Role of endosomes in simian virus 40 entry and infection

After binding to its cell surface receptor ganglioside GM1, simian virus 40 (SV40) is endocytosed by lipid raft-mediated endocytosis and slowly transported to the endoplasmic reticulum, where partial uncoating occurs. We analyzed the intracellular pathway taken by the virus in HeLa and CV-1 cells by using a targeted small interfering RNA (siRNA) silencing screen, electron microscopy, and live-cell imaging as well as by testing a variety of cellular inhibitors and other perturbants. We found that the virus entered early endosomes, late endosomes, and probably endolysosomes before reaching the endoplasmic reticulum and that this pathway was part of the infectious route. The virus was especially sensitive to a variety of perturbations that inhibited endosome acidification and maturation. Contrary to our previous models, which postulated the passage of the virus through caveolin-rich organelles that we called caveosomes, we conclude that SV40 depends on the classical endocytic pathway for infectious entry.     

Uncoating of influenza virus in endosomes

The intracellular uncoating site of influenza virus was studied by measuring the fluorescence intensity of probes conjugated to the virus or the isolated hemagglutinin and also by assaying virus replication under various incubation conditions. Acidification of the viral environment was monitored by the decrease in the fluorescence intensity of fluorescein isothiocyanate, and transport of the virus particles into secondary lysosomes was assayed by the increase in the fluorescence intensity of fluorescein isothiocyanate diphosphate. The intracellular pH was estimated by the ratio of fluorescence intensities excited at two different wavelengths. It was found that the viral environment became acidified to a pH value of 5.1 to 5.2 within 10 min at 37 degrees C or 1 h at 20 degrees C after endocytosis. Addition of ammonium chloride to the medium rapidly raised the pH to 6.7. Transport of the virus particles into the secondary lysosomes was slower and negligibly low during those incubation periods. Virus replication occurred when the cells were incubated for 10 min at 37 degrees C or for 1 h at 20 degrees C, followed by incubation in the presence of ammonium chloride for a total of 12 h. These results indicate the uncoating of influenza virus in endosomes before reaching the secondary lysosomes.     

Role for influenza virus envelope cholesterol in virus entry and infection

Enveloped viruses are highly dependent on their lipid envelopes for entry into and infection of host cells. Here, we have examined the role of cholesterol in the virus envelope, using methyl-beta-cyclodextrin depletion. Pretreatment of virions with methyl-beta-cyclodextrin efficiently depleted envelope cholesterol from influenza virus and significantly reduced virus infectivity in a dose-dependent manner. A nonenveloped virus, simian virus 40, was not affected by methyl-beta-cyclodextrin treatment. In the case of influenza virus, infectivity could be partially rescued by the addition of exogenous cholesterol. Influenza virus morphology, binding, and internalization were not affected by methyl-beta-cyclodextrin depletion, whereas envelope cholesterol depletion markedly affected influenza virus fusion, as measured by a specific reduction in the infectivity of viruses induced to fuse at the cell surface and by fluorescence-dequenching assays. These data suggest that envelope cholesterol is a critical factor in the fusion process of influenza virus.     

Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization

The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1)/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.     

Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles

Hepatitis C virus (HCV) is a major human pathogen associated with life-threatening liver disease. Entry into hepatocytes requires CD81 and a putative second receptor. In this study, we elucidated the postreceptor attachment stages of HCV entry using HCV pseudoparticles (HCVpp) as a model system. By means of dominant-negative mutants and short interfering RNAs of various cellular proteins, we showed that HCVpp enter via clathrin-coated vesicles and require delivery to early but not to late endosomes. However, the kinetics of HCV envelope glycoprotein-mediated fusion are delayed compared to those of other viruses that enter in early endosomes. Entry of HCVpp can be efficiently blocked by bafilomycin A1, which neutralizes the pH in early endosomes and impairs progression of endocytosis beyond this stage. However, low-pH exposure of bafilomycin A1-treated target cells does not induce entry of HCVpp at the plasma membrane or in the early stages of endocytosis. These observations indicate that, subsequent to internalization, HCVpp entry necessitates additional, low-pH-dependent interactions, modifications, or trafficking, and that these events are irreversibly disrupted by bafilomycin A1 treatment.     

Human biliverdin reductase, a previously unknown activator of protein kinase C betaII

Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) betaII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109-7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC betaII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC betaII, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC betaII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC betaII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC betaII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC betaII nor PKC zeta activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR.PKC betaII immunocomplex. The potential biological relevance of the hBVR activation of PKC betaII was suggested by the finding that in cells transfected with the PKC betaII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC betaII, as well as in untransfected cells, upon treatment with phorbol myristate acetate, the PKC translocated to the plasma membrane and co-localized with hBVR. hBVR activation of PKC betaII underscores its potential function in propagation of signals relayed through PKCs.     

Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.     

Palmitoylcarnitine modulates interaction between protein kinase C betaII and its receptor RACK1

Palmitoylcarnitine, known to promote differentiation of neuroblastoma NB-2a cells as well as to inhibit protein kinase C (PKC) activity and to decrease phorbol ester binding, was shown previously to diminish the amount of complex formed between PKCdelta and its substrate GAP-43. In the present work we studied the effect of palmitoylcarnitine on the interaction between PKCbetaII and its receptor RACK1. Palmitoylcarnitine was found to decrease autophosphorylation of PKCbetaII on serine in a concentration-dependent manner and to decrease the amount of PKCbetaII/RACK1 complex. The effect of palmitoylcarnitine on cellular localization was found to be dependent on the presence of ATP; palmitoylcarnitine lowered the amount of PKCbetaII in cytosol and decreased the amount of PKCbetaII-RACK1 complex in membrane in the absence of ATP. Palmitoylcarnitine also reversed the effect of phorbol ester on the increase in the amount of PKCbetaII in membrane. Palmitoylcarnitine binds to PKCbetaII through hydrophobic interactions, although acylation of PKCbetaII by the palmitate moiety has been excluded. The presence of palmitoylcarnitine did not have any additive effect on the diminution of PKCbetaII-RACK1 complex formation in the presence of a RACK1-binding peptide from within the C2 region of PKCbetaII. These results rather exclude a possibility of interaction of palmitoylcarnitine with the C2 domain and suggest a possible interaction with the V5 domain and a conformational change affecting the C1 region.     

Carboxyl-terminal phosphorylation regulates the function and subcellular localization of protein kinase C betaII

Protein kinase C is processed by three phosphorylation events before it is competent to respond to second messengers. Specifically, the enzyme is first phosphorylated at the activation loop by another kinase, followed by two ordered autophosphorylations at the carboxyl terminus (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This study examines the role of negative charge at the first conserved carboxyl-terminal phosphorylation position, Thr-641, in regulating the function and subcellular localization of protein kinase C betaII. Mutation of this residue to Ala results in compensating phosphorylations at adjacent sites, so that a triple Ala mutant was required to address the function of phosphate at Thr-641. Biochemical and immunolocalization analyses of phosphorylation site mutants reveal that negative charge at this position is required for the following: 1) to process catalytically competent protein kinase C; 2) to allow autophosphorylation of Ser-660; 3) for cytosolic localization of protein kinase C; and 4) to permit phorbol ester-dependent membrane translocation. Thus, phosphorylation of Thr-641 in protein kinase C betaII is essential for both the catalytic function and correct subcellular localization of protein kinase C. The conservation of this residue in every protein kinase C isozyme, as well as other members of the kinase superfamily such as protein kinase A, suggests that carboxyl-terminal phosphorylation serves as a key molecular switch for defining kinase function.     

Isoenzyme-specific translocation of protein kinase C (PKC)betaII and not PKCbetaI to a juxtanuclear subset of recycling endosomes: involvement of phospholipase D

Elucidation of isoenzyme-specific functions of individual protein kinase C (PKC) isoenzymes has emerged as an important goal in the study of this family of kinases, but this task has been complicated by modest substrate specificity and high homology among the individual members of each PKC subfamily. The classical PKCbetaI and PKCbetaII isoenzymes provide a unique opportunity because they are the alternatively spliced products of the beta gene and are 100% identical except for the last 50 of 52 amino acids. In this study, it is shown that green fluorescent protein-tagged PKCbetaII and not PKCbetaI translocates to a recently described juxtanuclear site of localization for PKCalpha and PKCbetaII isoenzymes that arises with sustained stimulation of PKC. Mechanistically, translocation of PKCbetaII to the juxtanuclear region required kinase activity. PKCbetaII, but not PKCbetaI, was found to activate phospholipase D within this time frame. Inhibitors of phospholipase D (1-butanol and a dominant negative construct) prevented the translocation of PKCbetaII to the juxtanuclear region but not to the plasma membrane, thus demonstrating a role for phospholipase D in the juxtanuclear translocation of PKCbetaII. Taken together, these results define specific biochemical and cellular actions of PKCbetaII when compared with PKCbetaI.     

Role of the CM2 protein in the influenza C virus replication cycle

CM2 is the second membrane protein of influenza C virus. Although its biochemical characteristics, coding strategy, and properties as an ion channel have been extensively studied, the role(s) of CM2 in the virus replication cycle remains to be clarified. In order to elucidate this role, in the present study we generated CM2-deficient influenza C virus-like particles (VLPs) and examined the VLP-producing 293T cells, VLPs, and VLP-infected HMV-II cells. Quantification of viral RNA (vRNA) in the VLPs by real-time PCR revealed that the CM2-deficient VLPs contain approximately one-third of the vRNA found in wild-type VLPs although no significant differences were detected in the expression levels of viral components in VLP-producing cells or in the number and morphology of the generated VLPs. This finding suggests that CM2 is involved in the genome packaging process into VLPs. Furthermore, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced reporter gene expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner. Taken together, the data obtained in the present study indicate that CM2 has a potential role in the genome packaging and uncoating processes of the virus replication cycle.     

Critical role of protein kinase C betaII in activation of mast cells by monomeric IgE

Accumulating evidence suggests that IgE-mediated activation of mast cells occurs even in the absence of antigen, which is referred to as "monomeric IgE" responses. Although monomeric IgE was found to induce a wide variety of responses, such as up-regulation of the FcepsilonRI, survival, cytokine production, histamine synthesis, and adhesion to fibronectin, it remains to be clarified how mast cells are activated in the absence of antigen. It has been controversial whether monomeric IgE responses are mediated by a similar signaling mechanism to antigen stimulation, although recent studies suggest that IgE can induce the FcepsilonRI aggregation even in the absence of antigen. In this study, we focused on the role of conventional protein kinase C (cPKC), since this response is suppressed by a specific inhibitor for cPKC. Monomeric IgE-induced Ca(2+) influx was not observed in a mouse mastocytoma cell line, which lacks the expression of PKCbetaII, although Ca(2+) influx induced by cross-linking of the FcepsilonRI was intact. Transfection of PKCbetaII cDNA was found to restore the Ca(2+) influx induced by monomeric IgE in this cell line. Furthermore, the dominant negative form of PKCbetaII (PKCbetaII/T500V) significantly suppressed the Ca(2+) influx, histamine synthesis, and interleukin-6 production in another mouse mast cell line, which is highly sensitive to monomeric IgE. Expression of PKCbetaII/T500V was found not to affect the antigen-induced responses. These results suggest that PKCbetaII plays a critical role in monomeric IgE responses, but not in antigen responses.     

TGF-beta induced hyaluronan synthesis in orbital fibroblasts involves protein kinase C betaII activation in vitro

Graves' ophthalmopathy is accompanied by hyaluronan (HA) accumulation in the orbital space and infiltration of immunocompetent cells and cytokines, including IFN-gamma, IL-1beta, and TGF-beta. We examined the signal transduction pathways by which TGF-beta induces HA synthesis in normal orbital fibroblasts, orbital fibroblasts from patients with Graves' ophthalmopathy, and abdominal fibroblasts. Calphostin C inhibited the stimulation of HA synthesis by TGF-beta. Phorbol 12-myristate 13-acetate (PMA) activation of PKC stimulated HA production. The effects of TGF-beta and PMA were not synergistic. Stimulation by TGF-beta and PMA were dependent on protein synthesis and their effects were inhibited by cycloheximide. Since TGF-beta-induced HA synthesis was inhibited by BAPTA or by PKC inhibitors, a calcium-dependent PKC was most likely involved. The PKA inhibitor H-89 enhanced TGF-beta- and PMA-induced HA synthesis, thus showing that communication between the PKA and PKC pathways was evident. TGF-beta stimulated the translocation of PKCbetaII to the cell membrane. PKCbetaII, a key enzyme in the regulation of HA synthesis by TGF-beta, might be an appropriate target for therapeutic compounds to be used to treat Graves' ophthalmopathy accompanied by inflammation.     

Mechanism of inhibition of sequestration of protein kinase C alpha/betaII by ceramide. Roles of ceramide-activated protein phosphatases and phosphorylation/dephosphorylation of protein kinase C alpha/betaII on threonine 638/641

Sustained activation of protein kinase C (PKC) isoenzymes alpha and betaII leads to their translocation to a perinuclear region and to the formation of the pericentrion, a PKC-dependent subset of recycling endosomes. In MCF-7 human breast cancer cells, the action of the PKC activator 4beta-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKCalpha/betaII translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKCalpha/betaII. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKCalpha/betaII at carboxyl-terminal sites (Thr-638/641 and Ser-657/660), whereas in MCF-7 cells PKCalpha/betaII were phosphorylated at Ser-657/660 but not Thr-638/641. Inhibition of ceramide synthesis by fumonisin B1 overcame the defect in PKC phosphorylation and restored translocation of PKCalpha/betaII to the pericentrion. To determine the involvement of ceramide-activated protein phosphatases in PKC regulation, we employed small interference RNA to silence individual Ser/Thr protein phosphatases. Knockdown of isoforms alpha or beta of the catalytic subunits of protein phosphatase 1 not only increased phosphorylation of PKCalpha/betaII at Thr-638/641 but also restored PKCbetaII translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKCbetaII abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKCalpha/betaII at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKCbetaII overcame the inhibitory effects of ceramide on PKC translocation in PMA-stimulated MCF-7 cells. Therefore, the results demonstrate a novel role for carboxyl-terminal phosphorylation of PKCalpha/betaII in the translocation of PKC to the pericentrion, and they disclose specific regulation of PKC autophosphorylation by ceramide through the activation of specific isoforms of protein phosphatase 1.     

Role of protein kinase C in transmembrane signaling

Many extracellular signals elicit Ca2+ mobilization and diacylglycerol formation in their target cells. Diacylglycerol is derived from the receptor-linked phosphoinositide turnover and serves as a second messenger for the activation of protein kinase C in the presence of Ca2+ and phosphatidylserine. Unique diacylglycerols such as 1-oleoyl-2-acetyl-glycerol, which activate intracellular protein kinase C when added to intact cells, have been synthesized. Tumor-promoting phorbol esters substitute for such diacylglycerols and directly activate protein kinase C in both intact cell and cell-free systems. Under appropriate conditions, the synthetic diacylglycerols and phorbol esters induce protein kinase C activation without Ca2+ mobilization, whereas Ca2+ ionophore A23187 induces Ca2+ mobilization without protein kinase C activation. Using these substances, we have obtained evidence that both protein C and Ca2+ are involved in and play a synergistic role in exocytosis, cell division, and other cellular functions. In this article, the role of protein kinase C in transmembrane signaling is discussed.     

Role of protein kinase C in cellular regulation

Protein kinase C (PKC) consists of a family of closely related enzymes ubiquitously present in animal tissues. These enzymes respond to second messengers, Ca2+, diacylglycerol and arachidonic acid, to express their activities at membrane locations. Numerous hormones, neurotransmitters, growth factors and antigens are believed to transmit their signals by activation of a variety of phospholipases to generate these messengers. The various PKC isozymes, which exhibit distinct biochemical characteristics and unique cellular and subcellular localizations, may be differentially stimulated depending on the duration and strength of these messengers. Activation of PKC has been linked to the regulation of cell surface receptors, ion channels, secretion, gene expression, and neuronal plasticity and toxicity. The mechanisms of action of PKC in the regulation of these cellular functions are not entirely clear. Further study to identify the target substrates relevant to the various cellular functions is essential to define the functional diversity of this enzyme family.     

Role of lipid metabolism in hepatitis C virus assembly and entry

HCV (hepatitis C virus) represents a major global health problem. A consistent body of evidence has been accumulating, suggesting a peculiar overlap between the HCV life cycle and lipid metabolism. This association becomes evident both for the clinical symptoms of HCV infection and the molecular mechanisms underlying the morphogenesis and entry process of this virus. The HCV core–lipid droplets association seems to be central to the HCV morphogenesis process. Moreover, the biogenesis pathway of very‐low‐density lipoproteins has been shown to be involved in HCV morphogenesis with MTP (microsomal triacylglycerol transfer protein), ApoB (apolipoprotein B) and ApoE (apolipoprotein E) as essential elements in the production of infectious HCV particles. HCV infectivity also correlates with the lipidation status of the particles. Furthermore, some HCV cellular receptors and the regulation of the entry process are also connected to lipoproteins and lipid metabolism. Specifically, lipoproteins modulate the entry process and the cholesterol transporter SR‐BI (scavenger receptor class B type I) is a cellular entry factor for HCV. The present review aims to summarize the advances in our understanding of the HCV–lipid metabolism association, which may open new therapeutic avenues.     

Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis.

Protein kinase C betaII (PKC betaII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC betaII function in vivo, we generated transgenic mice that overexpress PKC betaII in the intestinal epithelium. Transgenic PKC betaII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC betaII mice exhibit elevated colonic beta-catenin levels and decreased glycogen synthase kinase 3beta activity, indicating that PKC betaII stimulates the Wnt/adenomatous polyposis coli (APC)/beta-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC betaII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/beta-catenin signaling pathway.