of protein kinase CK2. An update

Keywords: CK2 inhibitors, drug design, selectivity, ATP-mimetics, PIM-1, HIPK2 Biochemical

The selectivity of inhibitors of protein kinase CK2. An update.

Introduction

CK2 (an acronym derived from the misnomer "casein kinase" 2) denotes one of the most pleiotropic Ser/Thr protein kinases, with more than 300 protein substrates already identified [START_REF] Meggio | One-thousand-and-one substrates of protein kinase CK2?[END_REF] and responsible for the generation of a substantial proportion of the eukaryotic phosphoproteome [START_REF] Pagano | Protein kinase CK2: a newcomer in the "druggable kinome[END_REF]. Such an extreme pleiotropy may, at least partially, account for the observation that CK2 catalytic activity is not controlled by either second messengers, phosphorylation events or association of its catalytic (α and/or α') with its regulatory βsubunits to give a "constitutively active" heterotetrameric holoenzyme [START_REF] Pinna | The raison d'etre of constitutively active protein kinases: the lesson of CK2[END_REF][START_REF] Litchfield | Protein kinase CK2: structure, regulation and role in cellular decisions of life and death[END_REF]. In turn constitutive activity and lack of physiological down-regulatory devices may underlie the pathogenic potential of CK2 whose abnormally elevated levels have been correlated to a number of global diseases, with special reference to neoplasia. Over-representation of CK2 has been documented in a number of cancers including kidney [START_REF] Stalter | Asymmetric expression of protein kinase CK2 subunits in human kidney tumors[END_REF], mammary gland [START_REF] Landesmann-Bollag | Protein kinase CK2 in mammary gland tumorigenesis[END_REF], lung [START_REF] Daya-Makin | Activation of a tumour-associated protein kinase (p40TAK) and casein kinase II in human squamous cell carcinomas and adenocarcinomas of the lung[END_REF], head and neck [START_REF] Faust | Antisense oligonucleotides against protein kinase CK2-a inhibit growth of squamous cell carcinoma of the head and neck in vivo[END_REF] and prostate [START_REF] Yenice | Nuclear casein kinase 2 (CK2) activity in human normal, benign hyperplastic and cancerous prostate[END_REF] and a number of experimental models and coincidental 4 presented here, support the view that the selectivity of TBB for CK2 is narrower than those of DMAT and TBI/TBBz which also are powerful inhibitors of kinases belonging to the PIM, HIPK, DYRK and PKD families, and led to the identification of new and more selective inhibitors of CK2.

Experimental

Enzymes. Native CK2 (nCK2) was purified from rat liver [26]. Human recombinant α and β subunits of CK2 were expressed in E. coli and the holoenzyme was reconstituted and purified as previously described [27]. Single and double mutants of CK2α subunit were generated as reported in [19,20]. The source of all the other protein kinases used for specificity assays is as previously either described or referenced [22].

Cell culture, treatment, and viability assay. The human leukemia Jurkat T-cell line was maintained in RPMI-1640, supplemented with 10% (v/v) foetal calf serum, 2mM Lglutamine, 100 units/ml penicillin and 100 µg/ml streptomycin. For the treatment, cells were suspended at a density of 10 6 cells/ml in a medium containing 1% (v/v) foetal calf serum, then incubated at 37 °C, in the presence of the compounds at the indicated concentrations. Control cells were treated with equal amounts of solvent (0.5 % v/v DMSO). At the end of incubations, cells were lysed by the addition of hypo-osmotic buffer, as previously described [20]. Cell viability was assessed by means of 3-(4,5-dimethylthiazol-2-yl)-3,5diphenyltriazolium bromide (MTT) reagent, while apoptosis was evaluated by means of the Cell Detection Elisa kit (Roche), based on the quantification of nucleosomes present in the cytosol of the apoptotic cells, following the manufacturer instructions. 10000 cells were used for each determination.

Phosphorylation assays. CK2 activity was tested in a final volume of 25 µl containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 12 mM MgCl 2 , 100 µM synthetic peptide substrate RRRADDSDDDDD and 0.02 mM [γ-33 P-ATP] (500-1000 cpm/pmole), unless otherwise indicated, and incubated for 10 minutes at 37 ºC. Native CK2 purified from rat liver (0.5-1 pmol) was usually the phosphorylating enzyme, unless otherwise indicated. Assays were stopped by addition of 5 µl of 0.5 M orthophosphoric acid before spotting aliquots onto phosphocellulose filters. Filters were washed in 75 mM phosphoric acid (5-10 ml/each) four times then once in methanol and dried before counting. PIM1 activity was determined by following the same procedure by incubating the kinase in the presence of 50 mM Tris-HCl pH 7.5, 0.1% (v/v) 2-mercaptoethanol, 0.1 mM EGTA, 30 µM synthetic peptide substrate RKRRQTSMTD [29] and 100 µM [γ-33 P-ATP]. HIPK2 activity was determined under the same conditions used for PIM1 assays except for ATP which was 20 µM and the use of MBP (10 µg) as phosphorylatable substrate. Conditions for the activity assays of all other protein kinases tested in selectivity experiments are described or referenced elsewhere [22]. Kinetic determination. Initial velocities were determined at each of the substrate concentration tested. K M values were calculated either in the absence or in the presence of increasing concentrations of inhibitor, from Lineweaver-Burk double-reciprocal plots of the data. Inhibition constants were then calculated by linear regression analysis of K M /V max versus inhibitor concentration plots. Alternatively inhibition constants were also deduced from the IC 50 /K i by determining IC 50 for each compound at 1 µM ATP concentration, i.e. much below Km value. The rationale underlying this approach, based on 5 the assumption that inhibition is competitive with respect to ATP, is illustrated by Burlingham and Widlanski [29].

Molecular modelling.

Human CK2α subunit and PIM1 catalytic subunit were retrieved from the PDB (PDB code: 1JWH and 3C4E, respectively) and processed in order to remove the ligands and water molecules. Hydrogen atoms were added to the protein structure using standard geometries with the MOE program. To minimize contacts between hydrogen's, the structures were subjected to Amber94 force field minimization until the rms of conjugate gradient was <0.1 kcal mol -1 Å -1 keeping the heavy atoms fixed at their crystallographic positions. To strictly validate the model generated and to calibrate the high-throughput docking protocol, a small database of known CK2 and PIM1 inhibitors was built and a set of docking runs was performed. After the calibration, phase flexible ligand-docking steps with three different programs, MOE-Dock, Glide, and Gold were performed essentially as previously described [START_REF] Cozza | Identification of ellagic acid as potent inhibitor of protein kinase CK2: a successful example of a virtual screening application[END_REF].

Synthesis of new analogues. All chemicals and solvents used for the syntheses were purchased from Sigma Aldrich. Deatails for the synthesis of most benzimidazoles and benzotriazoles have been described elsewhere [19,25,31,32]. New derivatives discussed in this paper have been synthesized as follows. The synthesis of 4,5,6,7-tetrabromo-1H-indazole (1, K59) was realized by the ring closure of the intermediate 3,4,5,6-tetrabromo-2-methyl-N-nitroso-N-acetylaniline [33]. The bromination of 1 was performed using bromine in anh. acetic acid, at elevated temperature, yielding the pentabromoderivative 4 (K64). Alkylation of 1 with 2-bromoethanol and 2dimethylaminoethylchloride provided, as expected, two regioisomers each, 2a, b (K60, K62) and 3a, b (K61, K63), respectively. The mixtures of N 1 -and N 2 -substituted derivatives were separated by flash chromatography and the structures of the derivatives were determined by comparing their 1 H NMR-and UV-spectra to those of the respective known N-substituted indazoles [34,35]. Dissociation constants of 1 (10.6) and 4 (8.40) measured in 50% aqueous ethanol showed the inductive effect of bromine substituents by lowering the respective pK a value by several units as compared to that of unsubstituted indazole (14.0). A similar effect was observed for TBB (pKa = 4.95) and 4,5,6,7-tetrabromobenzimidazole (pK a = 8.95) [17]. Similarly to tetrabromobenzimidazole, the two brominated indazoles 1 and 4 described here are practically neutral molecules at physiological pH (Scheme 1). The synthesis of 1-substituted acetic acid ethyl ester of 2-dimethylamino-4,5,6,7tetrabromobenzimidazole 6 (K67) was performed by N-alkylation of DMAT (5) with bromoacetic acid ethyl ester in acetone in presence of anh. K 2 CO 3 as base. The alkaline hydrolysis of 6 in ethanol-water solution gave the N 1 -carboxymethyl derivative of DMAT (7, K66) (Scheme 2). The other modified tetrabromobenzimidazoles were obtained from 1-methyl-4,5,6,7tetrabromobenzimidazole (8) as substrate. The heating of 8 with thiourea in 1-propanol gave respective 2-mercapto derivative 9 (K72). Alkylation of 9 with bromoacetic acid ethyl ester yielded 2-sulfanylacetic acid ethyl ester 10, which was substrate for further modifications. Aminolysis of 10 with methylamine gave respective N-methylamide (11, K74), whereas alkaline hydrolysis yielded 1-methyl-4,5,6,7-tetrabromobenzimidazole-2-sulfanylacetic acid (12, K75). (Scheme 3). TBB (4,5,6,7-tetrabromobenzotriazole) and its 2-dimethylamino derivative DMAT are among the most widely used, commercially available inhibitors of CK2. At variance with other compounds used to inhibit CK2, notably DRB and apigenin, both TBB and DMAT are believed to be very specific based on selectivity profiles run on panels of 30 and 33 protein kinases, respectively [18,19]. In the meantime, however, the size of the panel of kinases available for testing inhibitors' specificity has more than doubled [22] including now-a-days representatives of nearly all subfamilies of the kinome (Fig. 1). This prompted us to reevaluate the selectivity of TBB and DMAT toward CK2. Since DMAT shares its benzimidazole scaffold with the parent compound TBI (or TBBz) (see Fig. 2) also marketed as a CK2 inhibitor though its selectivity was not profiled before, TBI was also included in our analysis.

Results

Biochemical

The residual activity of a panel of 76 protein kinases tested in the presence of 10 µM TBB, DMAT and TBI and expressed as per cent of their activity in the absence of inhibitors is reported in Table 1 or mentioned in its legend. Even a cursory scrutiny of the data reveals that CK2 is not the only protein kinase drastically affected, several other kinases (values bold typed in Table 1) being more than 90% inhibited by one or more of the three compounds. A more detailed analysis allows the following conclusions to be reached: 1) Although the selectivity spectra of TBB, DMAT and TBI are similar they are not super-imposable, denoting higher similarity between DMAT and TBI (characterized by an imidazole ring) both of which also tend to be more promiscuous than TBB (composed instead of a triazole ring). This point is highlighted by drastic inhibition of PKD1 and CDK2/CyclinA by either DMAT or TBI, in contrast to fairly high residual activity observed in the presence of the same concentration of TBB. 2) There are three subfamilies of protein kinases, none of which was represented in the 30 protein kinases panel originally used to define the TBB specificity [18] whose components tend to be inhibited as drastically as CK2 by all the three compounds at 10 µM concentration, with residual activities below 10%; these are DYRKs (1a, 2 and 3), PIMs [START_REF] Meggio | One-thousand-and-one substrates of protein kinase CK2?[END_REF][START_REF] Pagano | Protein kinase CK2: a newcomer in the "druggable kinome[END_REF][START_REF] Pinna | The raison d'etre of constitutively active protein kinases: the lesson of CK2[END_REF] and HIPK2. A >90% inhibition of HIPK3 is also observed by DMAT and TBI, while TBB is significantly less effective in this respect. More detailed information about the relative efficiency of each inhibitor toward protein kinases whose activity is 90% or more inhibited by 10 µM concentration was obtained by determining their IC 50 values at 50 µM ATP. These data are reported in Table 2 and they strengthen the notion that among the three inhibitors the most selective one toward CK2 is TBB with a IC 50 value (0.15 µM) 3-5 fold lower than those of DYRK2, PIM1 and PIM3, and one order of magnitude or more below those of the other kinases. In contrast TBI appears to be the least selective toward CK2, as it inhibits PIM3, PIM1, PIM2, DYRK2 and PDK1 more efficiently than CK2 itself, and HIPK2 and CDK2 with comparable efficiency. This is partially due also to its relatively high IC 50 value with CK2 (0.60 µM). The overall superiority of TBB over DMAT is also highlighted by selectivity profiles performed with 1 µM instead of 10 µM inhibitor concentration (last two columns on the right in Table 1). Under these conditions the number of protein kinases inhibited ≥50% are 3 and 12 with TBB and DMAT, respectively (values underlined in Table 1). Note moreover that 7 out of the 12 PKs ≥50% inhibited by DMAT are inhibited as drastically as CK2 itself.

DMAT as a powerful inhibitor of kinases other than CK2.

Although the IC 50 value with CK2 is quite low (0.13 µM) DMAT displays submicromolar IC 50 values with almost all the other kinases in Table 2, with special reference to PKD1, PIM3 and PIM1 whose IC 50 value (148 nM) is comparable to that of the most potent PIM1 inhibitor (BIM-1) reported to date [36]. 3A. However, IC 50 determinations at increasing ATP concentration denote weak competition by ATP which, at almost physiological concentration (0.6 mM), causes only a 5.3-fold decrease in DMAT inhibition, as compared to 1 µM ATP concentration, while in the same range of ATP concentration the IC 50 with CK2 increases 22.1-fold, doubling the value calculated with PIM1 (1.2 µM) (Fig. 3B). It is expectable therefore that in the cellular environment (ATP ≥1 mM) PIM1 will represent the preferred target of DMAT, among all protein kinases probed in this respect.

Improvement of selectivity toward CK2

The observation that CK2 inhibitors tend to inhibit with comparable efficacy at least three other groups of kinases, notably PIMs, HIPK2 and DYRKs, prompted us to search for more selective compounds in the available library of TBB/TBI derivatives. This presently includes besides several dozens of tetrabromobenzimidazole congeners previously described [19,32] also a number of newly developed compounds with a pyrazole penta-atomic ring replaced for the imidazole one, whose prototype K59, 4,5,6,7-tetrabromoindazole, is shown in Fig. 2 together with its triazole (TBB) and imidazole (TBI) congeners. All the compounds of the library (68 altogether) were tested at 20 µM concentration on CK2, PIM1 and HIPK2. The assays were run at ATP concentrations around the K m values of the kinases (20 µM for CK2 and HIPK2, 100 µM for PIM1) and the residual activities are on display in Table S2 (see supplementary data). Focusing our attention only on compounds highly effective on CK2 (residual activity ≤15%, bold typed in the table) we observed that most of these are equally or even more potent inhibitors of PIM1, and two actually inhibit HIPK2 even more efficiently than CK2. A few compounds, however, namely K36, K59, K60, K62, K64, K66, K74 and K75, whose structures are shown in Table 3, displayed toward CK2 an inhibitory efficacy significantly higher than those observed with either PIM1 and HIPK2. The K i values of these compounds with respect to CK2, PIM1, HIPK2 and DYRK1a were calculated (Table 3). In all cases CK2 displays the lowest K i value as compared to the other three protein kinases. In particular two compounds, K59 and K64 display K i values below 100 nM with CK2 while their K i values with the second most sensitive protein kinase, which is PIM1 in both cases, are several fold higher. Both K59 and K64 are characterized by a pyrazole ring replaced for the triazole/imidazole rings of TBB and DMAT. K64 also contains a 5 th bromine atom on the pyrazole ring in addition to the 4 bromine atoms on the benzene ring which are a common denominator of all TBB derivatives described so far. Three other promising compounds belong to the benzimidazole class: these are K66, K74 and K75. In this case the K i calculated with CK2 is somewhat higher than 100 nM: this disadvantage however is amply compensated by proportionally higher K i values with PIM1, HIPK2 and DYRK1a. We focused further attention on those compounds whose K i values with CK2 are 10-fold or more lower than those with the second most inhibited kinase, notably K64, K66, K74 and K75. We firstly investigated if and to what extent the double mutation of CK2 residues Val-66 and Ile-174 to alanine, previously reported to decrease susceptibility to TBB and DMAT [19,20], also affected inhibition by the new compounds. This is clearly the case, as shown in Table 4; however responsiveness to double mutation is sharply variable: two inhibitors, K74 and K75, are somewhat more sensitive than TBB and DMAT to the double mutation whereas K66 is almost insensitive to it, with a just 6-fold increase in IC 50 value. By sharp contrast the pentabrominated inhibitor K64 has lost almost all its efficacy toward the CK2 double mutant, with a 600-fold raise in IC 50 . These differences clearly reflect diverse modes of binding of these compounds, as further indicated by the variable efficacy of the two single mutations, that of Val66 and that of Ile174. In particular the role of Ile174 is crucial for the binding of K64 whereas the binding of K66 relies more on interactions with Val66 as also confirmed by modelling (see Discussion). The behaviour of K66 attracted our attention because its scaffold is identical to that of DMAT, having in addition a short carboxylic side chain adjacent to the imidazole ring nitrogen (compare structures in Fig. 2). While this carboxylic adduct is still compatible with fairly potent inhibition of CK2 it makes its binding almost independent of Ile174, while more dependent on Val66 (Table 4), and, more important, it drastically reduces the ability to inhibit PIM1 and HIPK2 which are instead dramatically sensitive to DMAT (see Table 2). Once ascertained that the new CK2 inhibitors considered in Table 3, with special reference to K64, K66, and K75 have lost most of their potency toward those protein kinases, notably PIM1, HIPK2 and DYRK1a which display the tendency of being susceptible to CK2 inhibitors, we wanted to make sure that their overall selectivity was preserved. This analysis was performed on a panel of 78 protein kinases almost completely but not entirely overlapping the one of 70 protein kinases used for the screening presented in Table 1. The data are shown in Fig. 4. For sake of comparison also the data relative to DMAT are reported. The most remarkable outcome is indeed the dramatic gain of selectivity resulting from the derivatization of DMAT with a carboxymethyl side chain to give K66 (Fig 4): while, as noted above, DMAT under these conditions inhibits ≥50% 34 kinases out of 70 (i.e. almost 50%), this figure with K66 drops to just 5 kinases out of 78. Only 2 of these, moreover, a part from CK2, are drastically inhibited (≥80%) by K66: ERK8, and DYRK2. However, the IC 50 values for inhibition of DYRK2 is far above that of CK2 (8 µM as compared to 0.25, see legend of Fig. 4). Consequently the only kinase whose inhibition by K66 is comparable to that of CK2 is ERK8 (IC 50 =0.50 µM) thus placing K66 in the category of "dual kinase inhibitors" able to block with equal efficiency two unrelated kinases. Note in this respect that it would be easy to discriminate between ERK8 and CK2 using the inhibitor BAY439006 which at 10 µM concentration suppresses ERK8 activity without significantly affecting CK2 (27). The overall selectivity of K64 (Fig. 4) is also good, with 6 protein kinases (including CK2) inhibited ≥50% by 10 µM inhibitor. One of these is again ERK8, but in this case it is inhibited significantly less than CK2. The same applies to PIM2, PIM3, HIPK2 and PKD1, whose IC 50 values accordingly are at least one order of magnitude higher than that of CK2. Due to solubility problems the selectivity of K75 was profiled at 1 µM concentration (Fig. 4). Under these conditions only 3 kinases are ≥50% inhibited: CK2 and PIM3 (both 70% inhibited), and MSK1 (54% inhibited). K74 turned out to be unstable when kept for >2 hrs in DMSO solution, a circumstance which prevented its robotic profiling on the panel of protein kinases and probably also seriously hampers its usage for in cell experimentation.

Pro-apoptotic efficacy of K64 and K66

In the light of the data above the first choice novel CK2 inhibitors worthy to be tested on cell cultures were K64 and K66, whose selectivity is comparable. The former is somewhat more potent but the latter has the advantage that its only known target inhibited as drastically as CK2, ERK8, is absent in hematopoietic cells [37], which are those most frequently used for testing the pro-survival and oncogenic potential of CK2 (e.g. [16,[38][39][40]). An added value of K64 is instead its almost complete loss of efficacy toward the CK2 double mutant V66/I174AA (see Table 4), a property which can be exploited to provide the proof of concept As shown in Fig. 5A both K64 and K66 display cytotoxic activity comparable to that of TBB. Interestingly the cytotoxic efficacy of the three compounds roughly correlates to their potency as CK2 inhibitors calculated from their K i values (0.048, 0.12 and 0.25 for K64, TBB and K66, respectively). It should be also noted that while in principle the cytotoxic effect of TBB could be accounted for by the inhibition of protein kinases of the PIM and DYRK families having IC 50 values close to that of CK2 (Table 2), this doesn't apply to K64 and K66 (see above). As shown in Fig. 5B the cytotoxic effect of K64, similar to that of TBB [40] and of K66 is, at least partially, due to apoptosis as judged from the quantification of nucleosomes present in the cytosol of the apoptotic cells: these in cells treated with K64 5 µM are comparable to those induced by etoposide 25 µM.

Discussion

Considering that the human kinome is composed by more than 500 members whose catalytic site is highly conserved and that the most promising cell permeable protein kinase inhibitors are competitive with respect to the universal kinase co-substrate ATP, it is understandable that the development of really specific protein kinase inhibitors is a formidable challenge. While absolute selectivity may be not required for a kinase inhibitor to become a valuable drug and some promiscuity could even be an advantage in terms of therapeutic efficacy, as exemplified by clinical practice with Imatinib/Gleevec [41], lack of specificity is always a serious drawback whenever inhibitors are used to dissect the biological functions of a given protein kinase, where the unwanted blockage of other kinases may generate misleading results. This shortcoming can be attenuated by using more than one inhibitor of the same kinase and can be circumvented by the generation and cell transfection of kinase mutants which are refractory to a given inhibitor. Since however clusters of kinases often tend to be inhibited by the same classes of inhibitors and cell transfection not always is feasible and is likely to alter anyway cell functionality, the availability of pharmacological tools that are as selective as possible, invariably represents a critical advantage for dissecting signaling pathways. This is especially true in the case of protein kinases like CK2 which are constitutively active and cannot be easily up or down-regulated within the cell using physiological effectors. In the past considerable efforts have been made to develop CK2 specific inhibitors, belonging to different chemical classes (reviewed by Sarno et al. [13,14]). Apart from apigenin, a flavonoid devoid of narrow selectivity [20,42], the two most successful CK2 inhibitors were both derived from the nucleoside DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole), a rather weak CK2 inhibitor [43], by deleting its sugar moiety and replacing the chlorine and hydrogen atoms of its benzene ring with 4 bromine atoms. These were shown to be critical to encapsulate the inhibitor in a hydrophobic cavity which in CK2 is smaller than in the majority of other protein kinases [24,25]. This feature, which underlies the fairly narrow selectivity of these compounds toward CK2, has been exploited in many recent reports to probe the implication of this kinase in a variety of cellular functions [START_REF] Duncan | Too much of a good thing: the role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2[END_REF]. Despite their structural similarity however TBB and DMAT bind in different ways into the active site of CK2 [25], denoting a role of the negative charge present at neutral pH on the triazole ring of TBB, which is not present in DMAT nor in the other TBB derivatives [17,23]. In these latter the N 2 atom is replaced by a carbon, to give a tetrabromobenzimidazole derivative (TBI or TBBz) devoid of the negative charge at neutral pH [17]. The relevance of this difference to determine kinase selectivity was overlooked in our original studies because the panel of kinases used at that time for selectivity profiles was not large enough to disclose significant differences. It was concluded therefore that, apart from some minor differences, TBB and DMAT exhibited 10 quite super-imposable inhibition profiles and a similar, quite remarkable selectivity toward CK2, although DMAT is somewhat more potent, especially if used in cell culture [44]. Another advantage of DMAT, as well as of TBI and the other benzimidazole derivatives analysed in this respect [23] is that, owing to its high pK a value (8.9 for TBI and 10.6 for DMAT), at physiological pH the N-H of the imidazole ring is not dissociable and therefore these compounds are unable to depolarize mitochondria [19] by shuttling protons across the membrane. The same applies to the pyrazole ring of the indazole derivatives, e.g. K59 (pK a 10.6) and K64 (pK a 8.40) (see Materials and Methods). In contrast mitochondria depolarization is a drawback of TBB [20] whose triazole ring exhibits a much lower pH value, allowing proton shuttling under the effect of the pH gradient on the two sides of the mitochondrial membrane. In this paper, exploiting a panel of kinases more than twice as large as the original one, and including members of nearly all the kinome subfamily (Fig. 1) we have reached the conclusion that the selectivity of TBB is definitely narrower than those of DMAT and TBI. While in fact both DMAT and TBI drastically inhibit several kinases in addition to CK2, notably PIM 1, 2 and 3, DYRK1a, 2 and 3, PKD1, HIPK2 and 3 and CDK/CyclinA, TBB is significantly less effective on these kinases than it is on CK2 as highlighted by comparing both IC 50 values (Table 2) and selectivity profiles of TBB and DMAT performed at 1 µM inhibitor concentration (see Table 1). On the other hand it should be taken into account the possibility that besides protein kinases also non kinase enzymes interact with CK2 inhibitors: this applies to TBB, but, even more to TBI and DMAT [START_REF] Duncan | An unbiased evaluation of CK2 inhibitors by chemoproteomics: characterization of inhibitor effects on CK2 and identification of novel inhibitor targets[END_REF]. Incidentally the work presented here provides valuable hints for the development of PIM and HIPK inhibitors more potent and/or selective than those currently available. In the case of PIM1, the product of a proto-oncogene whose dysfunction has been implicated in diverse cancers with special reference to diffuse large cell lymphoma (DLCL) [START_REF] Pasqualucci | Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas[END_REF], some inhibitors are available [36], whose potency however is comparable to those of TBI and DMAT and whose selectivity has not been explored. Our data moreover disclose the possibility to discriminate among PIM isoforms exploiting their different susceptibility to TBI and DMAT (see Table 2). In contrast inhibitors of HIPK2, a kinase required for normal hematopoiesis and blood vessel formation [START_REF] Aikawa | Roles of HIPK1 and HIPK2 in AML1-and p300dependent transcription, hematopoiesis and blood vessel formation[END_REF], which plays a critical role in triggering p53-dependent apoptosis in response to the antineoplastic drug cisplatin [START_REF] Di Stefano | Homeodomain-interacting protein kinase-2 activity and p53 phosphorylation are critical events for cisplatin-mediated apoptosis[END_REF], are not available at all, to the best of our knowledge, a circumstance which makes DMAT and TBI the best tools presently available to downregulate HIPK2. Once established that TBB and, even more, DMAT and TBI are CK2 inhibitors less selective than it was previously thought, our next aim was the identification of DMAT/TBB derivatives which still are effective on CK2 having lost most of their efficacy toward PIM1, HIPK2 and DYRK1a. This goal turned out to be hard to attain since most compounds tested which are potent inhibitors of CK2 also tend to inhibit PIM1 and/or HIPK2 with similar potency (see Table S2 of supplementary data). A few notable exceptions however were provided by two compounds belonging to the class of indazoles (K59 and K64), having a pyrazole ring replaced for the triazole and imidazole pentatomic rings present in TBB and DMAT, respectively, and three new benzimidazole derivatives (K66, K74 and K75). These are all characterized by the ability to inhibit CK2 more potently than PIM1, HIPK2 and DYRK1a, with K i values which in the case of K64 and K66 are many-fold lower than those calculated with the second most inhibited kinase (PIM1 in both cases) Especially remarkable appears in this respect the behaviour of K66, considering its close structural relatedness with DMAT: while DMAT however inhibits PIM1 with the same potency as CK2, K66 has lost most of its efficacy toward PIM1, as judged from big differences in K i and IC 50 values. The structural similarity between DMAT and K66, in conjunction with availability of 3D structures of both PIM1 and CK2, this latter also in complex with DMAT [25] provide a key to rationalize the preferential binding of K66 to CK2. The structure of DMAT bound into the active site of CK2 together with the derived model in which DMAT has been replaced by K66 are shown in Fig. 6 A,C. Incidentally this accounts also very well for the finding that Ile174 is less important for the binding of K66 (see Table 4). Note in fact that K66, owing to its carboxylic group, is making electrostatic interactions with the positive area close to Lys68 thus displacing the dimethylamino group from its apolar contacts with Ile174 and placing it closer to Val66. In the case of PIM1, DMAT is rotated about 60° with respect the crystallographic data, due to the presence of Phe49. In fact, unlike its CK2 homologue Tyr50, which is exposed toward the solvent, Phe49 is instead oriented toward the hydrophobic cavity forcing the inhibitor to a new pose. As shown in figure 6B the dimethylamino group at position 2 is placed inside a new favourable hydrophobic pocket formed by PIM1 residues Val52, Leu120, Ile185 and Phe49, while in CK2 is simply oriented toward the outside (Figure 6A). These structural observations may explain the similar activity of DMAT in CK2 and PIM1, even in the presence of Val66/Ala65 natural substitution. On the other hand K66 interacts with PIM1 binding cleft in the same way showed for DMAT due to the presence of the dimethylamino group. Under these conditions its carboxyl side chain at position 1 cannot interact with Lys67, homologous to CK2 Lys-68, and thereby is placed in an unfavourable hydrophobic zone (Figure 6D). Importantly moreover the improved selectivity of K66 as compared to DMAT is not restricted to its ability to discriminate between CK2 and PIM1, but it also reflects in a reduced or abolished inhibitory efficiency toward a large number of kinases deeply affected by DMAT, as outlined by comparing their specificity profiles in Fig. 4: while 34 kinases are ≥50% inhibited by 10 µM DMAT, this figure drops to just 5 with K66, under identical condition. Only one of these latter moreover (ERK8) is inhibited as drastically as CK2 (i.e. >80%), while 12 are those >80% inhibited by DMAT. In an attempt to quantify these differences a "promiscuity score" has been devised, expressing "average" inhibition of all the kinases of the whole panel, by a given concentration of the inhibitor, sufficient to drastically inhibit (about 90%) the kinase considered (in our case CK2) (see legend of Table 5). Such a score, which reflects the promiscuity of the kinase, and is inversely proportional to its "average" selectivity, equals 18.36 and 46.66 with 10 µM K66 and DMAT, respectively. Interestingly a promiscuity score similar to that of K66 has been calculated for K64 (16.8) reinforcing the concept that both K64 and K66 are markedly more selective than the three related commercially available CK2 inhibitors, TBB, DMAT, and TBI, this latter having a score >50 (see Table 5). It should be also noted that while at 10 µM inhibitor the promiscuity scores of TBB and DMAT are similar, if the profiling is performed at 1 µM concentration, where CK2 is about 80% inhibited by either TBB or DMAT (see Table 1), the promiscuity score of DMAT (22.6) doubles that of TBB (11.14) confirming also by this criterion the concept that TBB is more selective than DMAT. Comparing however the selectivity of TBB with those, much higher, of K64 and K66 and considering that these latter are equally effective as TBB as inducers of apoptosis it is predictable that K64 and K66 will replace TBB (not to say about DMAT and TBI) as first choice tool for studying cellular functions of CK2. Since the chemical names of K64 and K66 are exceedingly long (3,4,5,6,7-pentabromo-1H-indazole and 4,5,6,7- Table 1: Selectivity profiles on large kinase panel of TBB, DMAT and K17. Activity assays were linear with respect to time and enzyme concentration and were performed as previously described [22]. Residual activity was determined in the presence of the indicated concentrations of inhibitor and expressed as a percentage of the control without inhibitor. Residual activities ≤10% are bold typed, those ≤50% are underlined. Other protein kinases not included in the selectivity panel, namely the isoforms α, γ and δ of CK1, the tyrosine kinases Lyn, c-Fgr and Syk, and the Golgi casein kinase (G-CK), were tested under conditions specified elsewhere [18] and found to be unaffected (<10% inhibited) by 10 µM TBB, DMAT and TBI. Activity assays were linear with respect to time and enzyme concentration and were performed as previously described [22]. Residual activity was determined in the presence of 10 µM inhibitor except in the case of K75 which was tested at 1 µM (see text) and expressed as a percentage of the control without inhibitor. DMAT is also shown for comparison (data drawn from Table 1). IC 50 values of K66, determined for ERK8 and DYRK2, were 0.5 and 8, respectively.

Kinase

  Journal Immediate Publication. Published on 30 Jun 2008 as manuscript BJ20080309 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080309 S t a g e 2 ( a ) P O S T -P R I N T 6 Re-evaluation of TBB, DMAT and TBI selectivity.

Biochemical

  Journal Immediate Publication. Published on 30 Jun 2008 as manuscript BJ20080309 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080309 S t a g e 2 ( a ) P O S T -P R I N T 7 Similar to CK2 [19] PIM1 is also inhibited by DMAT by a mechanism which is competitive with respect to ATP, as shown by the double reciprocal plot reported in Fig.

  Biochemical Journal Immediate Publication. Published on 30 Jun 2008 as manuscript BJ20080309 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080309 S t a g e 2 ( a ) P O S T -P R I N T

  Biochemical Journal Immediate Publication. Published on 30 Jun 2008 as manuscript BJ20080309 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080309 S t a g e 2 ( a ) P O S T -P R I N T that the cellular effects of a given inhibitor are actually mediated by CK2 rather than by other cellular targets.

Scheme 1 .Scheme 2 .Scheme 3 .

 123 Scheme 1. Synthesis of substituted tetrabromoindazoles and pentabromoindazole. Reagents: (a) 1) Ac 2 O, 2) nitrous gases; (b) Br 2 , AcOH; (c) K 2 CO 3 , 2-butanone, 2-BrC 2 H 4 OH or (CH 3 ) 2 NCH 2 CH 2 Cl.Br

Fig. 1 :

 1 Fig. 1: Distribution within the human kinome of protein kinases tested for selectivity against CK2 inhibitors. The human kinome [50] is shown and the protein kinases included in the selectivity profiling tested for selectivity are indicated by blue circles. Protein kinases included in the 2001 TBB selectivity test [18] are marked in red. In yellow additional protein kinases tested only in Padova under conditions previously described [18].

Fig. 2 :Fig. 3 :

 23 Fig. 2: Molecular structure of representative benzotriazole, benzimidazole and benzopyrazole derivatives dealt with in this paper. Fig. 3: Kinetic analysis of PIM-1 inhibition by DMAT. Phosphorylation assays were performed as described in the Experimental section in the absence and in the presence of the indicated concentrations of DMAT. The data represent the mean of values obtained in triplicate with SD never exceeding 15%. Lineweaver-Burk double reciprocal plots obtained in the absence and in the presence of the indicated DMAT concentrations are shown in A. The inset refers to the K m /V max vs. [I] replot the value of K i has been determined from. B: IC 50 values for the inhibition of PIM1 and CK2 by DMAT calculated at increasing ATP concentrations.

Fig. 4 :

 4 Fig. 4: Comparison between the selectivity profiles of DMAT and some new CK2 inhibitors.Activity assays were linear with respect to time and enzyme concentration and were performed as previously described[22]. Residual activity was determined in the presence of 10 µM inhibitor except in the case of K75 which was tested at 1 µM (see text) and expressed as a percentage of the control without inhibitor. DMAT is also shown for comparison (data drawn from Table1). IC 50 values of K66, determined for ERK8 and DYRK2, were 0.5 and 8, respectively.

Fig. 5 :

 5 Fig. 5: Proapoptotic efficacy of new CK2 inhibitors on Jurkat cells.A. Jurkat cells were treated for 24 h with increasing concentration of the indicated inhibitors. Cell viability was assessed by the MTT method, assigning 100% value to the control cells, treated with the solvent. Reported values represent the means +S.E.M. from four separate experiments. B. Jurkat cells were treated with the indicated concentrations of inhibitor for 4h. Apoptosis was evaluated by quantification of nucleosomes present in the cytosol of the apoptotic cells, using the Cell Detection Elisa kit (Roche). Treatment with 25 µM etoposide (Eto) was used as a positive control for apoptosis induction. Nucleosome enrichment was calculated from the ratio between the signal in control cells and treated cells.

Fig. 6 :Figure 1 Biochemical

 61 Fig. 6: Molecular Docking of DMAT (A, B) and K66 (C, D) bound to the active site of CK2 (left) and PIM1 (right).Molecular docking was performed as described in the Experimental Section. Active LP distribution surface of CK2 and PIM1 binding pockets were represented. Green indicates hydrophobic regions, while in violet are hydrogen bonding or metal-LonePair regions.

Figure 5 Figure 6

 56 Figure 3

Table 2 :

 2 IC 50 (µM) of selected protein kinases for TBB, DMAT and TBI (TBBz). Phosphorylation conditions are either described in the Experimental section or referenced elsewhere[22]. ATP concentration was 50 µM. Data with CK2 were obtained using native holoenzyme purified from rat liver. Replacement of native CK2 with recombinant human CK2 holoenzyme (α 2 β 2 ) or with the isolated α subunit caused only minor alterations in the IC 50 values.

	Biochemical Journal Immediate Publication. Published on 30 Jun 2008 as manuscript BJ20080309
	Kinase CK2 PIM1 PIM2 PIM3 HIPK2 HIPK3 DYRK1a DYRK2 DYRK3 PKD1 CDK2 S t a g e 2 ( a ) P O S T -P R I N T TBI DMAT TBB 0.60 0.13 0.15 0.115 0.148 1.04 0.18 1.6 4.3 0.07 0.097 0.86 0.7 0.37 5.3 1.22 0.59 4.9 2.1 0.41 4.36 0.34 0.35 0.99 3.7 1.7 5.3 0.34 0.18 5.9 0.58 0.64 14.0	THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080309

Table 3 .

 3 Ki values (µM) of selected compounds for CK2, PIM1, HIPK2 and DYRK1a protein kinases. Kinetic determinations were performed as described in the Experimental section (with native CK2 holoenzyme) according to the Cheng-Prusoff equation[28].

	Kinase	K36			K59			K60		K62				K64			K66			K74						K75
		Br Br	Br Br	N N C H 3	S C H 3	Br Br	Br Br	N H N	Br Br	Br Br	N O H N	Br Br	Br Br	N N	N	Br Br	Br Br	N H N Br	Br Br	Br Br	N N	OH O CH 3 N CH 3	Br Br	Br Br	N N CH 3	S	C H 3	O NH	Br Br	Br Br	N N CH 3	S	O OH
	CK2	0.08			0.068		0.71		0.40				0.048		0.25			0.28						0.16
	PIM1	0.56			0.55			3.66		4.50				0.738		8.65			4.00						2.55
	HIPK2	2.29			1.53			11.33		8.88				1.594		15.25			60.31					12.77
	DYRK1a >25.00			1.00			3.45		2.85				1.64			11.90			>40.00					12.10

Biochemical Journal Immediate Publication. Published on 30 Jun 2008 as manuscript BJ20080309 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080309 S t a g e 2 ( a ) P O S T -P R I N T

Table 4 :

 4 IC 50 (µM) of selected indazole and benzimidazole derivatives for wild type and mutated CK2. Recombinant CK2 holoenzyme was reconstituted in vitro by mixing at 1:1 molar ratio β subunit with wild type or mutated CK2α subunit. The data represent the mean of three independent experiments with SD never exceeding 12%. dm= double mutant in which both Val66 and Ile174 have been replaced by Ala.

	Compound CK2 wt	Val66Ala	Ile174Ala	CK2 dm	Ratio
						dm/wt
	K64	0.052	2.60	>40.00	31.28	601.5
	K66	0.50	22.37	1.14	3.32	6.6
	K74	0.60	>40.00	>40.00	40.64	67.3
	K75	0.31	40.00	>40.00	15.22	49.0
	DMAT a	0.14	1.78	3.30	5.79	30.47

a Data obtained from ref.

[19]

. Biochemical Journal Immediate Publication. Published on 30 Jun 2008 as manuscript BJ20080309 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080309 S t a g e 2 ( a ) P O S T -P R I N T

Table 5 :

 5 "Promiscuity score" of selected CK2 inhibitors. Promiscuity score was calculated from the selectivity profiles (Fig.4and Table1) as the mean of % inhibitions observed with all the kinases tested, excluding the kinase of interest (CK2). Inhibition of CK2 was 93-95% in the presence of 10 µM inhibitors, 80% in the presence of 1 µM DMAT and TBB. Values of inhibition <0 (i.e. stimulation) were adjusted to 0%.

	Inhibitor	Concentration	Score (%)
		(µM)
	K64	10	17.00
	K66	10	19.33
	DMAT	10	48.60
	TBB	10	47.37
	DMAT	1	23.10
	TBB	1	10.18

Biochemical Journal Immediate Publication. Published on 30 Jun 2008 as manuscript BJ20080309 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080309 S t a g e 2 ( a ) P O S T -P R I N T

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgments

We thank Professor Sir Philip Cohen (Dundee, Scotland) for critical reading of the manuscript. The Molecular Modelling Section coordinated by Prof. Stefano Moro (Padova) is gratefully acknowledged. The work was supported by grants to L.A.P. from AIRC and European Commission (PRO-KINASERESEARCH 503467) and by grant to ZK and AO from Ministry of Education and Science PBZ-MIN 014/P05/2004 (Poland).

Supplementary Data Available

Detailed description of the synthesis and analytical data (Table S1) for the newly obtained compounds discussed in the present work. Profiling of a library of compounds on CK2, PIM1 and HIPK2 (Table S2). This material is available online.