Function and regulation of protein kinase C alpha in the intestinal epithelium
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The protein kinase C (PKC) family of serine/threonine kinases participates in regulation of cell proliferation and survival in a variety of systems. PKCα is a ubiquitously expressed member of the PKC family. In the intestinal epithelium, PKC/PKCα signaling is of key importance in maintenance of tissue homeostasis. Activation of PKCα in non-transformed IEC-18 intestinal crypt cells induces a program of molecular events leading to cell cycle withdrawal into G 0 . One of the earliest events in this program is down-regulation of the important mitogenic factor cyclin D1. Previous findings from this laboratory have demonstrated that PKCα signaling inhibits cap-dependent translation initiation of cyclin D1 protein by inducing hypophosphorylation/activation of the translational repressor 4E-BP1 (Hizli, Black et al. 2006). The activity of the protein translation machinery is often up-regulated during intestinal carcinogenesis, resulting in increased synthesis of growth-related proteins such as cyclin D1. Notably, the expression and activity of PKCα are markedly reduced or absent in a majority of intestinal tumors, suggesting a link between these events. Thus, the focus of this study was to elucidate the molecular pathways involved in PKCα-mediated translation inhibition in normal intestinal epithelial cells, and to explore the mechanisms underlying loss of PKCα expression during intestinal carcinogenesis. In the first part of this study, we characterized a novel phosphoinositide-3-kinase (PI3K)/Akt-independent, protein phosphatase 2A (PP2A)-dependent pathway as the mechanism responsible for PKCα-induced hypophosphorylation/activation of 4E-BP1 and translation inhibition in IEC-18 cells. PKCα signaling was found to promote dephosphorylation of Thr45 and Ser64 on 4E-BP1, residues directly involved in regulation of the translation-inhibitory activity of the molecule. The ability of H 2 O 2 and ceramide, two naturally occurring PKCα agonists that promote growth arrest in intestinal cells, to activate 4E-BP1 in these cells in a PKC/PKCα-dependent manner provided physiological significance to our findings using pharmacological agents. Consistent with the well-established role of the PI3K/Akt/mTOR signaling pathway in negative regulation of 4E-BP1, we demonstrated that PKC signaling transiently inhibits PI3K activity and Akt phosphorylation in IEC-18 cells. However, PKCα-induced activation of 4E-BP1 was not prevented by constitutively active mutants of PI3K or Akt, indicating that this PKCα-mediated response is independent of PI3K/Akt signaling. In keeping with the idea that inhibition of the PI3K/Akt/mTOR pathway is not the primary effector of 4E-BP1 activation, PKCα activation did not alter the activity of S6 kinase in intestinal cells. Instead, PKCα signaling was found to induce a ∼2-fold increase in PP2A activity, and inhibition of PP2A activity was found to abolish PKCα-induced 4E-BP1 hypophosphorylation and cyclin D1 down-regulation. Our studies, therefore, identify PP2A as a key mediator of PKCα-induced translational blockade and growth inhibition in intestinal epithelial cells. These findings may have important implications for understanding tumor development in the intestine, where a decrease in PKCα protein expression and activity suggests a role for the enzyme as an anti-oncogenic factor. Consistent with this role, PKCα knockout mice show increased intestinal tumor development (Oster and Leitges 2006), while exogenous expression of PKCα in colon adenocarcinoma cell lines promotes growth arrest and inhibits tumorigenicity (M. Pysz, J. Black, unpublished data). Loss of PKCα expression may provide a growth advantage to intestinal cells via disrupted control of the tumor suppressive activity of PP2A, increased translation and accumulation of growth-promoting molecules such as cyclin D1. In the second part of this study, several mechanisms were investigated and excluded as underlying loss of PKCα expression during intestinal carcinogenesis: (a) aberrant PKCα protein turnover or altered protein half-life; (b) epigenetic modifications, including hypermethylation and histone deacetylation, and silencing of the PKCα gene promoter; (c) disrupted TGFβ signaling; and (d) altered mRNA stability. Based on evidence for a direct correlation between levels of PKCα mRNA and protein in colon cancer cells (Leontieva O, Bateman N, and Black JD, unpublished data), future directions will involve the study of differential control of PKCα gene transcription by specific regions in the PKCα promoter. Studies of the underlying mechanisms responsible for loss of PKCα expression during intestinal carcinogenesis may lead to a better understanding of tumor development, while unveiling novel options for anti-cancer treatment.