A Growth Inhibitory PKCα‐ERK Signaling Pathway In the Intestine/Colon

Abstract

Background Increasing evidence points to growth inhibitory and tumor suppressive roles of theprotein kinase C (PKC) family of serine/threonine kinases. The conventional PKC isozyme, PKCα, has anti-proliferative and antitumor effects in the epithelium of the intestine and colon. Activation of PKCα in intestinal cells induces a program of cell cycle withdrawal that involves downregulation of the pro-proliferative proteins cyclin D1 and Id1 and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. These growth inhibitory effects of PKCα are dependent on activation of the MEK-ERK pathway, a signaling axis with established pro-proliferative effects in normal intestinal cells and in colon cancer. The objective of this study is to define the novel PKCα-induced growth suppressive MEK-ERK signaling cascade identified in intestinal epithelial cells. Hypothesis We hypothesize that PKCα modulates the activity of unique signaling intermediates to activate growth inhibitory ERK signaling. We will establish the point at which PKCα intersects the RAS-RAF-MEK-ERK pathway to induce growth arrest, identify the factors that link PKCα to the ERK pathway, and define effects that determine antiproliferative vs proliferative ERK signaling in intestinal cells. Results We have determined that PKCα activates RAS, RAF, MEK, and ERK in intestinal epithelial cells. The requirement for each of these pathway components in PKCα-mediated modulation of cyclin D1, Id1, and p21Cip1 and cell cycle withdrawal was confirmed using genetic approaches and inhibition of RAS, RAF, MEK, and ERK activity with the pharmacological agents Salirasib, LY3009120, PD0325901, and SCH772984, respectively. PKCα activation was shown to induce dimerization/activation of all three members of the RAF family (ARAF, BRAF and CRAF) and combinatorial knockdown indicated redundancy between these proteins. siRNA-mediated knockdown excluded a role for the RAF-MEK-ERK scaffolding protein KSR1. Analysis of the roles of Ras guanine nucleotide exchange factors identified at least two growth inhibitory pathways downstream of PKCα. Knockdown of the RasGRP isoform, RasGRP3, but not RasGRP1 or 2, inhibited the ability of PKCα activation to upregulate p21Cip1. However, RasGRP3 knockdown had no effect on PKCα-induced downregulation of cyclin D1 or Id1. Consistent with these findings, knockdown of RasGRP3 only partially inhibited PKCα-induced growth arrest. Based on evidence that SOS1 phosphorylation status is altered by PKCα signaling, we are currently exploring the potential involvement of SOS1 in the growth-inhibitory effects of PKCα. Conclusion PKCα activates RAS upstream of RAF, MEK and ERK to induce cell cycle arrest in intestinal cells. Activation of growth inhibitory ERK signaling by PKCα involves at least two mechanisms, one of which is RasGRP3-dependent and leads to p21Cip1 induction. Ongoing studies using siRNA knockdown strategies are exploring the role of Ras-GEFs such as RasGRP3, SOS1 and SOS2 in mediating PKCα-induced activation of RAS, to identify distinctive effects related to growth inhibitory versus growth promoting ERK signaling.