Regulation of Rnd3 localization and function by protein kinase Cα-mediated phosphorylation

Abstract

The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by posttranslational modifications such as prenylation and phosphorylation. We show here that, upon PKC agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift 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 to a nonphosphorylatable mutant of Rnd3. We further show that PKCα directly phosphorylates Rnd3 in an in vitro kinase assay. Additionally, we provide evidence that the phosphorylation status of Rnd3 has a direct effect on its ability to block signaling from the Rho-ROCK pathway. These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signaling to alter cytoskeletal organization.

The GTPase-deficient Rnd Proteins Are Stabilized by Their Effectors

“…2A), as assessed by Western blot analysis using an anti-Rnd3 Mab. Because Rnd3 is detected as a doublet (7,10,21,35), we sought clarify whether both bands correspond to Rnd3. Antibody reactivity toward overexpressed Rnd1, Rnd2, and Rnd3 suggests that this antibody is specific for Rnd3 (supplemental Fig.…”

“…Rnd3 exists largely in a GTP-bound state (1) and undergoes post-translational modifications such as phosphorylation (9,10), which might affect its localization. We show here that the levels of Rnd3 are strongly influenced by co-expression of either Syx or p190B RhoGAP.…”

“…For example, the oncogene B-Raf, genotoxic stress, and the mTOR pathway all promote increased Rnd3 expression (5)(6)(7)(8). Rnd3 phosphorylation by RhoA-associated kinase (ROCK1) 2 or protein kinase C (PKC␣) have been reported to be important in regulating its localization and function (9,10). Seven ROCK1 phosphorylation sites are identified in Rnd3, with Ser-7/Ser-11 reported to regulate Rnd3 stability (9).…”

Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors

“…Phosphorylation of serine 181 sufficiently weakens this interaction to cause K-Ras4B to dissociate from the plasma membrane. Since the initial report of this farnesyl-electrostatic switch (6), other small GTPases that associate with membranes via polybasic regions that operate in conjunction with prenylation, including RalA (22) and Rnd3 (23), have been shown to be substrates for kinases that modulate membrane association through a similar prenyl-electrostatic switch.…”

Rho GTPases: Regulation and roles in cancer cell biology

“…19 Rnd3 is known to be phosphorylated on 5 serines and 1 threonine by ROCK1 and on serine 210 by PKC. 19,41,117 Moreover, expression of Rnd3 is induced by activation of Raf, leading to changes in the actin cytoskeleton. 46 In mammals, RhoBTB1 and RhoBTB2 form the RhoBTB subfamily of Rho GTPases.…”