PKC-dependent phosphorylation of p27 at T198 contributes to p27 stabilization and cell cycle arrest

Abstract

In this manuscript, we present experimental evidence that PKCs phosphorylate p27 at T198 in vitro and in vivo, resulting in p27 stabilization and cell cycle arrest in MCF-7 and HeLa cells. Our findings indicate that (1) recombinant PKCα, βII, δ, η and θ isoforms phosphorylate, in in vitro kinase assays, wild-type recombinant p27 protein expressed in E. coli and wild-type p27 protein immunoprecpitated from transfected HEK-293 cells but not the T198A mutant, (2) adoptive expressed PKCα and δ phosphorylate both transfected and endogenous p27 at T198 in HEK-293 cells, (3) T198 phosphorylation of transfected and endogenous p27 is increased by PKC activators [Phorbol 12-myristate 13-acetate (PMA)] and suppressed by PKC inhibitors (Rottlerin A, G06976, Calphostin C), (4) in parallel with increased T198 phosphorylation, PMA induces stabilization of p27 protein in HeLa cells, whereas PKC inhibitors induce a decrease in p27 stability and, finally, (5) PMA-induced p27 upregulation is necessary for growth arrest of HeLa and MCF-7 cells induced by PKC activation by PMA.

 

Overall, these results suggest that PKC-dependent upregulation of p27 induced by its phosphorylation at T198 represents a mechanism that mediates growth arrest promoted by PMA and provide novel insights on the ability of different PKC isoforms to play a role in controlling cell cycle progression.

Keywords: :

• p27
• phosphorylation
• PKC
• stabilization
• T198

Introduction

The cyclin-dependent kinase (Cdk) inhibitor p27^Kip1 (p27) is an important regulator of the mammalian cell cycle.^1,2 p27 negatively regulates G₁ progression by binding to cyclin-Cdk1, cyclin-Cdk2 and cyclin-Cdk4/6 complexes and preventing their activity.^3-6 The activity of p27 is controlled by its concentration, its distribution among different cellular complexes and its subcellular localization.^7,8 One key mechanism involved in the regulation of p27 abundance is proteolysis by the ubiquitin-proteasome pathway.⁹ Accordingly, the levels of p27 protein are high in quiescent cells and decline upon mitogenic stimulation.^10,11

Both the intracellular concentration and the localization of p27 are regulated by extensive posttranslational modifications.^12,13 p27 protein is phosphorylated at multiple sites: serine 10, threonines 157, 187, 198 and tyrosines 74, 88, 89. Phosphorylation of p27 has been studied extensively, and for each of the phosphorylation sites, several kinases have been reported, and different functions have been ascribed.¹⁴ When phosphorylated on Thr187 by cyclin E-Cdk2,^15-18 p27 is recognized and targeted for ubiquitylation by the SCF Skp2 ubiquitin-protein ligase and its cofactor, the Cdk subunit 1.^19-24 In addition, p27 undergoes rapid translocation from the nucleus into the cytoplasm followed by degradation through the ubiquitin-proteasome pathway by undetermined mechanisms that involve phosphorylation of Ser10, Thr157 or Thr198 by hKIS, AKT or RSK, respectively.

As to Thr198, several papers have illustrated the role of its phosphorylation in the regulation of p27 localization and stability. Fujita et al.²⁵ and Motti and coworkers²⁶ identified Thr198 as a residue phosphorylated by Akt. Subsequent work demonstrated that Thr198 is phosphorylated also by p90 ribosomal protein S6 kinases (RSKs)²⁷ and by the AMP-activated protein kinase (LKB1-AMPK).²⁸ Phosphorylation of p27 at Thr198 apparently increases p27 stability²⁹ and regulates cell motility.³⁰

Bioinformatic analysis of p27 phosphorylation sites has suggested that Thr198 may be the target of other kinases, including protein kinase C. Protein kinase C (PKC) is a family of serine-threonine kinases that regulate several cellular processes, including cellular growth and proliferation, transformation, differentiation and apoptosis.^31,32 Numerous PKC isoenzymes have been described so far and are classified according to their structures and mechanisms of activation.³³ Accordingly, PKC isoforms can be classified into three groups based on their structure and cofactor requirement: classical PKC isoforms (α, β1, β2, γ), which are regulated by diacylglycerol (DAG) and Ca²⁺, novel PKC isoforms (δ, ε, η, θ) which are regulated by DAG but not by Ca²⁺, and atypical PKC isoforms (ζ, ι), which are not responsive to either DAG or Ca²⁺.^34,35

In this manuscript, we have identified PKC as a novel bona fide Thr198 kinase and characterized the effects of PKC-dependent phoshorylation of p27 on cell proliferation.

Results

PKCs phosphorylate p27 at T198 in vitro

It has been shown that the intracellular levels of p27 protein are tightly regulated by its phosphorylation status.⁹ A computational model based on kinase-specific phosphorylation site prediction group-based prediction system (GPS) version 2.1 software (http://gps.biocuckoo.org/)³⁶ was used to identify novel putative p27 kinases. GPS analysis identified threonine 198 as a potential phosphorylation site by the members of PKC kinase family (Fig. 1A).

Figure 1. PKC phosphorylates p27 in vitro. (A) p27 phosphorylation site prediction by the Group-based Prediction System software. (B) In vitro kinase assays. Recombinant p27 was incubated with several recombinant PKCs in presence of ³²P-labeled adenosine triphosphate (ATP), fractionated by SDS-PAGE, and transferred to nylon membrane. p27 phosphorylation was determined either by autoradiography. Histone H1 was used as control of PKCs’ activity.

Therefore, we performed in vitro kinase assays to determine whether PKCs were able to phosphorylate p27. We set up the kinase reactions, incubating PKCs and 6-His-tagged p27 recombinant proteins in presence of ³²P-labeled adenosine triphosphate (ATP). We tested different isoforms of PKC (α; βΙ; βΙΙ; γ; δ; η; ζ; θ) using recombinant histone H1 as control. We found that all the isoforms tested except PKCγ were able to phosphorylate p27, though with different efficacy (Fig. 1B).

In order to investigate whether PKC was able to phosphorylate p27 at T198, we performed immunoblots using an antibody that recognizes the phosphorylated Thr198 (pT198).²⁶ Filters were incubated with total α-p27 antibody in order to normalize the results (Fig. 2). We found that all PKCs that phosphorylate p27 induced a marked increase in pT198 staining. Data shown in Figure 2 are related to kinase assays performed with PKCα, PKCδ and PKCθ.

Figure 2. PKC phosphorylates p27 at T198. In vitro kinase assays. Recombinant p27 was incubated with recombinant PKCs in presence of ³²P-labeled adenosine triphosphate (ATP), fractionated by SDS-PAGE, and transferred to nylon membrane. p27 phosphorylation was determined either by autoradiography or by immunoblot using an antibody specific for phosphorylated T198 (anti-pT198).

We also performed an immunocomplex kinase assay to confirm that PKCs were able to phosphorylate p27. To this aim, HAp27-wt and HAp27-T198A were transiently transfected into HEK-293 cells, and total cell extracts were immunoprecipitated with anti-HA antibody. The immunocomplexes were incubated with recombinant PKCs proteins (PKCα and PKCδ) in the presence of [γ-³²P]-ATP; T198 phosphorylation was detected by western blotting with pT198 antibody. Results were similar to those obtained with recombinant p27 and also indicated that PKCs were able to phosphorylate HAp27-wt but not the mutant HAp27-T198A (data not shown).

PKCs phosphorylate at T198 p27 in vivo

We also performed transfection experiments to determine whether PKCs were able to phosphorylate p27 in vivo. To this aim, HEK-293 cells were transiently transfected with HAp27-wt in the presence or absence of PKCα, PKCδ (Fig. 3A) or PKCθ (not shown). Total cell extracts were analyzed for the phosphorylation status at p27-T198A by immunoblot with the pT198 antibody and then with an antibody anti-p27 to normalize for protein levels.

Figure 3. PKC phosphorylates p27 at T198 in vivo. In vivo kinase assays. (A) Active PKCα or PKCδ were transfected with HAp27-wt or HAp27-T198A into HEK-293 cells. P27 phosphorylation was determined by immunoblot with anti-pT198. (B) HEK-293 cells were stimulated with or without PMA. (C) HEK-293 cells were transiently transfected with HAp27-wt and/or active PKCα and cells were cultured with or without PMA. P27 phosphorylation was determined by immunoblot using an antibody specific for phosphorylated T198.

The phorbol ester PMA is a potent activator of PKC in eukaryotic cells.^37,38 Therefore, we analyzed the effect of PMA treatment on p27 phosphorylation. We found that treatment with PMA increased T198 phosphorylation of endogenous p27 in untransfected HEK-293 cells (Fig. 3B) or of transfected HAp27-wt (Fig. 3C, left panel). In addition, T198 phosphorylation of HAp27-wt transfected into HEK-293 cells was markedly stimulated by PMA treatment of HEK-293 cells transduced with PKCα (Fig. 3C, right panel). As expected, PMA treatment was accompained by activation of PKCα (Fig. 3).

To provide complementary evidence that PKC phosphorylates p27 at T198, we transiently transfected HAp27-wt into HEK-293 that had been prior treated with PKC inhibitors (calphostin C, G06976, Rottlerin) or the PI-3 kinase inhibitor (LY294002) as control (Fig. 4A). We found that PMA-dependent T198 phosphorylation of p27 in HEK-293 cells was markedly reduced, if PMA treatment was preceded by use of PKC inhibitors. These results support the hypothesis that p27 is a novel substrate of PKC.

Figure 4. PMA induces T198 phosphorylation and increases p27 protein levels in HeLa and MCF7 cells. (A) Immunoblot analysis of phospho-T198 and p27 protein levels in HEK-293 cells transfected with HAp27-wt and treated with PMA with or without PKC inhibitors before adding PMA as indicated. LY294002 was used as control. (B) Kinetic analysis of p27 T198 phosphorylation in HeLa cells. Immunoblot analysis of phospho-T198 and p27 protein levels after stimulation with PMA at various time points. (C) Kinetic analysis of p27 T198 phosphorylation in MCF7 cells. Immunoblot analysis of phospho-T198 and p27 protein levels after stimulation with PMA at various time points.

PMA-dependent PKC activation increases p27 phosphorylation and stability

To further explore the effect of PMA on p27 phosphorylation, we determined the kinetic of p27 phosphorylation in MCF7 breast cancer cells and HeLa cervical cancer cells stimulated with PMA over a 24 h time course (Fig. 4B and C). Total cell extracts were loaded on SDS-page gel and were immunoblotted with antibodies specific for p27 and its T198 phosphorylated form. We found that PMA increases the levels of T198 phosphorylation of endogenous p27 at 1–12 h in MCF7 cells and at 6–24 h in HeLa cells, respectively. As expected, the increased phosphorylation observed in MCF7 and HeLa cells after PMA treatment was reflected into an increased levels of total p27 levels (Fig. 4B and C).

To test whether the stability of p27 was dependent on PKC-mediated phosphorylation at T198, we compared the half-life of p27 protein in the presence or absence of PMA. To this aim, HeLa cells after PMA stimulation, were treated with cycloheximide (CHX), a potent inhibitor of protein translation. The stability of p27 protein was determined by immunoblot performed on total protein extracts (Fig. 5). As shown in Figure 5, the stability of p27 was markedly higher when HeLa cells were treated with PMA. Similar results were obtained when we compared the half-life of transfected wild-type p27-HA protein in the presence or absence of PMA in HEK-293 cells (data not shown).

Figure 5. PMA stabilizes p27 in HeLa cells. Immunoblot analysis of p27 stability in HeLa treated with solvent or PMA for 1h before cycloheximide treatment as indicated. The intensities of WB signals were quantified by NIH ImageJ software; the results are showed in the graphics. Cells were cultured with or without PMA.

PMA inhibits cell proliferation in a p27-dependent manner

To evaluate the functional relevance of PMA-dependent p27 phosphorylation on cell cycle progression, we performed a BrdU incorporation assay in HeLa cells treated with PMA. As shown in Figure 6A, PMA inhibited BrdU incorporation in HeLa cells. To understand whether p27 plays a prominent role in the antiproliferative effects induced by PMA, Hela cell lines were transduced with a lentivirus expressing shp27 to knockdown the expression of p27. Cells infected with shSCR were used as control. We found that the depletion of p27 (shp27) resulted in slightly increased cell proliferation of HeLa cells compared with SCR cells. Notably, the antiproliferative effect exerted by PMA treatment of HeLa cells was suppressed by p27 knockdown (Fig. 6B). These data indicate that p27 contributes to the cell cycle arrest induced by PKC activation. Similar results were obtained with MCF7 cell lines (data not shown).

Figure 6. p27 induction is critical for PMA-dependent inhibition of proliferation of HeLa cells. (A) BrdU incorporation of HeLa cells in presence or absence of PMA. (B) BrdU incorporation of HeLa cells infected with a control lentivirus (SCR) or a virus encoding a short hairpin RNA directed against p27 (shp27) in presence or absence of PMA.

Discussion

Phorbol 12-myristate 13-acetate (PMA) is known to affect a variety of cellular processes, including cell proliferation, differentiation and migration, through the activation of PKC kinases. Accordingly, PMA has been shown to promote antiproliferative and antimigratory effects in some types of cancer cells, including HeLa cervical cancer cells. In the present study, we report that the strong PMA-dependent antiproliferative effect is mediated at least in part through the activation of PKC and the subsequent phosphorylation and stabilization of the cell cycle inhibitor p27.

Our findings indicate that (1) the PKCα, βII, δ, η and θ isoforms preferentially phosphorylate p27 in vitro and in vivo at the T198; no such phosphorylation was observed with PKCβΙ, ζ and, to a lesser degree γ; (2) PKCα and δ physically interact in vivo with p27 in HEK-293 and HeLa cells; (3) T198 phosphorylation can be detected in immunoprecipitates of transfected and/or endogenous p27, and it is increased by PMA and abolished by PKC inhibitors, such as Calphostin C, G06976 and Rottlerin; (4) in parallel with increased p27 phosphorylation at T198, PMA induces stabilization of p27 protein in HeLa cells, whereas PKC inhibitors induce a decrease in p27 stability; (5) PMA-induced p27 upregulation is necessary for growth arrest of HeLa cells following PKC activation by PMA. Overall, these results suggest that PKC-dependent phosphorylation of p27 at T198 represents one of the mechanisms that mediates the growth arrest promoted by PMA treatment in some cells, possibly by increasing p27 stability, and provide novel insights on the ability of different PKC isoforms to play a role in controlling cell cycle progression.

Phosphorylation of key amino acid residues appears to be a critical mechanism whereby both the stability and the localization of p27 are controlled. In cells arrested in the G₀ phase of the cell cycle, p27 is expressed at high levels in the nucleus and contributes to the maintenance of the quiescent state.^39,40 Such high expression levels are due to enhanced p27 mRNA translation rate and/or stabilization of the protein as a result of phosphorylation at serine 10 (S10) and threonine198 (T198).^29,41 Serine 10 represents the major phosphorylation site of p27 and accounts for about 80% of the total p27 phosphorylation. Myrk/Dyrk1B phosphorylates p27 during the G₀ phase of the cell-cycle, stabilizing and maintaining it within the nucleus, where it can bind to CDK2 and induce cell cycle arrest.⁴² Conversely, the Kis kinase phosphorylates p27 at ser10 in the G₁ phase, enabling it to bind CRM1 and be transported into the cytoplasm for degradation.⁴³

T198 is mainly phosphorylated in G₀. However, as with S10, T198 is also subject to modification early in G₁. At least four kinases have been demonstrated to phosphorylate p27 at T198, including AKT, RSK1, RSK2 and AMPK.^25,27 These kinases are activated in response to mitogenic stimuli or to nutrient deprivation and induce p27 cytoplasmic delocalization. However, phosphorylation of p27 at T198 also appears to increase p27 stability. Nutrient-deprived cells showed AMPK-dependent elevation of T198 phosphorylation and increased p27 stability.²⁸ Accordingly, cells treated with PMA or transfected with activated PKCα or δ show increased T198 phosphorylation and increased stability of p27 protein, though the subcellular localization of p27 is apparently unaltered. The findings summarized above suggest a model in which PKC-dependent phosphorylation of p27 at T198 early in G₁ controls the stability of p27 but not its subcellular localization. The difference with the reported effects of AKT and RSK phosphorylation of p27 localization may be due to different kinetics of PKC phosphorylation compared with AKT and RSK, the simultaneous phosphorylation of other residues (i.e., T157 or S10) or the different cellular contest.^25-27

The mechanism underlying the stabilization of p27 by T198 phosphorylation in G₀ and G₁ cells remains to be determined. Also, it is unclear whether phosphorylation at S10 and T198 cooperate in the stabilization of p27 in G₀/G₁ cells, or whether one or the other phosphorylation is sufficient to achieve maximal p27 stability. The finding that PKC phosphorylates T198 but not S10 or T157 suggests that the different phosphorylation events are not necessarily linked.

It is worth noting that the PKC isoforms that more efficiently phosphorylate p27 at T198 are PKCα and δ, whereas PKCβΙ and ζ and, to a lesser degree, PKCγ are relatively inefficient in their ability to phosphorylate p27 at T198. Such differential phosphorylation by PKC has often been observed; for example, only PKCα, βI, βII, γ, δ and θ, but not PKCε, ζ and η, phosphorylate S1674 of Cav1.2 α_1c,⁴⁴ and may be associated with the different roles that PKC isoforms play in transformation.

In fact, PKCs have long been recognized to have a role in regulating different aspects of tumor growth and development,⁴⁵ although this role is clearly complex and highly tissue-dependent, because in some cases they act as tumor promoters, and in others, they appear to function as tumor suppressors. For example, overexpression of some PKCs has been demonstrated in tissue samples of prostate, endometrial, high-grade urinary bladder and hepatocellular cancers,^46-49 while PKCα, βΙ, δ and ζ have been shown to be decreased at different stages of neoplastic progression.^50-56 It is of particular interest that in rat intestinal epithelial crypt cells, PKC signaling, and in particular PKCα, mediates cell cycle exit through rapid downregulation of G₁ cyclins and increased expression of Cip/Kip cyclin-dependent kinase inhibitors.^50,57-66 Similarly, PMA has been shown to be a potent inhibitor of thyroid cancer cell proliferation and migration by a mechanism involving an increase in the levels of the cell cycle regulators p21 and p27 and a decrease in the levels of cyclin D3, and the cyclin-dependent kinases Cdk4 and Cdk6 decreased.⁶⁷

In conclusion, we demonstrate that some PKC isoforms, including PKCα and δ, are novel bona fide p27 kinases, and that PKC-dependent p27 phosphorylation at T198 increases the stability of p27 protein, which, at least in part, mediates cell cycle arrest of MCF-7 and HeLa cells induced by PMA.

Experimental Procedures

In vitro kinase assay

The recombinant 6-His-tagged-p27 proteins (or the recombinant Histone H1 used as control), were incubated for 30 min at 30°C in kinase assay buffer containing recombinant PKC. The PKC kinase assay buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2, 0.1 mM CaCl2 or EGTA, 150 μM ATP, 0,1μl [γ³²-P]ATP per reaction) was completed with 10 μg of phosphatidylserine (PS) and 2 μg of diacylglycerol (DAG) (SIGMA-Aldrich). For reactions with PKC ζ, kinase buffer required only PS. After incubation, the reaction mixtures were boiled in 2x Laemmli buffer, separated by 12% SDS-PAGE, transferred to nitrocellulose membrane and exposed to X-ray film.

For the immune complex protein kinase assay, cell lysates were immunoprecipitated with anti-HA antibody as described above. The immunoprecipitates were washed three times with cold lysis buffer and once with PKC kinase assay buffer containing recombinant PKC. After incubation for 30 min at 30°C, the reaction mixtures were boiled in 2x Laemmli buffer, separated by 12% SDS-PAGE, transferred to nitrocellulose membrane, and the filters were immunoblotted with the specific antibodies.

The recombinant proteins used in this work were: histone H1 from calf thymus; human recombinant PKCα; human recombinant PKCβΙ; human recombinant PKCβΙΙ; human recombinant PKCγ; human recombinant PKCδ; human recombinant PKCη; human recombinant PKCζ; human recombinant PKCθ. All recombinant proteins were purchased from SIGMA-Aldrich. The 6-His-tagged-p27 fusion proteins used as substrates were produced as described previously.²⁶

Constructs and lentiviral production

HA-tagged wild-type p27 has already been described.⁶⁸ The HA-tagged p27T198A was generated with a site-specific mutagenesis kit (Stratagene). The PKC plasmids used in this work are: Myr-PKCalpha-FLAG, PKC-Δ-wt and pBS-PKCtheta, and were purchased from AddGene.

pLenti PKCδ was generated as follows: PKCδ cDNA was cloned into pEntry vector and then a recombination reaction with a pLenti-DEST vector (Gateway® Technology cloning method) was performed. shRNA-targeting p27 (shp27) and shSCR constructs were purchased from Sigma-Aldrich. For transient transfection, HEK-293T cells seeded at 60–80% confluency in 100-mm dishes were transfected with appropriate expression plasmids and harvested after 24 h.

For viral production of shRNA, HEK-293T were transfected with 12μg of VSVG, 18μg of Δ8.9 and 13μg of shRNA. Forty-eight hours after transfection, media containing viral particles were collected and filtered through a 0.45-μm cellulose acetate filter and stored at -80°C. HeLa and Mcf7 cells were infected with appropriate constructs 24 h after seeding and treated with or without PMA for the indicated times.

Cell cultures

HEK-293 (Human Embryonic Kidney), HeLa, MCF-7, wild-type and p27-T198A MEF cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) containing 10% fetal calf serum and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin, and glutamine) in a humidified incubator at 37°C. PMA (Phorbol 12-myristate 13-acetate, Sigma-Aldrich) was added to the medium at a concentration of 100 nM for the indicated times. Prior to PMA stimulation, the medium was replaced with DMEM containing 10% Fetal Bovine Serum Charcoal Stripped (Sigma-Aldrich).

LY294002 and PKC inhibitors were purchased from Calbiochem.

For determination of protein half-life, exponentially growing cells were seeded at 60% confluency, grown overnight and treated with 10 μg/mL cycloheximide for the indicated times.

BrdU incorporation

The BrdU (5-bromo-2′-deoxyuridine Labeling and Detection Kit I, Roche) incorporation assay was performed as follows. Cells (5 × 10⁵) were plated into 60-mm-diameter dishes on 12mm coverslips and allowed to attach for 24 h. Then cells were cultured in the presence or absence of PMA for the indicated times. The labeling procedure was performed for 2 h at 37°C as recommended by manufacturer; BrdU labeling reagent was used at a final concentration of 10μM. After fixation, the cultures were incubated with anti-BrdU antibody (1:10 – mouse) for 30 min at 37°C. Then cells were washed with PBS and incubated with the anti-mouse-Ig-fluorescein for 30 min at 37°C. The cells were washed four times with PBS. The nuclei were simultaneously stained with 10 μg/mL of 4V, 6-diamidino-2-phenylindole. Fluorescence was visualized with a Zeiss 140 epifluorescence microscope equipped with filters allowing discrimination between FITC and DAPI and cells were counted.

Protein extraction, western blotting and Immunoprecipitation

Total cell extracts were prepared with lysis buffer: 50 mM HEPES pH 7.5, 5 mM EDTA, 250 mM NaCl,, 1 mM dithiothreitol, 0,5% Nonidet P40, 1mM Na3VO4, 1mM NaF, 1mM PMSF and a mix of protease inhibitors (SIGMAFAST protease inhibitor tablets for general use).

Lysates were centrifuged at 13,000 rpm for 30 min at +4 ◦C and the supernatants were collected. The protein concentration was determined using the Biorad Protein Assay (BIORAD). Samples were subjected to SDS/PAGE (12% polyacrylamide). The proteins were transferred onto nitrocellulose membrane (Whatman Protran nitrocellulose transfer membrane) by western blotting. Western blot analysis was performed using specific antibodies to the indicated proteins. The secondary antibodies used were horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies (GE Healthcare). The proteins were detected by enhanced chemiluminescence (Amersham ECL Western Blotting Detection Reagents, GE Healthcare). Densitometric analysis was performed using ImageJ program for image analysis (NIH).

For immunoprecipitation experiments, the HA-fusion proteins were immunoprecipitated from 500 μg of the cell extracts incubating samples over night at 4°C with anti-HA antibody and recovering the beads by centrifugation at 8,000 rpm for 1 min at + 4°C.

The antibodies used in this study were anti-p27 C19 (Santa Cruz Biotechnology Inc.), anti-pT198 p27 (R&D Systems), anti-HA Affinity Matrix (Roche), anti-PKCα Η7 (Santa Cruz Biotechnology Inc.), anti-PKCδ (BD Transduction Laboratories^TM), anti-PKCθ (Cell Signaling Technology), anti-α-Tubulin Ab-2 (Neo-Markers).

Acknowledgments

This work was supported by MIUR (PRIN, 20087FSFFP_001) to GV. Marianna Scrima was supported by a fellowship from FIRC.

Disclosure of Potential Conflicts of Interest

The authors have no potential conflicts of interest to disclose.