The Rnd proteins (Rnd1, Rnd2, Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Rnd3 decreases RhoA activity and regulates both cytoskeletal organization (to cause cell rounding, hence the name Rnd) and cell cycle progression. How Rnd3 itself, is regulated to cause these changes is still under investigation. One possible mechanism is spatiotemporal regulation of this constitutively activated GTPase by dynamically modulated post-translational modifications. In this work, I have shown that, upon protein kinase C (PKC) agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and becomes metabolically labeled with 32P, and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCα-null cells or with a nonphosphorylatable mutant of Rnd3, indicating that Rnd3 is a target for PKCα-mediated phosphorylation. I have provided evidence that integrin engagement regulates the downstream signaling functions of Rnd3 by inducing PKCα-mediated phosphorylation. These processes result in increased downstream signaling to Rho-ROCK pathway targets such as cofilin and myosin light chain. Thus, integrin engagement is a physiological regulator of Rnd3 posttranslational modification by PKCα, and in turn is a mediator of Rnd3 subcellular localization and downstream signaling. Further, I have demonstrated that both localization and function of Rnd3 require post-prenyl processing. Rnd3 localization to the plasma membrane is inhibited both in cells devoid of the post-prenyl processing enzymes Ras converting enzyme1 (Rce1) and isoprenylcysteine carboxymethyltransferase (Icmt), and also upon mutation of the Rnd3 C-terminal tetrapeptide CAAX motif to one insensitive to Rce1/Icmt. The function of Rnd3 is also inhibited in the absence of post-prenyl processing. Ectopic expression of Rnd3 causes a smaller loss of stress fibers and less cell rounding in Rce1-/- and in Icmt-/- fibroblasts than in matched WT cells, and cells expressing the misprocessed Rnd3 CAAX mutant display more stress fibers than cells expressing WT Rnd3. Together, these results add an additional mechanism of Rnd3 regulation to those documented previously, and clarify how Rnd3 modulates Rho signaling to alter cytoskeletal organization.
|Advisor:||Cox, Adrienne D.|
|Commitee:||Burridge, Keith, Der, Channing J., Schaller, Michael D., Sharpless, Norman E.|
|School:||The University of North Carolina at Chapel Hill|
|Department:||Genetics & Molecular Biology|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 69/04, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Protein kinase C, Rho GTPases, Rnd3|
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