Satu H Helppolainen

Kirsi P Nurminen

Juha Ae Määttä

Katrin K Halling

Peter Slotte

Tuulia Huhtala

Timo Liimatainen

Seppo Ylä-Herttuala

Juha A E Määttä

J Peter Slotte

Seppo Ylä- Herttuala §

Ale NÄRVÄNEN ‡ Kari J Airenne §

Janne Jänis

Pirjo Vainiotalo

Jarkko Valjakka

Markku S Kulomaa

Henri R Nordlund
email: henri.nordlund@uta.fi

Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family

Keywords: avidin-biotin technology, biotin-binding protein, bacterial avidin Biochemical

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

among known avidins. In addition to its quaternary structure assembly, its ligand-binding properties, thermal stability, protease sensitivity, immunological cross-reactivity, and biodistribution were studied here.

EXPERIMENTAL Construction of the expression vector

The rhizavidin gene was amplified by PCR using the symbiotic plasmid DNA of Rhizobium etli as a template. The first construct was the full-length wild-type form (GenBank™ U80928, complement 293047-293586) in which the rare translation initiation codon UUG was converted to AUG for E. coli expression. The primer used for the 5`-end of the full-length form was CACCATGATTATTACGAGTTTATATGC. The second construct was N-terminally truncated by removing 24 amino acids from the putative signal peptide in comparison to the wild-type form. The PCR primer for the 5'-end of the truncated form was CACCATGATCCGTACTAATGCAGTTGC. Also the N-terminally truncated form possessed a putative signal peptide, 20 residues in length, and both forms were expected to produce identical mature peptide, since the predicted signal cleavage site was identical for both of them (SignalIP program). The primer for the 3'-end of both protein forms was TTAATCCTTCAAGAGGCTTTTGT. The 5'-ends of the forward primers included CACC, which is necessary for directional TOPO ® cloning. The PCR products were extracted from 1.5% agarose gel. The constructs were cloned to pET101/D-vector using the TOPO ® cloning protocol (Invitrogen). The expression vectors were validated by sequencing with an ABI Prism™ 310 Genetic Analyzer.

Production of rhizavidin

The pET101/D-based expression vectors were transformed into Escherichia coli BL21-(AI) cells (Invitrogen), of which fresh transformants were cultured in Luria-Bertani medium supplemented with 0.1% (w/v) glucose and 10 µg/ml ampicillin (Sigma/Aldrich) at 26 °C, 225 rpm. When the culture density reached an OD 600 of 0.2 -0.3, L-arabinose 0.2% (w/v) was added to induce protein expression. Cultivation was continued for approximately 22 h at 26 °C, where after the cells were collected by centrifugation (5000 g, at 4 °C, 10 min) and suspended as previously described [START_REF] Hytönen | Efficient production of active chicken avidin using a bacterial signal peptide in escherichia coli[END_REF].

The suspension was sonicated twice for 2 min (Vibra cell TM ). The protein was isolated by affinity chromatography on a 2-iminobiotin column (Affiland, Liège, Belgium) as described by Airenne et al. [START_REF] Airenne | Production of biologically active recombinant avidin in baculovirus-infected insect cells[END_REF] and subsequently eluted with 0.1 M acetic acid. Concentrations of the eluted protein fractions were determined using the theoretical extinction coefficient of rhizavidin at 280 nm (29160 M -1 cm -1 ). The fractions were analyzed by SDS-PAGE and gels were stained with Coomassie Brilliant Blue.

Primary structure analysis

The stereo representation of avidin was done by PyMol (DeLano Scientific LLC, Palo Alto, CA, USA). The sequence comparisons were made using the Needle program from the European Molecular Biology Open Software Suite (EMBOSS), CLUSTAL W and Swiss-Prot.

Structural analysis

The size of the purified protein was analyzed by gel filtration HPLC in the absence and presence of D-biotin, as previously described [START_REF] Nordlund | Novel avidin-like protein from a root nodule symbiotic bacterium, bradyrhizobium japonicum[END_REF], using a Superdex 200 10/300 GL column. Formation of intersubunit disulfide bridges was analyzed by SDS-PAGE using a sample buffer devoid of the reducing agent β-mercaptoethanol, and subsequent Coomassie Brilliant Blue staining.

Mass spectrometry

The protein samples were desalted for mass spectrometry using PD-10 columns. All mass spectrometric experiments were performed on a 4.7-T Bruker APEX IV Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker Daltonics, Billerica, MA) employing electrospray ionization (ESI). The instrument was interfaced to an external Bruker Apollo™ I ion source. The sample solutions were directly infused at a flow rate of 1.5 µL min -1 (end-plate at -3.8 kV). N 2 was used as drying (10 psi, 80 °C) and nebulizing gas. ESI-generated ions were passed through a dielectric Pyrex capillary (entrance and exit potentials -4200 and 22-300 V, respectively), accumulated in a radio-frequency (RF)-hexapole for 4.0 s and subsequently transferred to a cylindrical RF-shimmed ICR cell (Infinity Cell) within 2600 µs. Ions were captured inside the cell by a "Sidekick" trapping technique and excited to detectable cyclotron radii by an RF-sweep (∼18-90 kHz). A total of 128-256 co-added (512-kword) time-domain transients were fast Fourier transformed, followed by magnitude calculation and frequency-to-m/z calibration with respect to the ions of an ES Tuning Mix (Agilent Technologies, Santa Clara, CA).

Differential scanning calorimetry (DSC)

The melting temperatures of rhizavidin, both in the absence and presence of D-biotin, were studied by DSC, as previously described [START_REF] Nordlund | Tetravalent single-chain avidin: From subunits to protein domains via circularly permuted avidins[END_REF][START_REF] Hytönen | Characterization of poultry egg-white avidins and their potential as a tool in pretargeting cancer treatment[END_REF][START_REF] Gonzalez | Interaction of biotin with streptavidin. thermostability and conformational changes upon binding[END_REF]. The experiments were performed in the absence and presence of biotin in 0.1 M acetic acid. The excess of biotin was 2.5 per biotin-binding site. The thermograms (25-130 °C, 0.92 °C/min) were collected with a Nano II high-sensitivity differential scanning calorimeter (Calorimetry Sciences Corp., Lindon, UT, USA). Data analysis was carried out using CpCalc 2.1 (Calorimetry Sciences Corp.) and Microcal Origin 6.0 (MicroCal, Northampton, MA, USA) softwares.

Heat treatment

The heat stability of rhizavidin was tested by exposing it to high temperatures. Protein samples (0.35 µM in PBS) were heated for 1 to 30 min at 80, 90 and 100 °C and chilled. The samples were analyzed by ELISA as previously described in detail [START_REF] Nordlund | Enhancing the thermal stability of avidin. Introduction of disulfide bridges between subunit interfaces[END_REF].

Proteinase K assay

Sensitivity to proteinase K was tested in the presence and absence of biotin as previously described [START_REF] Hytönen | Characterization of poultry egg-white avidins and their potential as a tool in pretargeting cancer treatment[END_REF]. The final concentration of biotin was 61 nM and that of rhizavidin 6.9 nM. The samples were incubated for 0, 15, 30, 60 min and 20 h in the presence of proteinase K (1:25 w/w). After incubation, an equal volume of SDS-PAGE sample buffer was added; the samples were heated for 5 min at 100 °C and chilled on ice. All of the samples were analyzed at the same time by SDS-PAGE (17% gel) and subsequent Coomassie Brilliant Blue staining.

Isothermal titration calorimetry (ITC)

The thermodynamics of rhizavidin-biotin binding interaction were studied by VP-ITC (MicroCal, LLC, Northampton, MA, USA). Rhizavidin, in acetic acid, was dialyzed against a sodium phosphate buffer (50 mM NaH 2 PO 4 /Na 2 HPO 4 , 100 mM NaCl, at pH 7). After dialysis rhizavidin was diluted to a concentration of 31 µM with the dialysis buffer. D-Biotin (0.4 mM) was diluted with the same buffer. The measurement was conducted at 25 °C using 10 µl titration aliquots (510 rev. /min, 30 µcal/s). In ITC Gibbs free energy, G = -RTlnK b = H-T S, is used to determine the thermodynamic parameters. Binding constant (K b ) and enthalpy change ( H) are measured by ITC, temperature (T) and the universal gas constant (R) are known, and, therefore, change in entropy S) can be calculated.

Biotin dissociation studies

The dissociation rate constant (k diss ) of rhizavidin was determined by dissociation of D-[8,9-3 H]biotin (Amersham Biosciences) at 20, 30, 40 and 50 °C as previously described by Klumb et al. [START_REF] Klumb | Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex[END_REF]. The labeled biotin was incubated with rhizavidin (50 nM) in sodium phosphate buffer (50 mM NaH 2 PO 4 /Na 2 HPO 4 , 100 mM NaCl, 10 µg/ml BSA, pH 7), followed by addition of an excess of nonradioactive (or cold) biotin which displaced D-[8,9-3 H]biotin. Unbound and displaced biotin was separated by filtration (Microcon YM-30, Millipore Corporation, Bedford, MA, USA). The amount of displaced radio biotin was detected by scintillation counter (Wallac 1410). The dissociation rate constant was determined from the fraction of biotin bound at each time point from the equation -k off t = ln[(x t -x)/(x t -x 0 )], in which x is the free biotin at each time point, x t the total amount of ligand before addition of protein and x 0 the amount of free ligand in the presence of protein prior to addition of cold biotin [START_REF] Klumb | Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex[END_REF].

The dissociation rate of a fluorescent biotin conjugate (ArcDia BF560 TM ) was measured with a Quanta Master TM spectrofluorometer (Photon Technology International, Inc., Lawrenceville, NJ, USA) with PTI Fluorescence Master System, Felix 32 software as previously described in detail [START_REF] Hytönen | Efficient production of active chicken avidin using a bacterial signal peptide in escherichia coli[END_REF]. The fluorescent biotin conjugate was diluted with 50 mM sodium phosphate buffer containing 650 mM NaCl (pH 7) to a concentration of 50 nM, and the initial signal was measured at 560 nm for 100 sec. Rhizavidin was added to a final concentration of 50 nM and the signal of the complex was measured for 300 sec. Finally, free biotin was added to a final concentration of 5 µM, and emission of released fluorescent biotin measured at 575 nm for 1 hour. The release percentage was determined from the equation [(x diss -x ini )/(x b -x ini )]*100%, in which x diss is dissociation of fluorescent biotin, x ini the fluorescent signal caused by unbound fluorescent biotin, and x b the signal caused by the protein fluorescent biotin complex.

Antibody recognition

The cross-reactivity of polyclonal antibodies against (strept)avidin with rhizavidin was tested with cancer patient sera samples. Positive samples were gathered from patients who were exposed to (strept)avidin and negative sera samples from those exposed to neither of the proteins. All of the samples were obtained from the Division of Nuclear Medicine, European Institute of Oncology, Milan, Italy. Rhizavidin was also exposed to polyclonal rabbit antibodies produced against avidin (University of Oulu, Finland) and streptavidin (Weissman Institute, Jerusalem, Israel). Both experiments were conducted by ELISA as previously described in detail [START_REF] Nordlund | Novel avidin-like protein from a root nodule symbiotic bacterium, bradyrhizobium japonicum[END_REF]. Additionally, recognition of rhizavidin by polyclonal rabbit antibodies against (strept)avidin was studied by Western blot analysis essentially as previously described by Airenne et al. [START_REF] Airenne | Production of biologically active recombinant avidin in baculovirus-infected insect cells[END_REF].

DTPA-conjugation

Avidin and rhizavidin were labeled with pSCN-Bn-DTPA (Macrocyclics, Dallas, TX); 2 mg (50 nmol) of avidin or rhizavidin were dissolved in 1 ml of water. An approximately 25-fold molar excess (1 mg/ml of 10 mM carbonate buffer, pH 9.5) was added to both avidin and rhizavidin solutions and stirred overnight at room temperature. After incubation, the conjugates were concentrated with an Amicon ultra centrifugal filter device to 100 µl (Millipore, Bedford, MA). To remove the remaining free DTPA, 2 ml of PBS were added to the filter devices and the conjugates were concentrated again. Finally, the concentrates were diluted with PBS (1 ml) and frozen.

Radiolabeling

Two patches of Rhizavidin-DTPA (Rz-1 and Rz-2) and one patch of avidin-DTPA (Av-1) conjugates were labeled with 99m Tc-pertechnetate. 150 µl (1 mg/ml in water) DTPA-Rz-1, DTPA-Rz-2 or DTPA-Av was mixed with 50 µl fresh solution of tin(II)chloride (10 mg/ml in 10 mM HCl) in a nitrogen atmosphere; 200 µl of 99m Tc-pertechnetate solution containing 200 MBq was added to each conjugate and incubated at room temperature for 10 min. After incubation radiochemical purity was tested by instant thin-layer chromatography (ITLC SG, Pall Corporation, OEM Healthcare MI USA) in a 13 x 1.5 cm strip and saline as the mobile phase. The purity was 98.9% for Rz-1, 99.6% for Rz-2 and 100% for Av-1.

SPECT imaging

SPECT studies were conducted using an XSPECT TM system provided by Gamma Medica Inc.

(Northridge, CA). Healthy male Wistar rats were anesthetized with isofluran and the vena femoralis was cannulated. The animals were injected with 99m Tc labeled DTPA avidin or rhizavidin in 500 µl of saline. Rat 1 was injected with 117.6 MBq of Rz-1, rat 2 with 70 MBq of Rz-2 and rat 3 with 130.9 MBq of Av-1. After injection, each individual rat was immediately moved to the animal bed of the SPECT equipment and a nose cone was used to ensure that the animals remained sedated.

SPECT acquisition was performed with a high-resolution parallel-hole collimator with a FOV of 125 x 125 mm and a hole diameter of 1.2 mm. In all, 11 planar projections with a duration of 5 minutes were collected. The energy window for 99m Tc was set at 141 keV +/-10%. Post reconstruction filtering was performed using the Butterworth method. After SPECT imaging an Xray image of the rat skeleton was taken using a CMOS detector with 48 µm pitch and voltage 60 kV.

RESULTS

Production and purification of rhizavidin

The N-terminally truncated form of rhizavidin was produced in E. coli. The yield was on the average 4 mg/l. As the full-length, wild-type form of the protein was not produced efficiently under the same conditions as the truncated form, all the experiments described in the present study were performed with the N-terminally truncated rhizavidin. After affinity chromatography purification a both pure and homogenous protein was obtained, subsequently observed in Coomassie Brilliant Blue-stained SDS-PAGE gels as one band of the expected size.

Gel filtration and intersubunit disulfide bridges

In gel filtration chromatography (HPLC) only the dimeric form of rhizavidin was observed as a single peak both in the absence and presence of biotin, at a size of 29.0 kDa and 31.7 kDa, respectively (Table 1). According to non-reducing SDS-PAGE analysis, rhizavidin was not observed to form intersubunit disulfide bridges, although two cysteine residues are present in its sequence. Only a band of the expected size (14 kDa), indicating a monomer, was observed (data not shown).

Mass spectrometry

Mass spectrometric experiments demonstrated unequivocally that rhizavidin is indeed a noncovalent dimer. Figure 2 shows the ESI FT-ICR mass spectra of rhizavidin measured in 10 mM ammonium acetate buffer (pH 6.8) both in the absence (A) and presence (B) of D-biotin. To avoid dissociation of the weak noncovalent protein complexes in the mass spectrometer, the drying gas temperature was optimized to 80 °C and the capillary exit potential adjusted to 20-150 V. The peaks in figure 2A represent different charge states of the dimeric protein, with a determined average mass of 28888.5 ± 0.4 Da (theor. 28888.7 Da). In the presence of D-biotin (Figure 2B), the peaks shifted towards higher m/z, consistent with the binding of two D-biotin molecules, and the mass increased to 29376.1 ± 0.4 Da (theor. 29376.5 Da). A very small amount of a dimer lacking one D-biotin was observed in the mass spectrum (marked with asterisk), this being due to a partial dissociation of the complex during the hexapole accumulation and not related to its solution behavior. No other protein forms (e.g., tetramer) were detected. As the charge states appeared in the m/z-range of 2400-3700, isotopic resolution was not obtained for the dimers, and the masses are therefore reported as average masses. To determine the isotopic mass for the rhizavidin monomer, the capillary exit potential was increased to 300 V to dissociate noncovalent dimers into monomers. The observed charge state distribution in figure 2C represents the monomeric protein, with the most abundant isotopic mass determined as 14443.66 ± 0.02 Da, which well agrees with the theoretical value (14443.68 Da) calculated from the protein sequence with an intact intramolecular disulfide bridge Cys50-Cys79.

Thermal stability assays

In ELISA heat-treated rhizavidin seemed to be more stable than avidin. Avidin forfeited all of its activity within 15 minutes at 100 °C, while rhizavidin still retained half. Rhizavidin lost all of its activity within 30 minutes at 100 °C. In line with this, rhizavidin retained its activity better than avidin at 90 °C and 80 °C.

In DSC measurement the melting temperatures (T m ) of rhizavidin (in 0.1M acetic acid) in the absence and presence of biotin were 74.8 °C and 100.5 °C, respectively (Table 1), shown as single peaks in the thermogram (Figure 3). The previously measured T m -values for chicken avidin in sodium phosphate buffer (pH 7) were 83.5 °C and 117.0 °C in the absence and presence of biotin [START_REF] Hytönen | Chicken avidin-related protein 4/5 shows superior thermal stability when compared with avidin while retaining high affinity to biotin[END_REF]. In the presence of biotin avidin control in acetic acid was shown to act highly similarly, T m 114.2 °C. A slight decrease in T m -value was observed. In the absence of biotin, no transition was observed in 0.1 M acetic acid.

Proteinase K assay

In the presence of biotin avidin remained intact even under extended incubation with proteinase K, as has been previously shown [START_REF] Ellison | Limited proteolysis of native proteins: The interaction between avidin and proteinase K[END_REF]. Similar to avidin, rhizavidin evinced high resistance to proteinase K in the presence of biotin (Figure 4). In the absence of biotin, avidin was shown to be almost entirely cleaved under extended incubation, this being observed as the disappearance of the band of 16 kDa in size. Rhizavidin seemed not to be cleaved by proteinase K.

Isothermal titration calorimetry (ITC)

The ITC studies showed rhizavidin to have high affinity toward D-biotin. This experiment showed binding enthalpy ( H) to be -26.9 kcal/mol (Figure 5). The corresponding value for avidin was -26.7 kcal/mol. In both cases entropy was highly negative and the binding constant higher than what is possible to measure by ITC.

Biotin dissociation assays

D-[8,9-3 H]biotin was allowed to bind into the protein followed by addition of an excess amount of unlabeled D-biotin. The replaced D-[8,9-3 H]biotin was separated by filtration and the radioactivity of the solution was used to determine the dissociation rate constant (k diss ) of biotin. According to the assay, the k diss of rhizavidin was the same order of magnitude as that of bradavidin [START_REF] Nordlund | Novel avidin-like protein from a root nodule symbiotic bacterium, bradyrhizobium japonicum[END_REF], but significantly higher than those of avidin and streptavidin (Figure 6A).

The fluorescent biotin conjugate (ArcDia BF560 TM ) was allowed to bind to rhizavidin, which caused quenching of the fluorescence. Upon addition of unlabeled D-biotin the fluorescent biotin was displaced and the emission signal increased. The dissociation rate constant was determined from the initial signal of the unbound fluorescent biotin, the quenched signal of the bound protein complex and the emission signal caused by the released fluorescent biotin. The dissociation rate of rhizavidin was faster than those of avidin and streptavidin. However, the biotin release percentage of rhizavidin remained at 50% instead of achieving 100% as that of avidin (Figure 6B). In other words, only half of the ligand was released. The dissociation rate of rhizavidin still indicated high affinity toward the biotin derivative.

Immunological studies of rhizavidin

No significant cross-reactivity was observed when rhizavidin was analyzed with serum samples from human cancer patients exposed to (strept)avidin by ELISA. Two out of five positive serum samples, namely samples A and B (Figure 7), evinced a slightly increased response toward rhizavidin. Both signals were close to those of the negative samples. None of the negative samples induced a response with rhizavidin, avidin or streptavidin. A cross-reactivity assay conducted by ELISA clearly showed rhizavidin to be recognized by polyclonal rabbit antibodies against avidin (AVD), but only exiguously by those against streptavidin (SA) (Figure 7). In contrast to the ELISA, Western blot analysis showed rhizavidin to be recognized by polyclonal rabbit antibodies against streptavidin but not by those against avidin (data not shown).

Biodistribution in the rat

Imaging of the 99m Tc-labelled DTPA avidin or rhizavidin demonstrated the accumulation of the conjugates in the liver and spleen. Also the bladder can be seen, suggesting clearance of a small part of the tracer. DTPA-avidin or rhizavidin accumulated immediately after injection into the liver and spleen (Figure 8) and the biodistribution of the molecules did not change during the 55 min measurement period.

DISCUSSION

In the present work the structural and functional characteristics of a new avidin-like protein, rhizavidin, were studied. Since the avidin-like protein bradavidin, from B. japonicum, was discovered, new bacterial avidins have offered an interesting field of study, both facilitating the development of avidin-biotin technology and providing more detailed information on avidin-like proteins in general. These studies were successful in illustrating rhizavidin to share properties with avidin, streptavidin and bradavidin. However, it differs from them radically in being the first naturally occurring dimeric high-affinity biotin-binding protein in the avidin family, which came as a revelation to us.

The rhizavidin gene possesses an extraordinary long signal sequence with an exceptional translation initiation codon, UUG. In addition, it possesses an alternative signal sequence, 24 amino acid residues shorter than the full-length sequence. Both signals are expected to be functional and to produce an identical mature peptide. The biological role of rhizavidin is unknown, but based on its tight biotin-binding affinity it could serve as an antimicrobial agent. In our studies, the rare initiation codon UUG was replaced with AUG, because the rare codon was expected to significantly reduce the yield in E. coli production. Here both full-length and truncated signal forms of rhizavidin were produced. The yield of the latter was considerably better and was, therefore, used in all studies presented in this paper.

Rhizavidin clearly belongs to the avidin protein family and possesses the avidin fingerprint characteristics [START_REF] Nordlund | Novel avidin-like protein from a root nodule symbiotic bacterium, bradyrhizobium japonicum[END_REF] In contrast to all known avidins so far studied, the loop located between β-strands 7-8 in rhizavidin contains a proline after a flexible loop stretch, GGSG, in a position close to Trp-110 in avidin and Trp-120 in streptavidin. In avidins this tryptophan encloses the biotin-binding site in the neighboring subunit, strengthening binding affinity [START_REF] Livnah | Three-dimensional structures of avidin and the avidin-biotin complex[END_REF][START_REF] Sano | Intersubunit contacts made by tryphtophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin[END_REF]. The interaction of Trp-120 in the binding cleft of streptavidin was not assumed to be as tight as that of avidin in the absence of biotin [START_REF] Gonzalez | Interaction of biotin with streptavidin. thermostability and conformational changes upon binding[END_REF][START_REF] Sano | Intersubunit contacts made by tryphtophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin[END_REF]. We assume that the rhizavidin dimer is composed of monomers equivalent to those forming the structural subunit pair in (strept)avidin [START_REF] Livnah | Three-dimensional structures of avidin and the avidin-biotin complex[END_REF]. Therefore, only the interface corresponding to the (strept)avidin interface between subunits 1 and 4 might be present in rhizavidin. The biotin-binding mode of rhizavidin is unique, since the functional interplay [START_REF] Livnah | Three-dimensional structures of avidin and the avidin-biotin complex[END_REF][START_REF] Sano | Intersubunit contacts made by tryphtophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin[END_REF] of (strept)avidin monomers 1 and 2 seems to be absent. Instead of forming biotin contacts in the neighboring binding site, the function of the proline-containing flexible loop between -strands 7-8

(GGSGP) could be to provide novel intramonomeric contacts with biotin. The difference between the biotin-binding affinities of (strept)avidin arises from the divergence of the binding pockets [START_REF] Livnah | Three-dimensional structures of avidin and the avidin-biotin complex[END_REF].

Particularly, the differences between aromatic residues in the binding pocket and the number of formed hydrogen bonds with biotin are in a key role, as well as differences in certain loop compositions and lengths [START_REF] Livnah | Three-dimensional structures of avidin and the avidin-biotin complex[END_REF][START_REF] Pazy | Ligand exchange between proteins. exchange of biotin and biotin derivatives between avidin and streptavidin[END_REF]. All known bacterial avidins, including rhizavidin, have aromatic residue analogues with streptavidin. The majority of the putative residues which form hydrogen bonds with biotin in rhizavidin are similar to streptavidin. Unlike (strept)avidin, the putative loop between -strands 3 and 4 of rhizavidin contains one cysteine residue which forms an intrasubunit disulfide bridge with the cysteine in the loop between -strands 5 and 6, as indicated by mass spectrometry.

The DSC studies proved rhizavidin to be less thermostable than avidin [START_REF] Hytönen | Chicken avidin-related protein 4/5 shows superior thermal stability when compared with avidin while retaining high affinity to biotin[END_REF], both in the presence and absence of biotin. In this sense it resembles streptavidin, which has T m -values of 75 °C and 112

°C in the absence and presence of biotin, respectively, in a phosphate buffer [START_REF] Gonzalez | Interaction of biotin with streptavidin. thermostability and conformational changes upon binding[END_REF]. When measured by ELISA, rhizavidin appeared to be more thermostable than avidin. However, the different buffers used in ELISA and DSC assays may explain the difference. In the ELISA experiment the samples were diluted with PBS; in DSC they were diluted in acetic acid. In harsh acidic conditions proteins may be more susceptible to heat-induced denaturation due to the possible dissociation of monomers or protonation of residues exposed to a solvent and, consequent changes in charge interactions.

Rhizavidin was seen to be stable in acidic and harsh conditions. Furthermore, the binding mode of avidin and rhizavidin onto an ELISA plate might be dissimilar. Thus, upon binding they may form dissimilar conformations, which could explain the differences in their ligand-binding ability. In this sense, rhizavidin showed good performance when immobilized to a surface, which makes it a useful substitute for avidin in such applications.

The insensitivity of rhizavidin to proteolytic activity was shown to be similar to that of streptavidin [START_REF] Wu | Engineering soluble monomeric streptavidin with reversible biotin binding capability[END_REF]. In avidin the loop between -strands 3 and 4 is located within a flexible region having no secondary structure. The region displays high accessibility to solvents and it is therefore most likely to be exposed to proteinase K [START_REF] Ellison | Limited proteolysis of native proteins: The interaction between avidin and proteinase K[END_REF]. The high proteolytic resistance of rhizavidin may arise from a 3-D structure of the loop between -strands 3 and 4 similar to that of streptavidin.

The biotin-binding thermodynamics of rhizavidin as measured by ITC were similar to those of streptavidin [START_REF] Klumb | Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex[END_REF] and avidin [START_REF] Hytönen | Chicken avidin-related protein 4/5 shows superior thermal stability when compared with avidin while retaining high affinity to biotin[END_REF]. The sensitivity of ITC is adequate for measuring nanomolar affinities. The affinity of rhizavidin-biotin interaction exceeds the sensitivity of ITC, wherefore only preliminary knowledge of the tight binding affinity was obtained.

The serum samples analyzed in immunological studies were gathered from patients subjected to pretargeting radioimmunotheraphy treatment, using both avidin and streptavidin. In the present study, two out of five positive samples caused a higher response to rhizavidin in comparison to that to avidin. In both cases the responses were small, and therefore counted as insignificant. Overall, rhizavidin clearly induced a smaller response than avidin, but slightly higher than that of bradavidin [START_REF] Nordlund | Novel avidin-like protein from a root nodule symbiotic bacterium, bradyrhizobium japonicum[END_REF]. Rhizavidin was clearly shown to cause a smaller response to all positive serum samples when set against streptavidin, which is known to be more antigenic than avidin [START_REF] Chinol | Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity[END_REF][START_REF] Paganelli | The three-step pretargeting approach reduces the human anti-mouse antibody response in patients submitted to radioimmunoscintigraphy and radioimmunotherapy[END_REF]. None of the negative serum samples was shown to set up a significant response to the proteins studied.

In cross-reactivity studies performed by ELISA, rhizavidin was shown to be recognized by polyclonal rabbit antibodies against avidin, in contrast to bradavidin [START_REF] Nordlund | Novel avidin-like protein from a root nodule symbiotic bacterium, bradyrhizobium japonicum[END_REF], whereas it was hardly at all recognized by polyclonal rabbit antibodies against streptavidin. In the experiment performed by Western blot, rhizavidin was clearly recognized by antibodies against streptavidin, but not by those against avidin. Rhizavidin may possess a few streptavidin-like linear epitopes which in denatured state are accessible in Western blot and are responsible for the recognition by polyclonal rabbit antibodies against streptavidin.

Rapid accumulation of rhizavidin and avidin into the liver was evident in SPECT imaging. Even a low dose of rhizavidin (70 MBq) was clearly detected in the liver (data not shown) five minutes after intravenous administration. This is in line with previous findings showing that (strept)avidin has a tendency to target the liver [START_REF] Wei | Dissociation of avidin-biotin complex in vivo[END_REF][START_REF] Schechter | Tissue distribution of avidin and streptavidin injected to mice. effect of avidin carbohydrate, streptavidin truncation and exogenous biotin[END_REF]. Streptavidin, which resembles rhizavidin more than avidin in respect of its physiological properties (pI, non-glycosylated), has been observed to accumulate in the kidney in variable magnitudes. Core streptavidin possesses an especially high tendency to accumulate in the kidney [START_REF] Wilbur | Streptavidin in antibody pretargeting. 2. Evaluation of methods for decreasing localization of streptavidin to kidney while retaining its tumor binding capacity[END_REF][START_REF] Forster | Pretargeted radioimmunotherapy with a single-chain Antibody/Streptavidin construct and radiolabeled DOTA-biotin: Strategies for reduction of the renal dose[END_REF], in contrast to avidin [START_REF] Schechter | Renal accumulation of streptavidin: Potential use for targeted therapy to the kidney[END_REF]. No kidney accumulation was detected either with rhizavidin or avidin (data not shown), both of which were used in the setup. This may be due to the inherent low pI of rhizavidin, which has been shown to inhibit accumulation in the kidney [START_REF] Wilbur | Streptavidin in antibody pretargeting. 2. Evaluation of methods for decreasing localization of streptavidin to kidney while retaining its tumor binding capacity[END_REF][START_REF] Forster | Pretargeted radioimmunotherapy with a single-chain Antibody/Streptavidin construct and radiolabeled DOTA-biotin: Strategies for reduction of the renal dose[END_REF][START_REF] Kang | Pharmacokinetics of 3H biotin bound to different avidin analogues[END_REF][START_REF] Rosebrough | Biochemical modification of streptavidin and avidin: In vitro and in vivo analysis[END_REF]. The labeling method used, in which the positive charges on the surface of the molecules are blocked with isothiocyanate and the net charge is changed due to the additional negative charges of DTPA, (diaminotriethylpentaacetic acid) may also explain the results [START_REF] Wilbur | Streptavidin in antibody pretargeting. Comparison of a recombinant streptavidin with two streptavidin mutant proteins and two commercially available streptavidin proteins[END_REF]. The localization of streptavidin in the kidney is problematic in pretargeting setups as it may lead to renal toxicity. Hence, rhizavidin provides a potential alternative to streptavidin in therapeutic applications [START_REF] Wilbur | Streptavidin in antibody pretargeting. 2. Evaluation of methods for decreasing localization of streptavidin to kidney while retaining its tumor binding capacity[END_REF][START_REF] Forster | Pretargeted radioimmunotherapy with a single-chain Antibody/Streptavidin construct and radiolabeled DOTA-biotin: Strategies for reduction of the renal dose[END_REF].

It would thus seem possible that rhizavidin could be used complementary to (strept)avidin in cancer treatments in that its cross-reactivity with antibodies against (strept)avidin was exiguous, this in addition to its favorable biodistribution. Furthermore, it possesses high resistance toward proteolysis, and its affinity toward biotin appears to be high enough for avidin-biotin technology applications.

Figures / tables Table 1 Protein characteristic

The apparent molecular masses are indicated according to gel filtration in the absence and presence of biotin, respectively, followed by calculated isoelectric points (pI) and the number of cysteine residues per monomer. The first T m -value (DSC) indicates the temperature at which half of the protein is in denatured state in the absence of biotin, the second value in the presence of biotin. Fluorescent biotin dissociation rate constants and one-hour release percentage value at 50 °C are also indicated. Biotin-binding studies of rhizavidin by isothermal titration calorimeter The binding enthalpy ( H) of the rhizavidin-biotin interaction was -26.9 kcal/mol, and entropy was highly negative. The sensitivity of the instrument was not adequate to measure the binding constant. However, the saturation of rhizavidin by biotin is clearly observed in the titration curve. Each peak in the upper part and each point in the lower part of the figure indicates a 10 µl aliquot of 0.4 mM biotin titrated into the rhizavidin solution. Molar ratio (x-axis) indicates the degree of saturation. Immunological assays Serum samples, labeled A through E, were gathered from patients exposed to (strept)avidin. Negative controls, labeled with N1-N3, were from individuals not exposed to either streptavidin or avidin. Cross-reactivity of polyclonal rabbit antibodies against avidin (AVD) and streptavidin (SA) was tested with rhizavidin. Both studies were conducted by ELISA.

Protein

Figure 8

Biodistribution SPECT studies

Serial planar images of a rat injected with 117.6 MBq of 99m Tc-DTPA-Rhizavidin and a corresponding X-ray image of rat skeleton. The imaging shows that the conjugates accumulated in the liver and spleen. A small part of the tracer is seen in the bladder.

  Figure 1Structural characteristics (A) A schematic representation of the tetrameric avidin structure (PDB file 1AVD), in which the eight successive -strands are indicated by arrows. The subunits are numbered according to Livnah et al.[START_REF] Livnah | Three-dimensional structures of avidin and the avidin-biotin complex[END_REF]. W110 from the loop between -strands 7 and 8 is labeled and its sidechain is shown for subunits one and two, in cyan and amber, respectively. Biotins in the binding pockets are shown in magenta.(B) The structure-based alignment of avidin and streptavidin was accomplished by SwissProt. Multiple sequence alignment was done by Clustal W for all avidins shown in the figure. Based on this bradavidin and rhizavidin sequences were manually inserted and adjusted to the alignment. The eight β-strands indicated by the arrows are positioned according to the structure of chicken avidin, and the biotin-binding residues in chicken avidin and streptavidin are underlined[START_REF] Livnah | Three-dimensional structures of avidin and the avidin-biotin complex[END_REF]. The conserved residues are highlighted by an asterisk. Sequence comparison between rhizavidin and chicken avidin revealed 22.4% sequence identity and 35.0% similarity, whereas the values were 28.1% / 40.4% when compared with streptavidin and 21.9% / 36.5% with bradavidin.

Figure 2 ESI

 2 Figure 2 ESI FT-ICR mass spectra of 10 µM rhizavidin In the absence (A) and presence (B, C) of D-biotin (15 µM), measured in 10 mM ammonium acetate buffer (pH 6.8). The ion source capillary exit potential was adjusted to 150 V (A), 22 V (B), and 300 V (C). M refers to the monomeric protein, whereas D and b-D refer to the dimeric proteins, with b designating the binding of two D-biotin molecules. Numbers denote different charge states as [M/D/b-D + zH] z+ . The peaks representing a dimer lacking one D-biotin are indicated with asterisks in panel B. The inset in panel C shows a magnified view of the isotopically resolved M5+, with a small arrow indicating the peak corresponding to the most abundant isotopic mass.

Figure 3

 3 Figure 3 Differential scanning calorimetry The thermograms of rhizavidin versus temperature were measured in the absence and presence of biotin in 0.1 M acetic acid. The T m -values (74.8 °C and 100.5 °C) are obtained at the highest point in the thermograms.

Figure 4

 4 Figure 4Proteinase K assays for rhizavidin (left) and avidin (right) The sensitivities of rhizavidin (14 kDa) and avidin (16 kDa) to proteinase K were analyzed both in the absence (-btn) and the presence of biotin (+btn). The samples were incubated for 0-20 hours followed by SDS-PAGE analysis. The number above each lane indicates the incubation time in hours. Molecular mass standards are indicated by M.

Figure 6 B

 6 Figure 6Ligand dissociation analysis (A) The dissociation rate constants were determined by displacement of D-[8, 9-3 H]biotin with free D-biotin. The constants were plotted as functions of temperatures and compared to the other avidinlike proteins. The values for streptavidin are from[START_REF] Klumb | Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex[END_REF] and those for bradavidin from[START_REF] Nordlund | Novel avidin-like protein from a root nodule symbiotic bacterium, bradyrhizobium japonicum[END_REF]. The scale of the y axis is logarithmic. (B) The release of the fluorescent biotin conjugate (ArcDia BF560 TM ) from avidins was determined by displacing it with unlabeled D-biotin. The measurements were conducted as a function of time in the presence of excess D-biotin at 50 °C. The release percentage represents the proportional amount of the fluorescent biotin conjugate which D-biotin had replaced.

  . Until the 3D structure of rhizavidin is elucidated, only assumptions

	can	be	made	as	to	its	exact	structural	properties	and	relations	to
	the other members of the calycin superfamily [29]. It seems that rhizavidin
	differs from most calycins, since, for example, it does not have an obvious last strand arginine or
	lysine		residue	equivalent	to	that	in	other	avidins.	The	typical
	calycin structural pattern is thus not present in rhizavidin as such, but it probably forms
	compensating interactions by some other means.

Biochemical Journal Immediate Publication. Published on 20 Apr 2007 as manuscript BJ20070076 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements-We thank Ulla Kiiskinen for her excellent assistance; Ritva Romppanen for performing the ESI FT-ICR experiments; Dr Victor Gonzales and Dr Guillermo Davila, Universidad Nacional Autonoma de Mexico, for providing the symbiotic plasmid of R. Etli; the research group of Professor Giovanni Paganelli, Division of Nuclear Medicine, European Institute of Oncology, Milan, Italy for providing cancer patients sera samples. This work was supported by the Academy of Finland.


Pdfauthor=georgios Giamas

Heidrun Hirner

Levani Shoshiashvili

Arnhild Grothey

Susanne Gessert

Michael Kühl

Doris Henne-Bruns

Constantinos E Vorgias

Uwe Knippschild
email: uwe.knippschild@uniklinik-ulm.de

Georgios Giamas

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d

Keywords: casein kinase 1 delta; CK1δCK: casein kinase 1 delta CK1δ, PKA, AKAP's, Akt, CLK2, PKCα 2

enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

CK1 represents an unique group within the superfamily of serine/threonine specific kinases found in all eukaryotic organisms. So far 7 mammalian CK1 isoforms (α, β, γ 1 , γ 2 , γ 3 , δ and ε) and their splice variants have been characterized. They are all highly conserved within their kinase domains, while significantly differing in length and primary structure of their N-terminal and C-terminal regulatory domains (reviewed in [START_REF] Knippschild | The casein kinase 1 family: participation in multiple cellular processes in eukaryotes[END_REF]).

CK1 isoforms can phosphorylate a wide range of substrates bearing either a canonical or a non canonical consensus sequence [START_REF] Flotow | Phosphate groups as substrate determinants for casein kinase I action[END_REF][START_REF] Flotow | Role of acidic residues as substrate determinants for casein kinase I[END_REF][START_REF] Meggio | A synthetic beta-casein phosphopeptide and analogues as model substrates for casein kinase-1, a ubiquitous, phosphate directed protein kinase[END_REF][START_REF] Pulgar | Optimal sequences for non-phosphate-directed phosphorylation by protein kinase CK1 (casein kinase-1)--a re-evaluation[END_REF][START_REF] Marin | A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins[END_REF][START_REF] Bustos | Generation of protein kinase Ck1alpha mutants which discriminate between canonical and non-canonical substrates[END_REF]. As a result, they can influence the activity of numerous key regulator proteins. Processes modulated by CK1 include cell cycle progression and cytokinesis [START_REF] Gross | A casein kinase I isoform is required for proper cell cycle progression in the fertilized mouse oocyte[END_REF], DNA repair and recombination processes [9-12], chromosome segregation, microtubule dynamics, kinetechore and centrosome specific functions [START_REF] Brockman | Cell cycle-dependent localization of casein kinase I to mitotic spindles[END_REF][START_REF] Behrend | Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus[END_REF][START_REF] Milne | Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization[END_REF][16] and apoptosis [START_REF] Beyaert | Casein kinase-1 phosphorylates the p75 tumor necrosis factor receptor and negatively regulates tumor necrosis factor signaling for apoptosis[END_REF][18][START_REF] Zhao | Casein kinase 1alpha interacts with retinoid X receptor and interferes with agonistinduced apoptosis[END_REF][START_REF] Izeradjene | Casein kinase I attenuates tumor necrosis factor-related apoptosis-inducing ligandinduced apoptosis by regulating the recruitment of fas-associated death domain and procaspase-8 to the death-inducing signaling complex[END_REF]. Consequently, the interest in developing inhibitors specifically targeting CK1 family members has significantly increased the last years.

Several mechanisms have been identified that modulate the activity of CK1 in vitro and in vivo. Alternative splicing represents a mechanism that influences substrate binding, turnover and subcellular localisation of certain CK1 isoforms ( [START_REF] Knippschild | The casein kinase 1 family: participation in multiple cellular processes in eukaryotes[END_REF] and references therein). In addition, interaction with cellular proteins influences the subcellular localisation of CK1 [START_REF] Mckenzie | Casein kinase I epsilon associates with and phosphorylates the tight junction protein occludin[END_REF][START_REF] Sillibourne | Centrosomal anchoring of the protein kinase CK1delta mediated by attachment to the large, coiled-coil scaffolding protein CG-NAP/AKAP450[END_REF][START_REF] Wolff | Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A)[END_REF] and can inhibit its activity [START_REF] Yin | Dysbindin structural homologue CK1BP is an isoform-selective binding partner of human casein kinase-1[END_REF]. It has been reported that the subcellular localisation of CK1δ is an important factor for the regulation of its kinasesubstrate interactions and depends on the enzymatic activity of the kinase [START_REF] Milne | Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization[END_REF].

Furthermore, autophosphorylation provides an additional mechanism to downregulate CK1 activity [START_REF] Carmel | Expression, purification, crystallization, and preliminary x-ray analysis of casein kinase-1 from Schizosaccharomyces pombe[END_REF][26][START_REF] Gietzen | Identification of inhibitory autophosphorylation sites in casein kinase I epsilon[END_REF]. However, mechanisms exist to overcome the inhibitory effect of autophosphorylation [START_REF] Cegielska | Autoinhibition of casein kinase I epsilon (CKI epsilon) is relieved by protein phosphatases and limited proteolysis[END_REF]. Other processes that can regulate CK1 activity include; i) stimulation of cells with insulin, ii) viral transformation, iii) treatment of cells with topoisomerase inhibitors, or iv) γ-irradiation, all resulting in elevated CK1 activity ( [START_REF] Knippschild | The casein kinase 1 family: participation in multiple cellular processes in eukaryotes[END_REF] and references therein). Recently, the increase of CK1ε kinase activity by Wnt-induced dephosphorylation of CK1ε autophosphorylation sites, probably by PP2A, was shown in vivo [START_REF] Swiatek | Regulation of casein kinase I epsilon activity by Wnt signaling[END_REF]. Similarly, in neostriatal neurons, the metabotrophic glutamate receptors activate CK1ε by PP2B [START_REF] Chergui | Physiological role for casein kinase 1 in glutamatergic synaptic transmission[END_REF][START_REF] Liu | Control of beta-catenin phosphorylation/degradation by a dualkinase mechanism[END_REF][START_REF] Liu | Regulation of cyclin-dependent kinase 5 and casein kinase 1 by metabotropic glutamate receptors[END_REF].

MATERIALS AND METHODS

Construction of plasmids

Various CK1δ fragments were amplified by PCR using wt rat CK1δ (NM 139060, gi 2053176) as a template and the primers listed in Table 1.

The PCR fragments were first cloned into the MCS of pcDNA-3.1/V5-His-TOPO (Invitrogen, Germany) and then subcloned into the NcoI and XhoI sites of pGEX4T-3 generating the GST-CK1δ fusion proteins shown in Table 2.

The plasmid pGEX2T-CK1δ 1-428/S370A containing mutations at base pairs 1108 and 1109 (ag gc) of rat CK1δ was constructed using the QuikChange site-directed mutagenesis kit according to manufacturers´ instructions (Stratagene, USA).The plasmid pGEX2T-CK1δ 1-428 served as template and the complementary primers used were CK1δ20 (5' primer) and CK1δ21 (3' primer) (see Tables 1 and2).

Rat wt CK1δ was amplified by PCR using pGEX2T-CK1δ 1-428 (449, [34]) as template and the primers CK1δ22 (5' primer) and CK1δ23 (3' primer) (see Tables 1 and2) and cloned into the pcDNA-3.1/V5-His-TOPO vector generating pcDNA-3.1-HA-CK1δ (1101).

Rat mt CK1δS370A was amplified by PCR using pGEX2T-CK1δ 1-428/S370A (1021) as template and the primers CK1δ22 (5' primer) and CK1δ23 (3' primer) (see Tables 1 and2) and cloned into the pcDNA-3.1/V5-His-TOPO vector generating pcDNA-3.1-HA-mtCK1δ S370A (1102).

The kinase dead rat CK1δ (mt CK1δK38M) was amplified by PCR using pGBKT7-CK1δ 1-428/K38M (737, [START_REF] Wolff | Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A)[END_REF]) as template and the primers CK1δ24 (5' primer / myctag) and CK1δ25 (3' primer) (see Tables 1 and2) and then cloned into the pcDNA-3.1/V5-His-TOPO vector, generating pcDNA-3.1-myc-mtCK1δ K38M .

All plasmids were sequenced at GATC Biotech AG (Germany).

Cell lines

Cells (MiaPaCa-2 [START_REF] Yunis | Human pancreatic carcinoma (MIA PaCa-2) in continuous culture: sensitivity to asparaginase[END_REF], BxPc3 [START_REF] Tan | Characterization of a new primary human pancreatic tumor line[END_REF], Colo357 [37] and PancTu1 [START_REF] Moore | Genetic profile of 22 pancreatic carcinoma cell lines. Analysis of K-ras, p53, p16 and DPC4/Smad4[END_REF]) were grown in an 1:1 (vol/vol) mixture of DMEM and RPMI medium supplemented with 10% FCS, 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified 5% carbon dioxide atmosphere at 37°C.

Antibodies

The polyclonal antibody against PKA (Upstate, GB) and the monoclonal CK1δ specific antibody 128A (ICOS Corporation, USA) were used. Horseradish peroxidase conjugated with either anti-rabbit or anti mouse IgG was purchased from GE Healthcare, GB.

Treatment of cells, labelling of cells and cell lysis

MiaPaCa-2 cells were treated with either the PKA specific inhibitor H89 (5 µM;

Calbiochem, Germany), the PKA activator forskolin (20 µM; Calbiochem, Germany), the St-Ht31 inhibitory peptide (10 µM; Promega, USA) or the St-Ht31P control peptide (10 µM; Promega, USA). Subconfluent MiaPaCa-2 cells were labeled with 2 mCi/ml of [

Fractionation of cell extracts

MiaPaCa-2, BxPc3, Colo357 and PancTu1 cells were lysed in sucrose lysis buffer. Cell lysates were passed through a 0.40 µm filter and in each case 3 mg of total protein was applied to an anion exchange column (Resource-Q) attached to an ETTAN purifier (GE Healthcare, GB). The proteins were eluted with a linear ascending gradient between 0-1000 mM NaCl in 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 5% glycerol, 0.04% Brij, 1 mM benzamidine, 4 µg/ml leupeptin and 0.1% β-mercaptoethanol.

Western blot analysis

For the detection of CK1δ and PKA cells were lysed in NP40 lysis buffer. Extracts were clarified by centrifugation and the protein concentration of the lysates was determined. transferred onto a nitrocellulose blotting membrane (Hybond C super, GE Healthcare, GB). The membranes were probed with the appropriate antibodies. Immunocomplexes were detected using anti-mouse or anti-rabbit peroxidase conjugated IgG, followed by chemiluminescence detection (ECL; GE Healthcare, GB).

Overproduction and purification of Glutathione S-Transferase fusion proteins

The production and purification of the GST-fusion proteins FP267, FP380, FP449, FP882, FP894, FP897, FP898, FP1001, FP1003, FP1004, FP1006, FP1011, FP1012, FP1013, FP1014, FP1020, FP1021, FP1022, FP1023 were carried out as described elsewhere [START_REF] Wolff | Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A)[END_REF].

In vitro kinase assays and phosphopeptide analysis

In vitro kinase assays were carried out in the absence or presence of specific kinase inhibitors (5 µΜ H89 (PKA), 200 nM deguelin (Akt), or 1 µΜ calphostin C (PKCα)) as described previously [34].Different GST-CK1δ fusion proteins;GST-p53 1-64 (FP267), GST-p53 1-85/S4,6,9A (FP380), the CK1 peptide substrate (Asp-Asp-Asp-Glu-Glu-Ser-Ile-Thr-Arg-Arg, (Promega, UK), [START_REF] Agostinis | A synthetic peptide substrate specific for casein kinase I[END_REF] and β-casein (Sigma, Germany) were used as substrates. Recombinant PKA (Calbiochem, Germany), purified PKA catalytic subunit from porcine heart (Sigma, Germany), recombinant human PKCα kinase/aa 1-672 (Biomol, Germany), recombinant human Akt kinase/aa 106_480 (Biomol, Germany), recombinant human CLK2 kinase (Invitrogen, Germany) or single fractions of fractionated MiaPaCa-2 cell extracts were used as sources of enzyme.

Phosphorylated proteins were separated by SDS-PAGE and the protein bands were visualised on dried gels by autoradiography. Where indicated, the phosphorylated protein bands were excised and quantified by Cherenkov counting.

Phosphopeptide analyses of in vitro and in vivo labeled proteins were performed as described earlier [START_REF] Wolff | Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A)[END_REF].

RNA synthesis

For microinjection experiments, run-off transcripts of rat wt HA tagged CK1δ plasmid 1101 , HA tagged mutant CK1δS370A (plasmid 1102) and myc tagged kinase dead CK1δK38M (plasmid 737) were synthesised using the T7 Message Machine Kit from Ambion.

Xenopus microinjection

Xenopus embryos were microinjected into both ventral blastomeres at 4 cell stage with the indicated amount of RNA (total amount). Secondary axis formation was analysed at (see supplemental material, Figure 1S). These results suggest that post-translational modifications of CK1δ,-especially site-specific phosphorylation, are involved in modulating the activity of CK1δ.

Cellular kinases phosphorylate CK1δ at Ser370

The C-terminal domain of CK1δ plays an important role for the regulation of the kinase activity of CK1δ. In order to identify cellular kinases, which were able to phosphorylate CK1δ within its C-terminal domain, protein extracts of MiaPaCa-2 cells were prepared and fractionated by ion exchange chromatography using a Resource-Q column. Each eluted fraction was tested for kinase activity using GST-CK1δ 305-375 as substrate. A major kinase peak was detected between 160 and 190 mM NaCl (Figure 1A), indicating that GST-CK1δ 305-375 can be phosphorylated by cellular kinases in vitro.

To localise the CK1δCK dependent phosphorylation site(s) within the C-terminal region of CK1δ, additional GST-CK1δ fusion proteins comprising amino acids 330-375, 305-357 and 353-375 were constructed (Tables 1 and2) and used as substrates for phosphorylation by the CK1δCK activity. While the cellular CK1δCK activity (peaking in fraction 10) phosphorylated the truncated GST-CK1δ fusion proteins GST-CK1δ 330-375 and GST-CK1δ 353-375 to a similar extend (96% and 98%, respectively) compared to GST-CK1δ 305-375 (100%), this activity phosphorylated GST-CK1δ 2A).

Incubation of a series of GST-CK1δ fusion proteins (GST-CK1δ 305-375 , GST-CK1δ 330-375 , GST-CK1δ 305-357 , GST-CK1δ 318-351S318A , GST-CK1δ 305-343 , GST-CK1δ 341-375 , GST-CK1δ 353-375/S370A and GST-CK1δ 353-375 ) with the catalytic subunit of PKA purified from porcine heart and [γ-32 P] ATP confirmed that Ser370 is the major phosphorylation site targeted by PKA in vitro (Figure 2B).

To prove the stoichiometry of the phosphorylation by PKA in vitro kinase assays were performed using purified PKA as enzyme and GST-CK1δ 305-375 as substrate. The data of three independent experiments indicated that approximately 1.5 moles of phosphate were incorporated per mole of protein suggesting that there is one major phosphorylation site within amino acids 305 to 375 being phosphorylated by PKA in vitro (Figure 2C).

To examine whether additional serine/threonine residues of CK1δ are phosphorylated by PKA in vitro, the GST-CK1δ fusion proteins GST-CK1δ 1-428 , GST-CK1δ 1-375 , GST-CK1δ 1-350 , GST-CK1δ 52-304 and GST-CK1δ 149-428 were used as substrates. All GST-CK1δ fusion proteins containing Ser370 (GST-CK1δ 1-428 , CK1δ 149-428 , GST-CK1δ 1-375 ) were phosphorylated by PKA. Even though GST-CK1δ 1-428 and GST-CK1δ 1-375 were highly autophosphorylated, their degree of phosphorylation increased up to 82% in the presence of purified PKA. In contrast, GST-CK1δ fusion proteins that did not encompass Ser370 (GST-CK1δ 1-350 and GST-CK1δ 52-304 ) were either weakly or not phosphorylated. Interestingly, when the GST-CK1δ 1-428S370A fusion protein was used as substrate, no changes in the phosphorylation status of CK1δ were observed underlining that PKA mediated phosphorylation occurs predominantly at Ser370 in vitro (Figure 2D).

To gain more information regarding the identity of the CK1δCK activity, the CK1δCK activity was assayed in the absence and presence of kinase inhibitors specific for PKA, PKCα or Akt using GST-CK1δ 305-375 as substrate. As presented in Figure 3A, a 65% reduction of the CK1δCK activity was observed in the presence of the PKA specific inhibitor H89 (5 µΜ), whereas in the presence of the PKC specific inhibitor, calphostin C, (1 µΜ) or the Akt specific inhibitor, degeulin (200 nM),-the reduction of the CK1δCK activity was much lower (25% and 9%, respectively).

As PKA seems to be the major CK1δCK activity present in the kinase peak fractions (Figure 1A) and as PKA could be clearly detected in the kinase peak fractions 10 and 11

by Western blot analysis (Figure 3B), the kinetic parameters of the CK1δCK activity were compared with those of recombinant PKA using the GST-CK1δ 353-375 fusion protein as substrate. The results indicate that the K m and V max values for CK1δCK and PKA are nearly identical (Figure 3C). Taken together, all these experiments strongly point to PKA being the principal kinase responsible for the phosphorylation of CK1δ at Ser370.

Ser370 plays an important role in modulating the substrate phosphorylation of

CK1δ in vitro

In order to examine the importance of Ser370 for the catalytic activity of CK1δ in vitro, the kinetic constants of GST-CK1δ 1-428 and GST-CK1δ phosphorylation of β-casein and GST-p53 1-64 were experimentally calculated. As presented in Figure 2S (see supplemental material) GST-CK1δ 1-428S370A has lower K m and higher V max values compared to those of GST-CK1δ 1-428 . These observed differences revealed that CK1δ 1-428S370A has a higher affinity to phosphorylate β-casein and GST-p53 1-64 compared to that of GST-CK1δ 1-428 , which is furthermore indicated by the efficiency parameter (V max /K m ratio) (Figure 2S,C).

Phosphorylation of GST-CK1δ by PKA affects its activity in vitro

So far, our results clearly indicate that S370, which plays an important role in the modulation of the activity of CK1δ, is the major phosphorylation site targeted by PKA in vitro. To examine the influence of PKA phosphorylation on the activity of CK1δ, in vitro kinase assays were performed in the presence or absence of the catalytic subunit of PKA purified from porcine heart. GST-CK1δ 1-428 (FP 449) was used as source of enzyme activity. CK1δ was preincubated with [γ-32 P] ATP for 15 min before adding GST-p53 1-64 (FP267) as substrate either alone or in combination with PKA for the indicated time points (see supplemental material, Figure 3S).

As presented in Figure 3S, CK1δ mediated phosphorylation of GST-p53 1-64 (FP267) was reduced by up to 80% in the presence of PKA compared to that observed in the absence of the catalytic subunit of PKA. Phosphorylation of GST-p53 1-64 by PKA could be excluded since in vitro kinase assays demonstrated that PKA does not phosphorylate GST-p53 1-64 (data not shown).

Effects of CK1δS370A in Xenopus embryogenesis

Different CK1 isoforms have been implicated in the Wnt signaling pathway. Considering the high affinity of CK1δS370A to phosphorylate ß-casein and GS-p53 of an ectopic dorsal axis (Figure 4 A, B) as well as gastrulation movements defects. To identify any difference in biological activity of both constructs, we lowered the amount of injected RNA in subsequent experiments and selected for experiments with a low gastrulation phenotype (i.e. not more than 10% as this can occur non-specifically in egg batches of low quality or eventually after injection of higher RNA amounts). At lower amounts of microinjected mRNA the secondary axis phenotype was seen more often in embryos expressing exogenous HA-tagged CK1δS370A than in embryos expressing wt CK1δ (Figure 4 C).

PKA phosphorylates CK1δ in vivo

To examine whether CK1δ is phosphorylated by PKA in tissue culture cells, experiments were performed on the basis that modulating the activity of PKA should influence the phosphorylation status of CK1δ. For this purpose, MiaPaCa-2 cells were treated either with the PKA specific inhibitor H89 (5 µΜ) or with the PKA activator forskolin (20 µΜ) in the presence of 2 mCi [ 32 P]-orthophosphate for two hours. Cells were lysed and phosphorylated CK1δ was immunoprecipitated, isolated, digested with trypsin and oxidised. The resulting tryptic phosphopeptides were separated by two dimensional tryptic phosphopeptide mapping and compared with the phosphopeptides derived from the GST-CK1δ 305-375 fusion protein, which was phosphorylated by PKA in vitro.

Phosphopeptides 1 and 2 were present in CK1δ isolated from MiaPaCa-2 cells treated with forskolin (Figure 5A) and in GST-CK1δ 305-375 (Figure 5C), whereas these peptides were not detectable in CK1δ isolated from MiaPaCa-2 cells treated with H89 (Figure 5B) pointing to PKA as the cellular kinase responsible for the phosphorylation of amino acid residues within these two peptides. Other migrated peptides (labeled 3 and 4) could be detected in the phosphopeptide analyses of CK1δ isolated from both, forskolin and H89 treated cells indicating the existence of additional phosphorylation sites. Recently, it has been reported that interaction of PKA and CK1δ with AKAP450 docks both kinases to centrosomes [START_REF] Sillibourne | Centrosomal anchoring of the protein kinase CK1delta mediated by attachment to the large, coiled-coil scaffolding protein CG-NAP/AKAP450[END_REF][START_REF] Carr | Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79[END_REF][START_REF] Glantz | cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase II beta[END_REF] along with other signal transduction molecules.

The binding of PKA to AKAP´s can be blocked by a membrane-permeable AKAP St-Ht31 inhibitory peptide [START_REF] Carr | Localization of the cAMP-dependent protein kinase to the postsynaptic densities by A-kinase anchoring proteins. Characterization of AKAP 79[END_REF]44]. Therefore, we investigated whether disruption of the PKA-AKAP complex by the anchoring disrupting peptide St-Ht31 [START_REF] Carr | Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain[END_REF]46] can influence the activity levels of CK1δ. For this purpose, MiaPaCa-2 cells were treated with the St-Ht31 specific inhibitory peptide (10µΜ) or the St-Ht31P control peptide [47] for 3 h. Cell lysates were fractionated by anion exchange chromatography and the CK1δ kinase activity was determined. When the CK1 substrate peptide (Figure 6B) or GST-p53 1-64 (FP267) (Figure 6A) were used as substrates, the CK1δ activity increased 3 to 6 fold in cells treated with the St-Ht31 inhibitory peptide compared to untreated cells and cells treated with the St-Ht31P control peptide (Figure 6).

Respectively, differences in the CK1δ kinase activity were observed in cells treated with the St-Ht31 inhibitory peptide when GST-p53 1-85/S4, 6, 9A (FP380) was used as substrate, compared to untreated cells and cells treated with the St-Ht31P control peptide (Figure 6A). However, as expected, the CK1δ mediated phosphorylation of FP380 was significantly lower compared to that of FP267 due to the mutation of most of the CK1δ phosphorylation sites (S4, 6, 9A) within the N-terminal domain of p53.

DISCUSSION

CK1δ, a member of the CK1 family, is involved in the regulation of many different cellular processes including cell proliferation and cell death. Therefore, several mechanisms ensure a tight regulation of CK1δ on different levels [START_REF] Milne | Catalytic activity of protein kinase CK1 delta (casein kinase 1delta) is essential for its normal subcellular localization[END_REF][START_REF] Yin | Dysbindin structural homologue CK1BP is an isoform-selective binding partner of human casein kinase-1[END_REF]26,[START_REF] Cegielska | Autoinhibition of casein kinase I epsilon (CKI epsilon) is relieved by protein phosphatases and limited proteolysis[END_REF][START_REF] Fish | Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family[END_REF][START_REF] Longenecker | Crystallographic studies of casein kinase I delta toward a structural understanding of autoinhibition[END_REF]. In addition, phosphorylation of CK1δ by PKA reduced its ability to phosphorylate GST-p53 1-64 up to 80%. This result indicates that site specific phosphorylation is implicated in modulating the activity of CK1δ.

The importance of Ser370 for the substrate phosphorylation of CK1δ was indicated by comparing the kinetic parameters of GST-CK1δ 1-428 with those of GST-CK1δ 1-428S370A .

Our results show that the GST-CK1δ 1-428S370A mutant exhibits lower K m and higher V max values compared to those of GST-CK1δ 1-428 when β-casein and GST-p53 1-64 were used as substrates.

Furthermore, the role of Ser370 appears to be physiologically important since it has been shown that mutation of this site can interfere with normal development of Xenopus embryos. Expression of the mutant kinase (CK1δS370A) in Xenopus embryos resulted in a higher rate of ectopic dorsal axis formation compared to the expression of wtCK1δ whereas the kinase dead mutant (CK1δK38M) failed in the axis induction assay.

In addition, our data strongly support the idea that CK1δ and PKA interact physiologically based on the observation that inhibition of the interaction of PKA with AKAP's by the St-Ht31 inhibitory peptide [START_REF] Carr | Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain[END_REF]51] resulted in an increase of CK1δ activity. The increased activity of CK1δ could be explained by changes in the phosphorylation status of CK1δ. Loss of the interaction between CK1δ and PKA could lead to a reduced phosphorylation of Ser370. Furthermore, cellular phosphatases associated with AKAP's [START_REF] Bridges | Identification and characterization of D-AKAP1 as a major adipocyte PKA and PP1 binding protein[END_REF][START_REF] Kuntziger | Association of PP1 with its regulatory subunit AKAP149 is regulated by serine phosphorylation flanking the RVXF motif of AKAP149[END_REF] could more efficiently dephosphorylate the autophosphorylation sites of CK1δ in the absence of PKA thereby activating CK1δ.

However, St-Ht31 treatment of cells can also decrease the activity of signalling molecules as it has been shown that St-Ht31 could disrupt the activity of RhoA [START_REF] Wang | Table 1 Sequences of CK1δ-specific primers primer sequence CK1δ-1 5´-CGAATTCCATGGAGAGCAAGATCTACAAAATGAT-3´ CK1δ-2 5´-ACTCGAGTCGACAGCATCATCTGCAGCC-3´ CK1δ-3 5´-CGAATTCCATGGACTTTGGGCTGGCCAAG-3´ CK1δ-4 5´-ACTCGAGTCGACGTAGGTGGTACGTCGTG-3´ CK1δ-5 5´-CGAATTCCATGGAGCTGAGGGTCGGGAAT-3´ CK1δ-6 5´-ACTCGAGTCGACTCAACGGTGCAGCCGCA-3´ CK1δ-7 5´-ACTCGAGTCGACTGAGGTAGGGGTAAGG-3´ CK1δ-8 5´-CGAATTCCATGGAGCGGGAACGCCG-3´ CK1δ-9 5´-CCATGGCTTCCGGCCGTCTGCGGGGAACC-3´ CK1δ-10 5´-ACTCGAGTCGACTCAAGGAGAGGTGTTGGC-3´ CK1δ-11 5´-CCATGGCCCGGAATCCAGCCACTCGTGGC-3´ CK1δ-12 5´-ACTCGAGTCGACTCAATGTGAGGTAGGGGT-3´ CK1δ-13 5´-ACTCGAGTCGACTCATGGGGGAGCCACTTC-3´ CK1δ-14 5´-CCATGGCTCCCCCAACGCCCCTTACCCCT-3´ CK1δ-15 5´-CCATGGCCAACACCTCTCCTAGACCCGTC-3´ CK1δ-16 5´-ACTCGAGTCGACTCAACGGTGCAGCCGCATAGCCAC-3´ CK1δ-17 5´-CCATGGCCCGGAATCCAGCCGCTCGTGGC-3´ CK1δ-18 5´-CCATGGCTCGTGGCCTCCCTTCTGCAGCT-3´ CK1δ-19 5´-CCATGGCCGGCCGTCTGCGGGGAACCCAG-3´ CK1δ-20 5'-CGAGAACGGAAAGTGGCTATGCGGCTGCACCGTGGG-3' CK1δ-21 5'-GCTCTTGCCTTTCACCGATACGCCGACGTGGCACCC-3' CK1δ-22 5'-GATATCATGTACCCATACGATGTTCCAGATTACGCTC TTCATATGGCGATGGAGCTGAGG-3' CK1δ-23 5'-AGATCTTCAGTAGGTGGTACG-3' CK1δ-24 5'-GGTACCATGGAGGAGCAGAAGCTG-3' CK1δ-25 5'-GTCGACTCAGTAGGTGGTACGTCGTGG-3' CK1δ1 + CK1δ2 154-912 pGEX4T-3-CK1δ 52-304 GST-CK1δ 52-304 (FP882) CK1δ3 + CK1δ4 445-1284 pGEX4T-3-CK1δ 149-428 GSTCK1δ 149-428 (FP894) CK1δ5 + CK1δ6[END_REF].

In summary, these results show for the first time that CK1δ is phosphorylated by several kinases and that site specific phosphorylation, especially at Ser370 by PKA, plays an important role in modulating the activity of CK1δ. In vitro kinase assays were performed using fractions 7-12 of fractionated MiaPaCa-2 cell extracts as source of enzyme activity and fusion proteins GST-CK1δ 305-375 (FP1006), GST-CK1δ 330-375 (FP1011), GST-CK1δ 305-357 (FP1012), GST-CK1δ 353-375 (FP1022) and GST-CK1δ 305-375S370A (FP1021) as substrates. The highest cellular kinase activity (100%) was detected in fraction 10 when using the GST-CK1δ

TABLES AND FIGURES

8 Biochemical

 8 stage 22 and stage 26 of development. Staging was according to Nieuwkoop and Faber [40]. Journal Immediate Publication. Published on 27 Jun 2007 as manuscript BJ20070091 © 2007 The Authors Journal compilation © 2007 Biochemical SocietyRESULTSCK1δ activity differs in pancreatic tumour cell linesRecently, it has been suggested that the differences in the activity of CK1δ in extracts of various organs of young adult Balb/C mice might be influenced by site specific phosphorylation of CK1δ[START_REF] Maritzen | Casein kinase I delta (CKIdelta) is involved in lymphocyte physiology[END_REF]. To further follow up this idea the activity of CK1δ was analysed in fractionated extracts of highly apoptosis-resistant pancreatic tumour cell lines, all expressing similar protein amounts of CK1δ. The kinase activity in fractionated BxPc3, MiaPaCa2, PancTu1 and Colo357 cell extracts, which eluted at 220-240 mM NaCl and was inhibited by the CK1 specific inhibitor IC261 [41], differed up to six fold

  The subcellular localisation of PKA is important for modulating the activity of CK1δ Compartimensation of signal transduction molecules (e.g. kinases and phosphatases) occurs by anchoring to scaffold proteins. This then allows substrate 13 Biochemical Journal Immediate Publication. Published on 27 Jun 2007 as manuscript BJ20070091 © 2007 The Authors Journal compilation © 2007 Biochemical Society phosphorylation/dephosphorylation in concert with various kinases and phosphatases resulting in the regulation of the functions of the substrate, specific for a certain compartment.

  Using a biochemical approach to clarify the role of cellular kinases in modulating the activity of CK1δ, a CK1δCK activity eluting between 160-190 mM NaCl was detected in fractionated MiaPaCa-2 cell lysates. The use of various C-terminal truncated wt and mt GST-CK1δ fusion proteins as substrates revealed that mainly Ser370 is targeted by CK1δCK. Ser370 is a potential phosphoaceptor site for phosphorylation by several putative kinases among them Akt, PKA, CLK2 and PKCα. In vitro kinase assays using various GST-CK1δ fusion proteins as substrates revealed that PKA and Akt mainly phosphorylate Ser370, while the key targeted sites of PKCα and CLK2 lie within aa 305-353 of CK1δ. However, additional experiments are necessary to identify the phosphorylation sites targeted by PKCα and CLK2 and to elucidate their role in regulating the activity of CK1δ.Foccussing on the phosphorylation of Ser370, our data strongly suggest that this serine residue is predominantly phosphorylated by PKA, since (i) PKA co-eluted with the peak of the CK1δCK activity as verified by Western blot analysis, (ii) the PKA specific inhibitor H89 inhibited the kinase activity present in the kinase peak fraction up to 65% whereas in the presence of the Akt specific inhibitor degeulin or in the presence of the PKC specific inhibitor calphostin C a much weaker reduction was observed, (iii) the comparison of the kinetic parameters of recombinant PKA with those of CK1δCK present in the kinase peak fraction indicated nearly identical values and (iv) the purified catalytic subunit of PKA, as well as recombinant PKA, phosphorylated all GST-CK1δ fusion proteins containing Ser370 in vitro, whereas all GST-CK1δ fusion proteins lacking Ser370 or containing a serine to alanine substitution at Ser370 were not phosphorylated. Furthermore, time course experiments revealed that there is one major phosphorylation site for PKA within amino acids 305-375. The presence of arginine and lysine residues at positions n-3 and n-2 on the N-terminal side of Ser370 (R265ERKVS370) resembles a perfect consensus recognition motif for PKA [50] thereby favouring Ser370 as the main phosphorylation site targeted by PKA. Moreover, comparison of our phosphopeptide analyses suggests that CK1δ is phosphorylated in vivo at the same sites being phosphorylated by PKA in vitro.

  Figure 1 Characterisation of CK1δCK activities in MiaPaCa-2 cells (A) Fractionation of CK1δCK activities present in MiaPaCa-2 cells by anion exchange chromatography. A soluble protein extract (3 mg) derived from the lysis of MiaPaCa-2 cells was fractionated on a Resouce Q column with a linear ascending gradient of NaCl. Kinase assays were performed using equal amounts of GST-CK1δ 305-375 as substrate (see Coomassie gel) and single fractions of fractionated MiaPaCa2 cell extracts as source of enzyme. Proteins were separated by SDS-PAGE and the phosphorylated proteins were detected by autoradiography. Quantification of phosphate incorporation of phosphorylated GST-p53 1-64 was measured by Cerenkov counting. A major kinase activity phosphorylating GST-CK1δ 305-375 (FP1006) eluted between 160-190 mM NaCl (fractions 9-11). (B) Localisation of phosphorylation sites targeted by CK1δCK activities.

Figure 3 PKA

 3 Figure 3

Figure 4

 4 Figure 4 Secondary axis induction in Xenopus laevis embryos. Xenopus laevis embryos were injected with wt or mt CK1δ RNA at 4-cell stage as indicated. Evaluation of secondary axis formation was performed at stage 22 or 26. Different views of embryos are shown (A). Quantification of experiments shows that injection of higher amounts results in secondary axis formation and gastrulation movements defects in case of wt CK1δ and CK1δS370A. The kinase dead mutant CK1δK38M is inactive in this assay (B). Injection of lower amounts and selection of embryo batches with low gastrulation movement phenotype indicates that CK1δS370A has a higher capacity to induce secondary axis in comparison to wt CK1δ (C).

Figure 5

 5 Figure 5 Phosphopeptide analyses of CK1δ and GST-CK1δ 305-375 GST-CK1δ 305-375 and [ 32 P]-labeled CK1δ isolated from MiaPaCa-2 cells treated either with 20 µM forskolin (A) or with 5 µΜ H89 (B) for 2 h were oxidised and digested with

Figure 6

 6 Figure 6 Inhibition of the interaction of PKA with AKAP's is accompanied by an increased CK1δ activity Equal protein amounts (3 mg) of lysates derived from MiaPaCa-2 cells, untreated, treated with 10 µΜ of the St-Ht31 control peptide or with 10 µΜ of the St-Ht31 specific inhibitory peptide were fractionated as described in Material and Methods. The proteins were eluted with a linear gradient of increasing NaCl concentrations (represented as a solid diagonal line). (A) The CK1δ specific kinase activity was determined using the GST-p53 1-64 (FP267) and the GST-p53 1-84 S4, 6, 9A fusion proteins (FP380) as substrates. The kinase activity is shown as i) open circles: in cells treated with the St-Ht31 inhibitory peptide, ii) open rombs: in cells treated with the St-Ht31 control and iii) closed triangles: in untreated cells when FP267 was used as substrate The kinase activity is shown as i) closed circles: in cells treated with the St-Ht31 inhibitory peptide, ii) closed rombs: in cells treated with the St-Ht31 control and iii) open triangles: in untreated cells, when FP380 was used as substrate. (B) The CK1δ specific kinase activity was also determined using the CK1 peptide substrate Asp-Asp-Asp-Glu-Glu-Ser-Ile-Thr-Arg-Arg. Open circles: kinase activity in cells treated with the St-Ht31 inhibitory peptide, open rombs: kinase activity in cells treated with the St-Ht31 control peptide and closed triangles: kinase activity in untreated cells.

Figure 1 30

 1 Figure 1

Figure 2 31

 2 Figure 2 31

Figure 3 32

 3 Figure 3

Figure 4 33

 4 Figure 4

Figure 5 34

 5 Figure 5

Figure 6

 6 Figure 6

6 Biochemical Journal Immediate Publication. Published on 27 Jun 2007 as manuscript BJ20070091

  305-357 to approximately site(s) targeted by cellular kinases are localised within amino acids 353-375 of CK1δ.A Scansite research (http://scansite.mit.edu) revealed that within amino acids 353-375

	of CK1δ Ser370 might be the major phosphorylation site targeted by cellular kinases
	among them Akt, PKA, PKCα/β/γ/ζ and CLK2. Therefore, a GST-CK1δ 353-375/S370A fusion
	protein bearing a serine to alanine mutation at position 370 was generated and used as
	substrate in in vitro kinase assays. As shown in Figure 1B, the degree of
	phosphorylation of the GST-CK1δ 353-375/S370A fusion protein was approximately 4 fold
	lower compared to that of the GST-CK1δ 353-375 fusion protein. These data strongly
	suggest that the major phosphorylation site of CK1δ targeted by the CK1δCK activity is
	Ser370.
	To prove the ability of PKA, Akt, PKCα and CLK2 to phosphorylate CK1δ at Ser370
	several	GST-CK1δ	fusion	proteins	(GST-CK1δ 305-375 ,	GST-CK1δ 353-375 ,

GST-CK1δ 353-375/S370A , GST-CK1δ

305-357

, GST-CK1δ 330-375 , GST-CK1δ 318-375,S318A/T323A , GST-CK1δ 353-375, T323A/T329A and GST-CK1δ 331-375,S331A ) were used in in vitro kinase assays. The results presented in Figure

2A

indicate that Akt predominantly phosphorylates Ser370 in vitro. In contrast, Ser370 is only weakly phosphorylated by PKCα and Clk2 in vitro. The major phosphorylation sites targeted by these two kinases are located within aa 305 to aa 353 of CK1δ (Figure

Biochemical Journal Immediate Publication. Published on 27 Jun 2007 as manuscript BJ20070091 © 2007 The Authors Journal compilation © 2007 Biochemical Society

1-428S370A mediated 11

Biochemical Journal Immediate Publication. Published on 27 Jun 2007 as manuscript BJ20070091 © 2007 The Authors Journal compilation © 2007 Biochemical Society

	1-64 in vitro we
	now analysed the effects of this mutant on WNT/ß-catenin signaling in vivo using the
	Xenopus secondary axis induction assay.
	In vitro transcribed RNA encoding either HA-tagged rat wt CK1δ, mt CK1δS370A or

myc-tagged mt CK1δK38M were microinjected into both ventral cells of 4 cell-stage Xenopus embryos. The kinase dead mutant CK1δK38M did not effect embryonic development whereas high doses (2 ng) of wtCK1δ or CK1δS370A caused the formation 12

Table 1 Sequences of CK1δ-specific primers Table 2 Generation of CK1δ-specific fragments and nomenclature of pGEX- CK1δ plasmids and fusion proteins

 1

Table 2 Generation of CK1δ-specific fragments and nomenclature of pGEX- CK1δ plasmids and fusion proteins Primer pair bp of CK1δ pGEX vector name GST-CK1δ fusion protein

 2

Biochemical Journal Immediate Publication. Published on 27 Jun 2007 as manuscript BJ20070091 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung (10-2237-KN3) to Uwe Knippschild. Work in the lab of Michael Kühl is supported by the Deutsche Forschungsgemeinschaft (SFB 497/A6).

We would like to thank Tony DeMaggio (ICOS Corporation, USA) for providing us with the CK1δ specific monoclonal antibody 128A, David Meek for pGEX2T-CK1δ (rat), pGEX-p53 1-64 (mouse), and pGEX-p53 1-84S4, 6, 9A (mouse), Tilo Patschinsky for helpful discussions and reading the manuscript, Annette Blatz and Corinna Patzina for technical assistance.