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Originally published In Press as doi:10.1074/jbc.M512167200 on December 7, 2005

J. Biol. Chem., Vol. 281, Issue 6, 3237-3243, February 10, 2006
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Protein Kinase C Negatively Regulates Akt Activity and Modifies UVC-induced Apoptosis in Mouse Keratinocytes*

Luowei Li1, Keeran Sampat, Nancy Hu, Julia Zakari, and Stuart H. Yuspa

From the Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, November 11, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKC{delta} and PKC{epsilon} from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKC{delta} and PKC{epsilon} were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKC{alpha} enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKC{delta} and PKC{epsilon} provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Akt/PKB,2 a serine/threonine kinase, is an important regulator of cell proliferation and survival. Activation of Akt by growth factor receptors is mediated by activation of phosphatidylinositol 3-kinase (PI3K) and membrane translocation (1, 2), followed by phosphorylation on two key regulatory sites, threonine 308 and serine 473 (3). Akt delivers cell survival signals by phosphorylation of several pro-apoptotic proteins including BAD (4, 5), caspase-9 (6), and the Forkhead family of transcription factors that regulate a subset of death genes (7). Akt also phosphorylates mdm-2 and promotes its translocation into the nucleus, where mdm2 can interact with the p53 tumor suppressor protein, inhibit its transcriptional activity, and target it for degradation (8). In addition, Akt alters cell cycle checkpoint control by either phosphorylating and inactivating p21Cip/WAF1 (9, 10) or indirectly regulating the transcription of cyclin D1 and p27Kip1 (11). Overexpression of Akt has been reported in a number of human cancers, including ovarian, breast, colon, prostate, and pancreas (12, 13). In addition, activation of Akt in tumor cells can be mediated by the inactivation of PTEN, a tumor suppressor protein that dephosphorylates phosphatidylinositol(3,4,5)-triphosphate (PIP3), the second messenger produced by PI3K (14). Deletion and mutation of PTEN have been reported in Cowden syndrome (15), prostate cancer, glioma, endometrial carcinoma, melanoma, and breast cancer (16). In experimental mouse multistage skin carcinogenesis, Akt activity increases in benign squamous papillomas, and this increase persists through premalignant progression and malignant conversion (17). Overexpression of Akt in neoplastic keratinocyte cell lines enhances their tumorigenicity and produces a more aggressive malignant phenotype (17). Enhanced Akt kinase activity was also observed in skin-targeted IGF-1 transgenic mice (18). Skin-targeted deletion of PTEN, which leads to activation of Akt, showed accelerated tumor formation (19).

PKC is a family of well studied serine-threonine kinases that are involved in many cell functions, including proliferation, differentiation, apoptosis, and gene expression in multiple cell types. Upon activation, receptor-mediated activation of phospholipase C generates second messengers calcium and diacylglycerol, and these contribute to activation of PKC (20). As a cellular target for the tumor-promoting phorbol esters, PKC has been implicated in tumor development for decades (21). Among 12 members of PKC isozymes, PKC{alpha} and PKC{epsilon} have been implicated in cell proliferation (22, 23), whereas PKC{delta} and PKC{eta} have been associated with apoptosis and terminal differentiation (24, 25). Skin PKC isozymes, PKC{alpha}, PKC{delta}, PKC{epsilon}, PKC{eta}, PKC{zeta}, and PKCµ, have been localized in epidermis (26, 27). PKC{eta} is found in the granular cell compartment, whereas the others are distributed throughout the stratified epidermal compartments (28, 29). In cultured mouse keratinocytes, the activation of PKC{alpha} is associated with regulating gene expression of differentiation markers in the epidermis, where it inhibits expression of keratins 1 and 10 and induces expression of loricrin, filaggrin, and transglutaminase in the transition from the spinous to granular cell compartment (30). Skin targeting of PKC isozymes in transgenic mice has revealed that overexpression and activation of PKC{delta} and PKC{epsilon} reduces the number of benign tumors induced by chemical carcinogenesis, whereas PKC{alpha} targeting does not alter the tumor yield (31).

In our current report, we explored the interaction of the Akt survival pathway and PKC pathway in epidermal keratinocytes. Previous studies have shown that IGF-1 activates both the PKC and the Akt/PKB pathways in other cell types (32–36). Since IGF-1 and other growth factors influence the growth, differentiation, and survival of keratinocytes, we reasoned that an interaction between PKC and Akt pathways may underlie some of these growth factor responses. Further, the study of these two pathways may help us gain an understanding of the mechanisms responsible for diseases caused by loss of control in cell growth or death.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Adenoviral Infection—Primary mouse keratinocytes were freshly isolated and cultured in Eagle's minimum essential medium supplemented with 0.05 mM CaCl2 and 8% Chelex-treated fetal bovine serum (Gemini BioProducts, Calabasas, CA) according to previously published protocols (37). A tumorigenic mouse keratinocyte cell line, SP-1, was also maintained in the same medium (38). Adenoviruses carrying cDNA coding for PKC{alpha}, PKC{delta},PKC{epsilon}, and PKC{eta} (AdPKC{alpha}, AdPKC{delta}, AdPKC{epsilon}, and AdPKC{eta}) were reported previously (25). The infection of adenovirus was carried out in serum-free medium containing 2.5 µg/ml Polybrene (Sigma) at indicated multiplicity of infections (m.o.i.) for 30 min at room temperature. Fresh serum-containing medium was added thereafter. The transducing efficiency of adenovirus is over 90%. All experiments described were replicated at least twice with similar results.

Antibodies and Chemicals—Antibodies for Akt, biotin-Akt, phospho-Serine 473-Akt, phospho-Threonine 308-Akt, phospho-Akt substrates, phospho-PKC pan, and phospho-GSK3 were purchased from Cell Signaling Inc. (Beverly, MA). PKC-pan, PKC{alpha}, PKC{delta}, PKC{eta}, and PKC{epsilon} antibodies were form Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphatase 2A (PP2A) catalytic subunit (PP2A.C) or PP2A A subunit (PP2A.A) were purchased from Upstate%20Biotechnology">Upstate Biotechnology Inc. (Lake Placid, NY). 12-O-tetradecanoylphorbol-13-acetate (TPA), rottlerin, Gö6976, and GF109203X, okadaic acid (OA), and calyculin A (CA) were purchased from Calbiochem. Sodium orthovanadate and other chemicals were purchased from Sigma.

Immunoprecipitation and Immunoblotting—Cell lysates were collected in lysis buffer (Cell Signaling) containing protease inhibitors and phosphatase inhibitors. For immunoprecipitation, cell lysates were incubated with either PP2A.C antibody or Biotin-Akt antibody overnight and then followed by immobilized protein A/G (Santa Cruz Biotechnology) or streptavidin (Pierce), respectively. Total cell lysates or immunoprecipitants were separated by electrophoresis on SDS-PAGE and then transferred onto nitrocellulose membranes. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibodies were used as secondary antibodies (Bio-Rad). The immunoblots were visualized by enhanced chemiluminescence (ECL, Pierce). The intensities of immunoblots were quantified, and the relative expression of targeted protein were normalized.

Isolation of Soluble and Particulate Fractions—Cells treated with TPA were lysed in 20 mM Hepes, pH 7.4, buffer containing 250 mM sucrose, 150 mM NaCl, 0.5 mM EDTA, and 0.5 mM EGTA with freshly added 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, and 1 µg/ml aprotinin. The cell lysates were sonicated and applied for ultracentrifugation at 100,000 x g for 1 h. The soluble fraction was collected as cytosol, and the particulate fraction was collected as plasma membrane. Both fractions were further analyzed by immunoblotting for PKC isozyme translocation.

In Vitro Akt Kinase Assay—Akt kinase activity was examined using an in vitro kinase assay kit from Cell Signaling according to manufacturer's instructions. In brief, Akt was immunoprecipitated from cell lysates using an anti-Akt antibody, and a recombinant GSK-3 fusion protein was used as a substrate for the Akt kinase activity. Phosphorylation of GSK-3 fusion protein was measured by immunoblotting using phospho-GSK-3{alpha}/beta (Ser-21/Ser-9) antibody.


Figure 1
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  		FIGURE 1.
PKC activation induces dephosphorylation of Akt at serine 473. Primary mouse keratinocytes were treated as indicated in each figure. Total Akt and the phosphorylation of Akt on Ser-473 or Thr-308 were examined in cell lysates using specific antibodies as described under "Experimental Procedures." A, serum-starved primary mouse keratinocytes were treated with TPA (250 nM) for 15 min and then with or without IGF-1 (25 ng/ml) for an additional 10 min. P-Atk-S473, phoso-P-Akt-Ser-473; P-Akt-T308, phoso-P-Akt-Thr-308. B, Akt dephosphorylation induced by TPA in primary mouse keratinocytes. C, PKC inhibitor GF109203X blocks TPA-induced Akt dephosphorylation. Five µM GF109203X was added 15 min before the addition of TPA in mouse keratinocytes. Relative levels of phospho-Akt-Ser-473 or phospho-Akt-Thr-308 are indicated in each figure. Each experiment was repeated at least twice with similar results.

 
Apoptosis Assay—To prepare cells for UVC irradiation, primary mouse keratinocytes in 60-mm dishes or 12-well plates were overlaid with phosphate-buffered saline containing 0.05 mM CaCl2 to replace the culture medium. The cultures were irradiated with UVC at 20 J/cm2. Immediately after UV irradiation, the cells were then incubated with serum-free medium containing 0.05 mM CaCl2 supplemented with IGF-1, TPA, GF109203X, rottlerin, or 8% serum. After 22 h, both attached and floating cells were collected and stained with annexin V-APC or fluorescein isothiocyanate (BD Biosciences). Apoptotic cells were quantified as annexin V-positive cells using flow cytometry (BD Biosciences). A control sample of cells cultured in serum-containing medium without UVC exposure was included as negative control. A background level, determined from the negative control samples, was subtracted from each sample before the data were plotted. Data were analyzed using the Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of PKC Reduces Akt Phosphorylation on Ser-473 and Akt Kinase Activity—To evaluate the effect of activation of PKC on Akt kinase activity and phosphorylation, primary mouse keratinocytes grown in serum-free medium were treated with 250 nM TPA for 15 min and with IGF-1 (25 ng/ml) for an additional 10 min. As shown in Fig. 1A, IGF-1 induced substantial phosphorylation of Akt on Ser-473, and this was reduced by TPA in both basal and IGF-1-stimulated status. The levels of total Akt and phosphorylated Akt-Thr-308 were not affected by TPA treatment. A time course study indicated that treatment with 100 nM TPA caused dephosphorylation of constitutively active Akt as early as 10 min (Fig. 1B) and almost abolished the phosphorylation of Akt on Ser-473 after 60 min. One µM TPA abolished Akt phosphorylation on Ser-473 after 10 min. Fig. 1C indicates that TPA-induced dephosphorylation of Akt on Ser-473 was dose-dependent. Pretreating cells with the broad PKC inhibitor GF109203X completely blocked TPA-induced dephosphorylation on Akt-Ser-473 (Fig. 1C). We examined the direct effect of GF109203X on Akt kinase activity and found that GF109203X could only inhibit Akt kinase activity at concentrations 50-fold higher than it inhibits PKC (data not shown). These results suggested that activation of the PKC signaling pathway negatively regulates basal and stimulated Akt activity and could potentially modify the cell survival pathway in keratinocytes.

Activation of PKC Attenuates Growth Factor-induced Phosphorylation and Activation of Akt in Mouse Keratinocytes—Several growth factors have been shown to activate the Akt pathway in various cell types. In mouse primary keratinocytes, at concentrations shown to stimulate cell proliferation, EGF, IGF-1, IGF-2, and hepatocyte growth factor, but not basic FGF and keratinocyte growth factor, induced Akt phosphorylation on Ser-473 as shown in Fig. 2A. When keratinocytes were pretreated with TPA (250 nM) for 15 min, IGF-1- and EGF-induced Akt phosphorylation (Fig. 2B) on Ser-473 was substantially reduced. When TPA was added 15 min after IGF-1 or EGF treatment, further induction of Akt phosphorylation by IGF-1 and EGF was blocked. Constitutive and IGF-1-induced Akt kinase activities were also inhibited by TPA pretreatment of keratinocytes when tested in an in vitro kinase assay from total cell lysates (Fig. 2C). The contribution of activation of protein phosphatase to TPA-induced dephosphorylation of Akt was examined by pretreating cells with phosphatase inhibitors OA and CA before the addition of TPA. As shown in Fig. 2D, OA at 1 µM and CA at 50 µM blocked PKC-mediated reduction of Akt phosphorylation at Ser-473, and both inhibitors enhanced the basal phosphorylation level of Akt at Ser-473. Furthermore, co-immunoprecipitation studies indicated that Akt and the PP2A catalytic subunit were associated in keratinocytes to a limited extent, and the association was substantially enhanced by TPA treatment (Fig. 2E). The A subunit of PP2A was co-immunoprecipitated with the PP2A catalytic subunit, but not Akt, and TPA treatment also promoted the association of PP2A A and C subunits. These results suggested that PP2A participates in both basal and PKC-regulated Akt activity in keratinocytes by directly associating with Akt.

Inhibition of PKC by GF109203X Enhances IGF-1-induced Akt Activation and the Anti-apoptotic Function of IGF-1 in Mouse Keratinocytes—To investigate the biological consequences of PKC activation on the Akt cell survival pathway, primary mouse keratinocytes were irradiated with UVC (20 J/cm2) and then incubated with serum-free medium supplemented with IGF-1 in the presence or absence of either TPA or GF109203X. UVC-induced apoptosis of keratinocytes was assessed as cells that stained positive for annexin V-APC. As shown in Fig. 3A, UVC irradiation induced apoptosis in 40% of the primary keratinocytes in the absence of IGF-1. Inclusion of IGF-1 at 10 and 30 ng/ml, but not 3 ng/ml, in the medium following UVC exposure significantly reduced the number of apoptotic cells to less than 25%. TPA at 10 and 100 nM did not further increase the number of apoptotic cells induced by UVC irradiation; however, it completely blocked IGF-1 protection from UVC-induced apoptosis. The PKC inhibitor GF109203X alone at 1 µM and IGF-1 at 3 ng/ml had little effect on the number of apoptotic cells after UVC irradiation (Fig. 3B). However, the number of apoptotic cells was reduced by about 25% with combined treatment of 3 ng/ml IGF-1 and 1 µM GF109203X. A major reduction in the number of apoptotic cells was observed with combined treatment of GF109203X and 10 ng/ml IGF-1 when compared with cells treated only with UVC or 10 ng/ml IGF-1 alone. These results suggested that inhibition of PKC and stimulation with IGF-1 cooperated to protect mouse keratinocytes from UVC-induced apoptosis. We further examined the effect of inhibition of PKC on IGF-1-induced Akt activation. As shown in Fig. 3C, pretreatment of keratinocytes with GF109203X enhanced basal Akt phosphorylation and amplified the IGF-1-induced Akt phosphorylation on Ser-473. PKC inhibition also increased basal and IGF-1-stimulated Akt kinase activity as determined by the in vitro Akt kinase activity assay (Fig. 3D). These results suggested that unstimulated basal PKC activity may provide a constant negative regulation for the Akt signal transduction pathway.


Figure 2
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  		FIGURE 2.
TPA blocks growth factor-induced Akt phosphorylation. A, growth factors induce Akt phosphorylation on Ser-473. Serum-starved primary mouse keratinocytes were treated with EGF (10 ng/ml), IGF-1 (25 ng/ml), IGF-2 (25 ng/ml), basic FGF (bFGF) (10 ng/ml), keratinocyte growth factor (KGF) (5 ng/ml), hepatocyte growth factor (HGF) (10 ng/ml), or 10% serum for 15 min. Phospho-Akt Ser-473 (P-Akt S473) and Akt were analyzed by immunoblotting. B, TPA treatment attenuates IGF-1and EGF-induced Akt phosphorylation on Ser-473. Serum-starved mouse keratinocytes were treated with 25 ng/ml IGF-1 or 10 ng/ml EGF. 500 nM TPA was added either 15 min before or 15 min after IGF-1 or EGF treatment. Cell lysates were collected at the indicated times after IGF-1 or EGF treatment and subjected to immunoblotting. C, TPA attenuates IGF-1-stimulated Akt kinase activity. Serum-starved mouse keratinocytes were treated with 500 nM TPA for 15 min and then with 25 ng/ml IGF-1 for 20 min. The cell lysates were harvested and examined for Akt kinase activity after immunoprecipitation with Akt antibody as described under "Experimental Procedures." P-GSK, phospho-GSK. D, primary mouse keratinocytes were treated with protein phosphatase inhibitors OA and CA for 15 min and then treated with 200 nM TPA for an additional 30 min. The cell lysates were harvested and analyzed for phoso-P-Akt-Ser-473. Relative levels of phospho-Akt-Ser-473 are indicated in each figure normalized to the untreated cells. E, TPA-treated or control cell lysates from mouse keratinocytes were collected, and immunoprecipitation (IP) was performed using either Akt or PP2A.C antibodies. The immunoprecipitants or total lysates were then analyzed by immunoblotting for Akt, PP2A.A, and PP2A.C as indicated in the figure. Each experiment was repeated at least twice with similar results.

 


Figure 3
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  		FIGURE 3.
Activation of PKC blocks IGF-1 protection from UVC-induced apoptosis, whereas inhibition of PKC enhances Akt kinase activity and cell survival after UVC-irradiation. In A and B, primary mouse keratinocytes were irradiated with UVC at 20 J/cm2 and incubated with medium containing 8% serum, IGF-1, TPA, or GF109203X (GF) as indicated. After 22 h, the cells were harvested and incubated with annexin V-APC. The number of annexin V-APC-positive cells was determined using flow cytometry. Data presented are mean ± S.E. (n = 3). The data in panels A and B were statistically analyzed. c indicates the treated groups that are significantly different (p < 0.05) from the control groups (a) within each figure. In C and D, primary mouse keratinocytes were treated with 10 µM GF109203X for 15 min and then with IGF-1 for 20 min. The phosphorylation of Akt on Ser-473 (C) was assessed by immunoblotting, and Akt kinase activity (D) was measured in immunoprecipitates using GSK fusion peptide as substrate. Relative levels of phospho-Akt-Ser-473 (P-Akt-S473) and phospho-GSK (P-GSK) are indicated in the figure. Each experiment was repeated at least twice with similar results.

 
Activation of PKC by IGF-1 May Provide Feedback Regulation for Akt Activation—Activation of PKC by IGF-1 has been reported in other cell types. We asked whether IGF-1 could activate PKC in primary mouse keratinocytes by examining the phosphorylation status and translocation of PKC after IGF-1 exposure. Previous studies have indicated that phosphorylation of a C-terminal residue in the hydrophobic motif of PKC isozymes, homologous to Ser-660 of PKCbetaII, is common to activation of PKC isozymes (20). IGF-1 at several concentrations was added to serum-starved primary mouse keratinocytes for 30 min, and cell lysates were collected and analyzed for phosphorylation on both Akt Ser-473 and PKC. As shown in Fig. 4A, IGF-1 induced phosphorylation of PKC at concentrations similar to those that induce phosphorylation of Akt on Ser-473. The extent of PKC phosphorylation by IGF-1 was not as great as that induced by TPA, but the time course of induction was rapid and similar to the time course seen for IGF-1-induced Akt phosphorylation. It is well established that inactive cytosolic PKC translocates to membranes and is activated by diacylglycerol upon stimulation. To further support IGF-1 mediated PKC activation, we found that translocation of PKC{delta} and PKC{epsilon}, but not PKC{alpha} and PKC{eta}, from soluble to particulate fraction was induced by IGF-1 (Fig. 4B). In addition to translocation, IGF-1 also induced mobility shift of PKC{delta} and PKC{epsilon}, indicating phosphorylation of those PKC isozymes. To evaluate the consequences of PKC-mediated inhibition of Akt activity, a phosphoSer/Thr-containing motif common to multiple Akt substrates was probed by specific antibody (39) in cells treated with TPA, IGF-1, or both (Fig. 4C). This approach revealed a number of specifically phosphorylated substrates after IGF-1 receptor activation in keratinocytes, and a unique set of Akt substrates phosphorylated after TPA treatment alone. Although the identities of these individual substrates have not been determined, the co-treatment group indicates that PKC activation inhibits phosphorylation of several putative Akt substrates, but phosphorylation of several common targets is enhanced (Fig. 4C). Thus, the downstream consequences of the interaction of the PKC and Akt pathways are complex. Potential functions for the activated PKC pathway may be to provide both a negative control on the Akt signaling pathway and an altered response pattern.


Figure 4
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  		FIGURE 4.
IGF-1 induces phosphorylation of both Akt and PKC and stimulates translocation of PKC from cytosol to membranes in mouse keratinocytes. A, serumstarved mouse keratinocytes were treated with IGF-1 at various concentration for 30 min or for various times at 25 ng/ml. The activation of Akt and PKC was estimated by detecting phosphorylation of either Akt on Ser-473 or PKC on its C-terminal hydrophobic motif. Total protein levels of Akt and PKC were determined by immunoblotting. Relative levels of phospho-Akt-Ser-473 (P-Akt-S473) or phospho-PKC (pan) (P-PKC(pan)) are indicated in each figure. B, serum-starved primary mouse keratinocytes were treated with IGF-1 for the indicated times. T indicates cells treated with 500 nM TPA for 15 min. Soluble and particulate fractions were separated by ultracentrifugation and blotted for various PKC isozymes as indicated. C, TPA alters the profile of phosphorylated Akt substrates induced by IGF-1. Serum-starved primary keratinocytes were treated with TPA (250 nM) for 15 min and then treated with IGF-1 (25 ng/ml) for an additional 20 min. Total cell lysates were collected and probed for phosphorylated Akt substrates using antibody that recognizes a common phosphorylated motif in Akt substrates. The phosphorylation of IGF-induced phospho-Akt substrates, either suppressed (a) or enhanced (b) by TPA treatment, is indicated. Each experiment was repeated at least twice with similar results.

 


Figure 5
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  		FIGURE 5.
Individual PKC isozymes modify Akt phosphorylation on Ser-473 distinctively. A, SP-1 cells were infected with increasing m.o.i. of AdPKC{alpha}, AdPKC{delta}, AdPKC{epsilon}, AdPKC{eta}, or AdVector. After 36 h, the cell lysates were collected, and the levels of Akt and phospho-Akt Ser-473 (P-Akt S473) were determined by immunoblotting. Relative expression of P-Akt Ser-473 was normalized with the level of total Akt first and then compared with the relative level of phospho-Akt-Ser-473 from AdVector-infected samples (average values of the two samples). B, SP-1 cells were infected with 10 m.o.i. of AdPKC{alpha}, AdPKC{delta}, AdPKC{epsilon}, AdPKC{eta}, or AdVector for 36 h and then treated with 10, 30, 300, and 1,000 nM TPA for 30 min. The level of Akt and phospho-Akt-Ser-473 was analyzed by immunoblotting. The intensity of the phospho-Akt-Ser-473 was first normalized by the level of total Akt in the same group, and then the relative level of TPA treated samples was compared with samples without TPA treatment. Data presented are averages from two independent experiments (mean ± S.D., n = 2).

 
Isozyme-specific Effects of PKC on Akt Ser-473 Phosphorylation—To determine the effects of individual PKC isozymes on Akt signaling, we employed adenoviruses and SP-1 cells to overexpress PKC{alpha}, PKC{delta}, PKC{epsilon}, and PKC{eta}, the major TPA-activated PKC isozymes present in mouse keratinocytes. As shown in Fig. 5A, an empty adenoviral vector had little effect on the total Akt protein as well as the Ser-473 phosphorylated Akt protein. In the absence of TPA, AdPKC{alpha} enhanced phosphorylation of Akt on Ser-473 (up to 12-fold of control at the highest concentration of AdPKC{alpha}) and decreased the constitutive level of total Akt protein. Overexpression of PKC{delta} and PKC{epsilon} reduced the level of phospho-Akt-Ser-473, with PKC{eta} having a major effect even at low m.o.i. PKC{eta} showed no obvious dose-dependent effect on the level of phospho-Akt-Ser-473. When keratinocytes overexpressing various PKC isozymes were treated with TPA to activate the specific overexpressed isoform, the resulting Akt dephosphorylation was magnified. As seen before, a 50% reduction of Akt-Ser-473 phosphorylation was induced by TPA at the tested concentrations in control keratinocytes lacking exogenous PKC (Fig. 5B). PKC{delta} and PKC{epsilon} overexpression and activation reduced Akt-Ser-473 phosphorylation by 75% after TPA treatment. After an initial decrease of phospho-Akt-Ser-473 with a low concentration of TPA, PKC{alpha}-overexpressing keratinocytes has a substantial increase in Akt-Ser-473 phosphorylation at higher TPA concentrations. Activation of overexpressed PKC{eta} also reduced Akt-Ser-473 phosphorylation but only at the higher concentrations of TPA. These dose-response results, combined with the results from Fig. 5A, suggest that PKC{delta} and PKC{epsilon} are responsible for TPA-induced dephosphorylation of Akt in mouse keratinocytes. To further investigate the involvement of PKC isozymes in this process, selective inhibitors of PKC{alpha}, Gö6976 (40, 41), and PKC{zeta}, rottlerin (42), were evaluated. As shown in Fig. 6A, rottlerin and GF109203X inhibited TPA-induced dephosphorylation of Akt, but Gö6976 had only a minimal effect relative to TPA alone.


Figure 6
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  		FIGURE 6.
A, Pretreatment with PKC inhibitors, GF109203X and rottlerin, reduced PKC-mediated Akt dephosphorylation on Ser-473. Primary keratinocytes were first treated with GF109203X (5 µM), rottlerin (10 µM), and Gö6976 (5 µM) for 15 min, treated with TPA (250 nM) for 15 min, and then treated with IGF-1 (25 ng/ml) for 20 min. Total cell lysates were collected and blotted with antibodies for Akt and phospho-Akt Ser-473 (P-AktS473). The relative level of P-Akt-Ser-473 was normalized with total Akt protein as indicated in the figure. B, rottlerin protects primary mouse keratinocytes from UVC-induced apoptosis. Primary mouse keratinocytes were irradiated with UVC (20 J/cm2) and then incubated with serum-free medium containing either rottlerin and/or IGF-1 (5 ng/ml) for 24 h. The cells were harvested and stained with annexin-V-fluorescein isothiocyanate. The number of apoptotic cells were determined by flow cytometry. Data presented are mean ± S.E. (n = 3). Each experiment was repeated at least twice with similar results.

 
Additional support for the role of the PKC{zeta} isoform in the inhibition of Akt function comes from UVC cell survival studies. The PKC{zeta}-specific inhibitor rottlerin was tested for its effect on IGF-1 protection of UVC-induced-apoptosis. As shown in Fig. 6B, rottlerin alone at 5 and 10 µM protected against UVC-induced apoptosis and enhanced IGF-1 protection. However, Gö6976, the PKC{alpha}-selective inhibitor, did not protect against UVC-induced apoptosis and had no effect on IGF-1 protection (not shown). These results support previous data that activation of PKC{delta} mediates UV-induced apoptosis (43) and suggest that inhibition of PKC{delta} in concert with IGF-1 enhances cell survival.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our present study has indicated that activation of PKC reduces both phosphorylation of Akt on Ser-473 and Akt catalytic activity in mouse keratinocytes under both basal and IGF-1-stimulated conditions. Inhibition of PKC by GF109203X not only blocks TPA-induced dephosphorylation of Akt and enhances Akt kinase activity but also potentiates IGF-1 protection of UVC-induced apoptosis. Both positive and negative regulation of Akt phosphorylation by PKC have been reported previously (44–50). PKC-specific inhibitors elevated Akt phosphorylation on Ser-473 and its kinase activity in human epithelial cell lines A549 and HEK293 (46) but inhibited vascular endothelial growth factor-induced Akt activation (49). However, these studies did not explore whether Akt-mediated cellular function was altered by the negative regulation provided by PKC on Akt phosphorylation and kinase activity. Our study, for the first time, demonstrated that suppressing PKC activity enhances the anti-apoptotic function of the Akt pathway in ultravioletirradiated mouse keratinocytes. Additional studies in keratinocytes in which individual PKC isozymes are overexpressed or selectively inhibited have indicated that PKC{delta} is a major isoform involved in both inhibiting Akt activity and enhancing UV-induced apoptosis in keratinocytes. This is consistent with published results in human keratinocytes, where UVB-induced apoptosis is mediated by PKC{delta} (43). Based on these results, we propose that one function for PKC in keratinocytes is to provide feedback regulation for Akt activity in both basal and stimulated conditions. For example, exposure to ultraviolet light activates both PKC and Akt in keratinocytes (43, 51, 52). This may be particularly important in a tissue exposed to a toxic agent such as ultraviolet light, where local life and death decisions must be made to protect the organism.

Full activation of Akt requires multiple steps that are mediated by PI3K and PDK1 (1, 2). Following receptor-ligand interaction from a variety of growth factors that activate tyrosine kinase receptors, PI3K is activated, resulting in an increase in PIP3. Elevated PIP3 binds the pleckstrin homology domain on Akt and reverses the inhibitory effect of this domain on the activation loop. Once the activation loop is exposed, PDK1, a constitutively active protein kinase, is available to phosphorylate Akt on Thr-308. After Thr-308 is phosphorylated, Ser-473 is phosphorylated by either Akt autophosphorylation or a yet unidentified PDK2. Once Ser-473 is phosphorylated, Akt is fully activated regardless of the phosphorylation status of Thr-308 (3). Our studies showed that TPA has little effect on Thr-308 phosphorylation, suggesting that the upstream regulators of Akt, PI3K and PDK1, are not targets for PKC in mouse keratinocytes. The direct effect of PKC activation on PI3K has been reported. In human fibrosarcoma HT-1080 cells, TPA slightly reduces PI3K activity but enhances Ser-473 phosphorylation of Akt (53). In contrast, TPA stimulated PI3K kinase activity and enhanced insulin-induced PI3K activity in mouse JB6 cells (54). The discrepancy among these reports may reflect both the different tissue and the cell types in each study and the distribution of PKC isozymes in these tissues and cell lines.

Using adenovirus-mediated overexpression, PKC{delta} and PKC{epsilon} appear to be the isozymes that most determined the sensitivity of Akt to TPAinduced dephosphorylation; in contrast, overexpressing PKC{alpha} increased Akt phosphorylation. The latter result is consistent with a previous report showing that overexpression of PKC{alpha} stimulated Akt activity in 32D myeloid progenitor cells (55). Our results suggested that individual PKC isozymes have specific functions in the regulation of Akt phosphorylation and kinase activity. The ratio of each isozyme in a particular cell type, a particular developmental status, or a particular disease state may determine the consequences of the interaction of PKC and Akt pathways. With regard to keratinocytes, it should be noted that PKC{delta} activity increases in differentiating keratinocytes (26), and this isoform has been linked to a keratinocyte death pathway (25). In contrast, PKC{delta} catalytic activity is reduced in neoplastic keratinocytes due to tyrosine phosphorylation, and this is associated with a defect in keratinocyte terminal differentiation (56). Although Akt has not yet been shown to be a downstream effector of these PKC{delta} modifications, the current report suggests that such a link may be worth exploring since Akt activity is reduced in differentiating keratinocytes (57), where PKC{delta} is active (26), and increased in keratinocyte tumors, where PKC{delta} is inactive (56).

In search of the protein phosphatase(s) that mediates PKC-induced dephosphorylation of Akt on Ser-473, the effects of a number of phosphatase inhibitors on phosphorylation of Akt-Ser-473 were examined. In keratinocytes, the selective PP2A inhibitor OA enhanced basal Akt phosphorylation and blocked TPA-induced Akt dephosphorylation at 0.5 and 1 µM in keratinocytes. In contrast, the equipotent PP2A and PP1 inhibitor CA only blocked PKC-mediated Akt dephosphorylation at high concentration (50 µM). The PP2B inhibitor cyclosporin A had no effect (data not shown). In addition to studies using phosphatase inhibitors, we also demonstrated that the PP2A catalytic unit interacts with Akt and that the association could be enhanced by TPA treatment. Our results suggested that PP2A is most likely to be the phosphatase that mediates activation of PKC-induced dephosphorylation of Akt. Phosphatase 2A has been identified as the phosphatase that dephosphorylates Akt on Ser-473 constitutively in other cell types (58–60). Direct association of Akt and PP2A has been detected in adipocytes and human fibroblasts (61, 62). Recently, the involvement of PP1 in regulating Akt dephosphorylation and direct association of PP1 with Akt has been observed in an ErbB2-positive human breast cancer cell line (63). The protein phosphatase(s) involved in modulating Akt phosphorylation or activity may depend on the tissue type and/or the upstream regulator being examined.

Cross-talk of multiple signaling pathways activated by growth factors and cytokines has been observed in many cell types (64–66). We have now shown that in keratinocytes, these disparate receptors, EGF receptor, IGF receptor, and c-Met, can activate the Akt pathway in common, whereas the major receptors for basic FGF and keratinocyte growth factor, FGFR1 and FGFR2 (67), do not. However, all of these receptors can stimulate growth and improve the survival of keratinocytes. For at least the IGF receptor, we have now shown that the response to receptor activation is modified by co-activation of the PKC pathway, and this appeared to be a quantitative relationship. In particular, IGF-1-induced translocation of PKC is limited to only two PKC isozymes, PKC{delta} and PKC{epsilon}. Thus, the downstream targets of Akt activation from IGF-1 stimulation were modified by PKC{delta} and PKC{epsilon} activation. If Akt is to be considered a therapeutic target for cancer (68), our results suggest that the profiles of PKC isozymes or activity need to be considered in the target tissue, and combined therapy with a PKC modifying drug might be beneficial.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, NCI, National Institutes of Health, Bldg. 37-Rm. 4060, 37 Convent Dr., Bethesda, MD 20892. Tel.: 301-496-2383; Fax: 301-496-8709; E-mail: lilu{at}mail.nih.gov.

2 The abbreviations used are: PKB, protein kinase B; PKC, protein kinase C; TPA, 12-O- tetradecanoylphorbol-13-acetate; IGF, insulin-like growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; PP2A, phosphatase 2A; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; m.o.i., multiplicity of infections; PIP3, phosphatidylinositol(3,4,5)-triphosphate; OA, okadaic acid; CA, calyculin A; Ad, adenovirus; GSK, glycogen synthase kinase; APC, allophycocyanin. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
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