Graduation Date

Fall 12-17-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Dr. Jennifer Black

MeSH Headings

PKCα, RasGRP3, H-Ras protein, Raf kinase, ERK, p21Cip1, cyclin D1, Id1, growth arrest, EGF

Abstract

Members of the protein kinase C (PKC) family of serine/threonine kinases are involved in regulation of fundamental cellular functions, including proliferation, differentiation, survival, migration, and transformation. Increasing evidence points to anti-proliferative and tumor suppressive role of PKCs. Our laboratory and others have reported that the classical PKC isozyme, PKCαnegatively regulates proliferation and tumorigenesis in the intestinal epithelium. Our laboratory has further determined that PKCα signaling induces a program of cell cycle withdrawal in intestinal epithelial cells that involves downregulation of the pro-proliferative proteins, cyclin D1 and Id1, and upregulation of the cyclin dependent kinase (CDK) inhibitor, p21Cip1. Unexpectedly, the growth inhibitory effects of PKCα are dependent on activation of the MEK-ERK pathway, which is also a well-established mediator of pro-proliferative signaling in normal intestinal cells and in colon cancer. The overall goal of this study was to define the mechanistic basis of this novel PKCα-induced growth suppressive ERK signaling cascade. Based on the tumor promoting effects of ERK signaling, ERK pathway inhibitors are being used in the clinic to manage a broad range of tumors including colon cancer, with intestinal toxicity being a major side effect. Thus, characterization of the growth/tumor suppressive PKCα→ERK signaling axis will (a) enhance our understanding of growth suppressive ERK signaling, (b) point to potential strategies to avoid and/or manage side-effects of ERK pathway inhibitors, and (c) open new avenues for therapeutic intervention in colon cancer.

This thesis explores (a) the involvement of canonical ERK pathway components, including Ras, Raf, MEK, and ERK, in PKCα growth inhibitory signaling, (b) point(s) of intersection between PKCα and the ERK pathway and the signaling intermediates that link PKCα activation to ERK, and (c) how PKCα engagement of the ERK pathway differs from that of a pro-proliferative signal such as epidermal growth factor (EGF).

These studies have revealed that PKCα activates each component of the canonical Ras–Raf–MEK–ERK cascade, as seen with pro-proliferative factors such as EGF, and that activation of these molecules is required for regulation of the downstream effector proteins, cyclin D1, Id1 and p21Cip1, and induction of cell cycle arrest. Thus, PKCα and EGF both intersect the ERK pathway at the level of Ras, a finding that clarifies longstanding confusion in the PKC field regarding the requirement for Ras in PKC-mediated ERK activation. However, PKCα and EGF diverge in the mechanism of Ras activation. While EGF is known to require the Ras guanine exchange factors (RasGEFs) SOS1/2 for Ras activation and increased proliferation, PKCα directly or indirectly phosphorylates and activates the RasGEF, RasGRP3, for Ras stimulation of growth-suppressive ERK-signaling. PKCα–RasGRP3 specifically activates H-Ras to induce ERK-dependent upregulation of the cell cycle inhibitory protein, p21Cip1, and K-Ras or N-Ras are not required for p21Cip1 induction. Thus, the data identify a novel growth inhibitory PKCα–RasGRP3–H-Ras–Raf–MEK–ERK–p21Cip1 pathway in intestinal epithelial cells. This pathway partially mediates PKCα-mediated growth arrest; however, complete cell cycle arrest requires additional regulatory events, such as downregulation of cyclin D1 and Id1, that can be mediated by K-Ras or N-Ras. Notably, the finding that effects of PKCα on cyclin D1 and Id1 are not dependent on the RasGRP3–H–Ras module indicates that PKCα activates at least two Ras–ERK-dependent signaling pathways to promote growth arrest in intestinal epithelial cells. This study also revealed that PKCα forms a complex with all three Raf proteins in unstimulated cells, suggesting a model in which PKCα activation serves to concentrate Raf at the plasma membrane for activation by Ras. The data further show that there is redundancy among Raf isoforms in mediating PKCα-induced growth arrest, while ruling out a requirement for the Raf-MEK-ERK scaffold proteins KSR1/2.

In conclusion, this study has identified a novel PKCα-induced growth suppressive MEK-ERK signaling cascade in normal intestinal epithelial cells that involves a RasGRP3–H-Ras–Raf–MEK–ERK–p21Cip1 signaling axis. However, p21Cip1 only partially contributes to PKCα-mediated G1→S arrest, and additional Ras–ERK signaling pathway(s) are clearly required for the effect. These findings set the stage for studies to identify additional regulatory events in this novel growth suppressive pathway.

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