(The FASEB Journal. 2004;18:627-636.)
© 2004 FASEB
The enigmatic protein kinase C
: complex roles in cell proliferation and survival
DESMOND N. JACKSON and
DAVID A. FOSTER1
Department of Biological Sciences, Hunter College of The City University of New York, New York, New York, USA
1Correspondence: Department of Biological Sciences, Hunter College of The City University of New York, 695 Park Ave., New York, NY 10021, USA. E-mail: foster{at}genectr.hunter.cuny.edu
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ABSTRACT
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Protein kinase C
(PKC) has been implicated both as a tumor suppressor and a positive regulator of cell cycle progression. PKC has also been reported to positively and negatively regulate apoptotic programs. This has led to conflicting hypotheses on the role of PKC in the control of cell proliferation and survival. Surprisingly, PKC mice develop normally and are fertile, indicating that PKC is not critical for normal cell proliferation during development. However, PKC may play important roles in neoplastic cell proliferation. In this review, we have summarized the apparent multifunctional properties of this enigmatic protein with regard to its role in the regulation of cell cycle progression and cell survival. It is proposed that PKC has both tumor suppressor and proliferation capabilities that can be recruited as a backup kinase for both gatekeeper tumor suppression and as an activator of the Ras/Raf/MEK/MAP kinase signaling pathway in cell proliferation.—Jackson, D. N., Foster, D. A. The enigmatic protein kinase C: complex roles in cell proliferation and survival.
Key Words: cell cycle • tumor promotion • tumor suppressor • tyrosine kinase
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INTRODUCTION
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THE ABILITY OF PHORBOL ESTERS to act as tumor promoters was first characterized using the mouse skin carcinogenesis model (1)
where initiation, promotion, and progression phase of tumor development can be studied. Phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) were shown to act at the promotion stage, where tumor formation was stimulated in mouse skin treated with mutagenic DNA damaging agents. The discovery by Nishizuka and colleagues that the target of TPA was protein kinase C (PKC) (2)
stimulated much interest in the role of PKC in regulating cell proliferation. However, studies were complicated by the discovery that there was a large family of PKC isoforms that were responsive to TPA (3)
. It was discovered that TPA not only activated PKC isoforms, but also stimulated the ubiquitination and degradation of PKC (4
, 5)
. Thus, it was not clear which PKC isoform(s) was/were responsible for the tumor-promoting effects of TPA, nor was it clear whether it was the activation or the degradation of PKC isoforms that was important. Since the initial studies revealing a connection between PKC and tumor promotion, many studies have provided evidence for the involvement of different PKC isoforms in regulating cell proliferation and survival. Unfortunately, no clear pattern has emerged as to the critical target of phorbol esters in tumor promotion or whether it is the up-regulation or down-regulation of PKC that is critical. The most confusing data have involved studies of the isoform of PKC where studies have implicated PKC as a tumor suppressor and as a positive regulator of cell cycle progression. Adding to the confusion, PKC has been reported to both positively and negatively regulate apoptosis (6)
. In this review, we summarize the apparent conflicting evidence for the role of PKC in the regulation of cell cycle progression and survival.
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PKC NULL MICE
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Before discussing the role PKC in cell proliferation, it is important to mention studies of PKC null mice. Two separate groups have generated PKC null mice strains (7
, 8)
. These mice developed normally and were fertile (7)
. These studies demonstrated that PKC is not required for cell proliferation during normal development. Although these mice did not show obvious increases in cancer, they had significantly higher numbers of smooth muscle cells than were found in wild-type animals, which correlated with decreased smooth muscle cell death (7)
. Smooth muscle cells derived from the PKC null aortas were also resistant to cell death induced by several stimuli (7)
, indicating that PKC is critical for certain stress responses. Also found in the PKC null mice were increased proliferation of B cells and autoimmunity (8)
. These studies demonstrate that PKC is not required for the proliferation of normal cells. Phenotypes shown by the PKC null mice are similar to that observed in mice with a defect in the tumor suppressor p53, which were also susceptible proliferative disorders (9)
. The possibility that PKC may act as a tumor suppressor under certain conditions is addressed below.
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REGULATION OF CELL PROLIFERATION
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PKC as a negative regulator of cell cycle progression
Most studies of the effect(s) of PKC on cell proliferation have found that PKC inhibits cell proliferation. However, as discussed below, several studies have found that PKC is required for the proliferation of some transformed and cancer cells. We will first describe studies that have shown an inhibitory effect of PKC on cell proliferation. Watanabe et al. (10)
first demonstrated a negative effect of PKC on cell proliferation in CHO cells. Mischak et al. (11)
followed up by demonstrating that elevated expression of PKC inhibited proliferation of NIH 3T3 mouse fibroblasts. Acs et al. (12)
demonstrated that PKC suppresses NIH 3T3 cell proliferation, but a mutation at Tyr155 of PKC led to increased proliferation. This study provided an interesting insight into the ability of PKC to both positively and negatively regulate cell proliferation possibly through the differential phosphorylation of PKC on tyrosines. The potential role of tyrosine phosphorylation is addressed further below. PKC has also been shown to inhibit the proliferation of neonatal rat cardiac fibroblasts (13)
. The reports cited above used artificially elevated expression of PKC to negatively affect cell proliferation in culture. Recently, Kambhampati et al. (14)
showed that the anti-proliferative effects of retinoic acid were dependent on PKC in leukemic and MCF-7 cells, indicating that endogenously expressed PKC can suppress cell proliferation.
Several studies have examined the effect of PKC on cell cycle progression. PKC was shown to inhibit the proliferation of rat vascular smooth muscle cells (15)
; FACS analysis indicated that these cells were blocked with a G0/G1 DNA content, indicating that cells were prevented from entering S-phase. Consistent with this finding, expression of G1 cyclins was suppressed by elevated expression of PKC (15)
. PKC also inhibited progression into S-phase of capillary endothelial cells (16
, 17)
. A431 cells were blocked at the G1/S transition by an anti-tumor somatostatin analog in a mechanism that required PKC (18)
, demonstrating that the G1 block was not an artifact of elevated expression of PKC. Similarly, induction of the G1 cyclin-dependent kinase inhibitor, p21Cip1, by epidermal growth factor (EGF) in A431 cells was dependent on PKC (19)
. PKC was required for expression of p21Cip1 in rat aortic smooth muscle cells (20)
and MCF-7 cells (21)
. PKC inhibited expression of the G1 cyclin (cyclin D1) in vascular smooth muscle cells (15)
and in NIH 3T3 cells (22)
, and PKC inhibited cyclin D1 promoter activity in primary bovine airway smooth muscle cells (23)
. Thus, PKC suppresses cell cycle progression into S-phase, at least in part, by inhibiting G1 cyclin expression and increasing expression of cyclin-dependent kinase inhibitors.
In addition to blocking progression into S-phase, elevated expression of PKC led to cell cycle arrest at the G2/M checkpoint in CHO cells (10)
. Kitamura et al. (24)
reported that PKC suppressed entrance to M-phase in 3Y1 rat fibroblasts. Bistratene A, which activates PKC with some specificity (25)
, induced cell cycle arrest at G2/M in a human melanoma cell line (26)
. These studies suggest that PKC is able to provide a "gatekeeper" function that prevents cell cycle progression through G1/S and G2/M cell cycle checkpoints.
PKC as a positive regulator of cell proliferation
Although a majority of studies of the effect of PKC on cell proliferation suggest that PKC suppresses proliferation, several reports have demonstrated a positive role for PKC in cell proliferation. PKC was required for insulin-like growth factor-I (IGF-I), receptor-mediated cell transformation, most likely via an association between the IGF-1 receptor and PKC- and activation via tyrosine phosphorylation (27)
. Consistent with a role for PKC in IGF-I signaling, the von-Hippel-Lindau tumor suppressor pVHL interacted directly with PKC in renal cell carcinoma cell lines and inhibited a required association between PKC and the IGF-I receptor for downstream signaling (28
, 29)
. Thus, there appears to be a PKC requirement in proliferation signals mediated by the IGF-I receptor in transformed and cancer cells. Does PKC play a physiological role in signaling through the IGF-I receptor? It would seem unlikely since IGF-I receptor null mice exhibit severe developmental abnormalities and die within minutes of birth (30
, 31)
, whereas PKC null mice develop normally (7
, 8)
. If PKC were required for normal IGF-I receptor-mediated signaling, it would be expected that PKC null mice would have a phenotype similar to the IGF-I receptor null mice. It is still possible, however, that since the IGF-I receptor can activate multiple intracellular signals, a compensatory signal that overcomes the need for PKC could be activated in the PKC null mice. PKC was not required for the anti-apoptotic effects of IGF-I on cultured myocytes (32)
, indicating that PKC is not required for this particular effect of IGF-I on normal cells. We would suggest that under conditions where the IGF-I receptor is overexpressed or the VHL gene is not present, the IGF-I receptor most likely is able to co-opt PKC and utilize it to activate mitogenic signals. Thus, while PKC may not play a critical role in normal cell proliferation, PKC may be critical for the proliferation of some cancers such as renal cell carcinoma.
Consistent with a positive role for PKC in some transformed cells, Jaken and co-workers have reported increased levels of PKC in progressively transformed rat embryo fibroblasts and that a dominant negative PKC could block anchorage independent growth of these cells (33)
. This group has also showed a correlation between PKC expression and metastatic potential in breast cancer cell lines and that elevated expression of PKC led to increased anchorage-independent growth of the poorly metastatic cells (34)
. Inhibiting PKC also inhibited the metastatic potential of breast cancer cells (35)
. Consistent with these observations, PKC was required for the migration of breast cancer cells with elevated expression of the EGF receptor (36)
. PKC was reported to act as a prosurvival factor in the MCF-7 human breast cancer cell line in vitro (37)
. Inhibition of PKC in these cells decreased survival in response to radiation-induced DNA damage, indicating that PKC was able to provide a survival signal. PKC can interact positively with mTOR (38)
, which has also been implicated in survival signaling (39)
. It remains to be determined whether survival signals generated by PKC in MCF-7 cells are dependent on mTOR.
Inhibition of PKC was reported to prevent estrogen-induced, mitogen-activated protein kinase (MAPK) activation while having no effect on transforming growth factor-
-induced MAPK activation in MCF-7 human breast cancer cells (40)
. This study revealed that the activation of MAPK by estrogen was mediated through a HER-2/PKC/Ras pathway. These data suggest that the estrogen receptor-positive MCF-7 cell line has used PKC to activate MAPK and that this may prevent apoptosis in these cells. Since MAPK is activated in response to most, if not all, mitogenic signals, the ability of PKC to activate MAPK (40
, 41)
may be an important component of PKC’s ability to positively regulate cell proliferation and survival. What is not clear is why and under what circumstances PKC activates MAPK. Although PKC apparently can be co-opted into oncogenic signaling pathways, aside from the interesting observation that a Tyr 
Phe mutation at Tyr155 stimulate proliferation of NIH 3T3 cells (12)
, there is no evidence that elevated expression of wild-type PKC itself can stimulate cell proliferation or activate an oncogenic signal. It is therefore doubtful that PKC can act as an oncogene, however it may act as a partner to an oncogene or as a "co-oncogene" under some circumstances, such as renal cell carcinoma, where the VHL gene is lost.
The ability of PKC to act as a co-oncogene may be the result of its ability to activate MAPK, a critical target of mitogenic signaling through the Ras/Raf/MEK/MAPK signal transduction pathway. An activated mutant of PKC was reported to activate MAPK in a mechanism that is independent of Ras, but dependent on Raf and MEK (41)
. In contrast, the PKC-dependent activation of MAPK by estrogen was dependent on Ras (40)
. The PKC-dependent activation of MAPK by neurogenic agents, including fibroblast growth factor (FGF) was dependent on MEK, but not Raf (42)
. Similarly, FGF-induced MAPK in rat pituitary tumor cells was independent of Ras and Raf (43)
. Thus, it would seem that PKC can contribute to MAPK activation at several points in the Ras/Raf/MEK/MAPK signaling pathway: 1) upstream of Ras (40)
; 2) downstream of Ras but upstream of Raf (41)
; and 3) downstream of Raf but upstream of MEK (42)
. The ability of PKC to activate the MAPK pathway at different places is shown schematically in Fig. 1
. How PKC can activate MAPK using several apparently different mechanisms is not clear.
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TYROSINE PHOSPHORYLATION
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The ability of PKC to perform very different tasks under different conditions leads to the question: How this is accomplished? A likely explanation is the ability of PKC to associate with different tyrosine kinases resulting in differential patterns of phosphorylation on the tyrosine residues of PKC. PKC has been reported to associate with Src (44
45
46
47
48)
, Lyn (47)
, Fyn (49)
, and c-Abl (50)
. PKC is able to interact with the IGF-I receptor in cancerous and transformed cells (27
, 29)
. PKC becomes phosphorylated on different tyrosines in response to different stimuli, and the effect of tyrosine phosphorylation has been reported to have many effects on PKC. There are 19 tyrosines in PKC, and at least 9 have been implicated as sites that become phosphorylated. Tyrosine phosphorylation on PKC has been discussed in two reviews (6
, 51)
. Both reviews concurred that the effect of tyrosine phosphorylation on PKC—as might be expected for a protein that can apparently be phosphorylated at so many different sites—is complex and confusing.
Of particular relevance for the role of PKC in regulating cell proliferation is that tyrosine kinase activity is commonly elevated in response to mitogenic and oncogenic signals (52)
and that PKC 
frequently phosphorylated on tyrosine in response to mitogenic and other stimuli. Tyrosine phosphorylation of PKC was first reported in keratinocytes expressing oncogenic H-Ras (53)
. The tyrosine phosphorylation on PKC rendered PKC insensitive to TPA, indicating that tyrosine phosphorylation was inhibiting PKC kinase activity. Src was able to phosphorylate PKC on tyrosine in vitro, but this phosphorylation was reported to increase PKC activity (54)
. PKC associates with activated Src and is phosphorylated on tyrosine (44)
, but the tyrosine-phosphorylated PKC in these cells, like that in H-Ras-transformed cells, showed reduced kinase activity. TPA treatment of MCF-7 cells led to the association of PKC and activated c-Src (46)
. Tamoxifen also stimulated an association between c-Src and PKC that led to the tyrosine phosphorylation of PKC (48)
. Tamoxifen treatment led to a reduction in PKC protein levels (48)
. PKC was reported to be tyrosine-phosphorylated in response to TPA, and the authors reported that tyrosine phosphorylation by Src family kinases led to increased kinase activity (55)
. Blake et al. (45)
also reported an association between activated Src and PKC that led to tyrosine phosphorylation in NIH-3T3 cells, but this led to elevated activity. Tyrosine-phosphorylated PKC, however, was less stable and reduced levels of PKC were observed in the cells with expression of activated Src (45)
. A Tyr Phe mutation at Tyr311 blocked tyrosine-phosphorylation of PKC and led to the stabilization of PKC, suggesting that activated Src phosphorylates PKC at Tyr311 and that phosphorylation at this site enhances degradation (45)
.
PDGF stimulated the tyrosine phosphorylation of PKC (56)
, which was subsequently shown to occur at Tyr187 (57)
. Phosphorylation of PKC at this site did not appear to affect the activity; however, elevated expression of PKC blocked PDGF-induced DNA synthesis (45)
. Tyrosine phosphorylation of PKC in response to transforming growth factor- and EGF led to decreased activity of PKC (58)
. It was suggested that since the receptors did not phosphorylate PKC in vitro, the phosphorylation was probably due to indirect stimulation of Src family kinases. In addition to Src, both Lyn and Fyn have been reported to interact with and phosphorylate PKC. Lyn phosphorylates PKC at Tyr52, leading to an association between the Lyn SH2 domain and PKC (47)
. Activation of human platelets led to a functional association between Fyn and PKC (59)
, and Fyn was shown to associate with phosphorylated Tyr187 of PKC (12)
. Unlike other tyrosine kinase receptors, activated IGF-I receptor was able to phosphorylate PKC in vitro (27)
.
IgE stimulation of leukemia cells led to the phosphorylation of PKC at Tyr52 (60)
. IgE-stimulated tyrosine phosphorylation reduced activity toward some, but not all, substrates, leading the investigators to propose that tyrosine phosphorylation modifies substrate recognition (61)
. This important observation indicated how differential tyrosine phosphorylation could be critical for the reported differential effects of PKC.
In response to ionizing radiation (50)
and oxidative stress (62)
, PKC is phosphorylated by c-Abl. Oxidative stress leads to tyrosine phosphorylation of PKC (63)
. Sites 512 and 523 in the catalytic domain were critical for activation of PKC by H2O2; subsequent studies using mass spectrometry revealed that sites 311, 323, and 512 were the sites actually phosphorylated in response to H202 and phosphorylation at Tyr311 was apparently the most critical for increased activity (64)
. Etoposide also induced tyrosine phosphorylation of PKC, and sites 64 and 187 were critical for this effect (65)
.
Anti-sense PKC inhibited differentiation of mouse keratinocytes whereas overexpression of PKC in the presence of a Src kinase inhibitor enhanced differentiation (66)
. Plasmids encoding phenylalanine mutants of PKC tyrosine residues 64 and 565 induced differentiation in the absence of an Src inhibitor, suggesting that phosphorylation of these sites by Src suppressed differentiation. In contrast, phenylalanine mutants of tyrosine residues 52, 155, and 187 were inactive, indicating these sites are required for differentiation. Thus, Src kinase mediated post-translational modification of PKC on specific tyrosine residues in mouse keratinocytes apparently suppresses PKC-mediated differentiation.
Many of the phosphorylation site studies have used Tyr Phe mutations to block tyrosine phosphorylation and thus implicate various sites as critical sites of tyrosine phosphorylation. It is possible that a mutation at a particular site blocks phosphorylation at other sites. Thus, it is not always clear which and how many sites are actually phosphorylated in response to a given stimulus. The ability to alter substrate specificity and phenotype with these mutations indicates that the many permutations of differential tyrosine phosphorylation by different tyrosine kinases in response to different stimulation would provide PKC the ability to be used in many different ways under different circumstances. The reported differential effects of tyrosine phosphorylation on the activity of PKC have also suggested that PKC can be manipulated in different ways through tyrosine phosphorylation. However, the PKC activity assays are performed in vitro and may not reflect the actual in vivo activity. There remains much to be learned about the apparent multiple effects of tyrosine phosphorylation on PKC activity, substrate specificity, and subcellular localization. Table 1
summarizes the available data on the tyrosines phosphorylated on PKC and the effects of phosphorylation.
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PKC AS A TUMOR SUPPRESSOR
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The first indication that suppression of PKC could contribute to a transformed phenotype came when it was shown that down-regulating PKC with prolonged TPA treatment transformed rat fibroblasts with elevated expression of the Src tyrosine kinase (67)
. Further studies indicated that elevated expression of the EGF receptor could cooperate with suppression of PKC to transform rat fibroblasts (68)
. The ability of PKC down-regulation, in concert with elevated tyrosine kinase expression, to cause transformation led to the conclusion that the tumor-promoting effects of phorbol esters are due largely to the down-regulation of a tumor-suppressing effect of PKC (67
, 68)
. This conclusion was supported by subsequent studies in mice engineered to express elevated PKC in their skin. These mice were resistant to the tumor-promoting effects of phorbol esters in the standard mouse skin carcinogenesis assay originally used to define the tumor promoting effects of phorbol esters (69)
. A study by Gschwendt et al. (70)
where inhibitors of PKC were unable to affect tumor promotion by phorbol esters also supported the hypothesis that TPA-induced tumor promotion was not due to the activation of PKC. Similarly, bryostatin 1, which protects protein kinase C- from down-regulation (71)
, inhibited phorbol ester-induced tumor promotion (72)
. These studies all indicated that the tumor-promoting capability of phorbol esters was due not to the activation of any PKC isoforms, but rather to the down-regulation of a tumor suppressing effect of the isoform of PKC. Although the existing data strongly support the hypothesis that suppression of PKC by phorbol esters are critical for the tumor promotion, positive effects on other PKC isoforms cannot be ruled out.
Consistent with a tumor suppressing effect for PKC, the PKC inhibitor rottlerin (73)
was shown to induce a transformed phenotype in human keratinocytes (74)
, further implicating PKC as suppressor of transformation. Rottlerin was able to induce a transformed phenotype in rat fibroblasts overexpressing either c-Src or the EGF receptor (67
, 68)
. Perletti et al. (75)
reported reduced levels of PKC in rat colonic epithelial cells transformed by Src. This group went on to show that elevated PKC expression in these cells suppressed the Src-induced transformed phenotype. Moreover, a dominant negative PKC mutant was able to induce a partial transformed phenotype in rat colonic epithelial cells (75)
. Transient transfection of NIH3T3 cells with a dominant negative PKC mutant induced a transformed phenotype (76)
. Inhibition of PKC was able to abolish contact inhibition in human and murine fibroblasts (76)
. Immunofluorescence revealed a rapid translocation of PKC to the nucleus when cultures reached confluence with a peak in early-mid G1 phase (76)
. These data suggested that the suppression of growth in response to cell–cell contact may be due to PKC substrates in the nucleus. Nuclear translocation of PKC to the nucleus is also required for etoposide-induced apoptosis (77)
. Collectively, these studies suggested that PKC has the potential to suppress cell proliferation and act as a tumor suppressor.
The ability of PKC down-regulation to cooperate with elevated expression of a tyrosine kinase is consistent with a model for transformation of primary cells postulated by Weinberg and colleagues (78)
. In their model, an oncogene that provides the equivalent of growth factor signal such as Ras or Src cooperates with Myc or large T antigen to transform primary cells. TPA was shown to cooperate with Ras to transform primary cells (79)
. It is reasonable this cooperation was due to TPA-induced down-regulation of PKC.
A model for complementing transforming and tumor-suppressing genes is presented in Fig. 2
. In this model, complementing transforming or tumor-suppressing genes are designated class I or class II oncogenes based on whether they facilitate passage through the restriction point (80)
, or through cell cycle checkpoints that monitor the readiness of cells to progress into a new phase of the cycle (81)
. The class I oncogenes mimic growth factor signals that facilitate passage through the growth factor-dependent restriction point (80)
. The early G1 phase prior to the restriction point has been referred to as the G1 PM(postmitotic) phase (82)
. The absence of growth factors stimulates exit from G1-pm to a quiescent G0 state (81
, 83)
and class I genes such as an activated Ras gene or a tyrosine kinase provide growth factor independence and avoid cell cycle exit to G0 and quiescence. The class II genes, in contrast, facilitate progression through cell cycle checkpoints and, most important, through the later part of G1 after the restriction point, referred to as the G1-ps (pre-S) phase (82)
, and require cyclins that along with their partner kinases prevent the inhibitory function of Rb (84)
. In this model, the ability of PKC down-regulation to complement elevated expression of a tyrosine kinase (67
, 68)
would suggest that PKC down-regulation is providing the equivalent of class II genes that facilitate progression through cell cycle checkpoints. Thus, down-regulating PKC is apparently able to provide what T antigen accomplishes. T antigen sequesters and inactivates p53 and Rb (85)
, both of which block passage through cell cycle checkpoints. Consistent with a role for PKC down-regulation in progression through cell cycle checkpoints, expression of G1 cyclins was suppressed by elevated expression of PKC (15
, 22)
. Moreover, PKC was required for expression of the G1 cyclin inhibitor p21Cip1 in rat aortic smooth muscle cells (20)
and MCF-7 cells (21)
. Elevated expression of PKC also led to cell cycle arrest at the G2/M checkpoint in CHO cells (10)
and rat fibroblasts (24)
. Preliminary data indicate that PKC is required for DNA damage induced increases in p53 expression and concomitant apoptosis (86)
. The ability of PKC to suppress the expression of factors required for progression through cell cycle checkpoints indicates that PKC has gatekeeper functions similar to those of other known tumor suppressor genes, such as Rb and p53.
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STRESS RESPONSE AND APOPTOSIS
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Many genes that regulate cell cycle checkpoint passage also regulate apoptosis—the most obvious example being p53 (85)
. PKC with an apparent gatekeeper function has been implicated in apoptosis. The role that PKC plays in apoptosis appears to be as complicated as the role it plays in cell proliferation. A large body of evidence implicates PKC in apoptotic responses; as with cell proliferation, however, PKC seems to have been co-opted in some cancer cells to be anti-apoptotic. Brodie and Blumberg (6)
recently reviewed the role of PKC in apoptosis, and we will not attempt to expand on their review other than to highlight an emerging paradigm whereby PKC is a critical component of the cellular stress response. PKC is activated in response to a variety of genotoxic stress (6)
and was required for apoptosis induced by serum withdrawal (87)
. In cells treated with DNA-damaging agents, PKC is cleaved to a 40 kDa catalytically active fragment by caspase-3 (88)
. Overexpression of this PKC catalytic fragment induced chromatin condensation and DNA fragmentation, indicating a role for PKC cleavage in the induction of apoptosis (89)
. The tumor suppressor p73ß is regulated by the PKC catalytic fragment generated in response to DNA damage (90)
. PKC also apparently is required for basal transcription levels of the p53 tumor suppressor gene (86)
. PKC interacts with c-Abl in response to genotoxic stress and is phosphorylated on tyrosine (50)
, which is critical for caspase-dependent cleavage in the apoptotic response to DNA damage (65)
. In addition to the stress response to factors that damage DNA and promote apoptosis, PKC was recently shown to be critical for the response to mechanical stress in smooth muscle cells (91)
. These studies have clearly implicated PKC in cellular stress responses and show that in some cases PKC is required for apoptosis. Thus, cell survival can depend on the regulation of PKC.
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DOES PKC PLAY A ROLE IN HUMAN CANCER?
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A role for PKC in human cancer has not been clearly established, but it is of interest that the human PKC gene is located on chromosome 3p (92)
in a region where there is considerable loss of heterozygosity (LOH) in a wide range of tumors (93)
. This LOH has led to speculation that several tumor suppressor genes may be present on chromosome 3p at the loci between 3p12 and 3p21 (93
, 94)
. In breast cancer, especially, LOH has been reported at 3p25, 3p22-24, 3p21.3, 3p21.2-21.3, 3p14.2, 3p14.3, and 3p12 (94)
. PKC is located at 3p14 (92)
; thus, it is not unreasonable that down-regulation of PKC could play a role in breast cancer progression. Down-regulation of PKC cooperates with overexpression of tyrosine kinase to transform cells and overexpression of tyrosine kinases is observed more often in breast cancer than any other tumor type (52)
. Suppressing PKC activity in breast epithelial cells that have acquired a mutation that elevates expression of a tyrosine kinase could contribute to tumor progression.
As described, PKC may also contribute to the proliferation in renal cell carcinoma and breast cancer. The tumor suppressor VHL interacts directly with PKC in renal cell carcinoma cells and inhibits association between PKC and the IGF-I receptor for downstream signaling (28
, 29)
. Expression of PKC in breast cancer cell lines correlated with their metastatic potential, and elevated expression of PKC led to increased anchorage-independent growth of poorly metastatic cells (34
, 35)
. PKC was also reported to promote cell survival and chemotherapeutic resistance in non-small cell lung cancer cells (95)
. Elevated expression of PKC was also reported in multiple myeloma cells, and its down-regulation caused apoptosis (96)
. PKC has been shown to interact with MUC1 (97)
, and the stimulatory effects of MUC1 on anchorage-independent growth were abrogated by mutation of the PKC phosphorylation site (97)
. Since MUC1 is elevated in a variety of cancers, a requirement for PKC to stimulate anchorage-independent growth provides evidence that PKC may stimulate proliferation in cancers driven by MUC1. Thus, there is evidence that PKC may play both positive and negative roles in the control of neoplastic proliferation and survival.
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CONCLUSIONS
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There are sufficient data to indicate that PKC is capable of influencing cell proliferation both positively and negatively. PKC has also been reported to both positively and negatively influence apoptosis (6)
. So, can the apparent conflicting roles of PKC on cell proliferation and survival be reconciled? Surprisingly, results with PKC null mice revealed that mice lacking PKC developed normally and were fertile (7
, 8)
. The PKC null mice, however, had some hyperproliferative phenotypes that could correlate with the role that PKC plays in suppression of cell proliferation and apoptosis (6)
. Nevertheless, these studies demonstrated that PKC is not required for normal cell proliferation during development. Why, then, is PKC so widely expressed in so many tissues, and why does PKC turn up so often as a critical regulator of proliferative responses. We would propose that PKC is a critical stress response gene that can provide backup support for both proliferative and nonproliferative crises. PKC has the properties of a tumor suppressor that prevents inappropriate cell proliferation under some stressful conditions. Consistent with a tumor suppressor function for PKC, mice strains with knockouts of other tumor suppressor genes have phenotypes similar to the PKC null mice in that they develop normally, but develop proliferative disorders (9)
. But what of the pro-proliferative effects of PKC that have been reported in many transformed and cancer cells? While PKC-dependent proliferation in normal cells has not been reported, the IGF-I receptor can apparently stimulate PKC-dependent proliferation in transformed and cancer cells. It is possible that the ability of PKC to activate the critical MAPK pathway by several different mechanisms (Fig. 1
) could be used as a backup for defects in needed cell proliferation. We would propose that PKC is a multitasking kinase that can be co-opted into variety of cellular functions that largely protect the cell from cancer, but also from a lack of needed proliferation. That such a protein, capable of both anti- and pro-proliferative signals, is also involved in apoptosis makes sense. If there is an inappropriate co-opting of PKC leading to either neoplastic or degenerative disease, PKC can be cleaved by caspase 3 to a catalytic fragment capable of stimulating apoptosis (88
, 89)
. A model for PKC as a stress response protein that can function as a backup for tumor suppressor and proliferation functions is presented in Fig. 3
. Although this model provides hypotheses for the apparently varied roles for PKC, clearly much needs to be learned about how PKC participates in the varied and sometimes contradictory effects seen with this enigmatic protein.
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ACKNOWLEDGMENTS
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We would like to acknowledge the many individuals from the laboratory who have contributed to our efforts on the role of PKC in cell proliferation. We would also like to thank Lu-Hai Wang for comments on the manuscript. We acknowledge the support to the laboratory from the National Cancer Institute, and institutional support from the Research Centers in Minority Institutions (RCMI) program of the National Institutes of Health.
Received for publication October 3, 2003.
Accepted for publication December 10, 2003.
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- Foncea, R., Galvez, A., Perez,
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ABSTRACT
INTRODUCTION
