Serine 34 Phosphorylation of Rho Guanine Dissociation Inhibitor (RhoGDIα) Links Signaling from Conventional Protein Kinase C to RhoGTPase in Cell Adhesion

Athanassios Dovas,Youngsil Choi,Atsuko Yoneda,Hinke A.B Multhaupt,Seung-Hae Kwon,Dongmin Kang,Eok-Soo Oh,John R Couchman

doi:10.1074/jbc.m109.098129

Abstract

Conventional protein kinase C (PKC) isoforms are essential serine/threonine kinases regulating many signaling networks. At cell adhesion sites, PKCα can impact the actin cytoskeleton through its influence on RhoGTPases, but the intermediate steps are not well known. One important regulator of RhoGTPase function is the multifunctional guanine nucleotide dissociation inhibitor RhoGDIα that sequesters several related RhoGTPases in an inactive form, but it may also target them through interactions with actin-associated proteins. Here, it is demonstrated that conventional PKC phosphorylates RhoGDIα on serine 34, resulting in a specific decrease in affinity for RhoA but not Rac1 or Cdc42. The mechanism of RhoGDIα phosphorylation is distinct, requiring the kinase and phosphatidylinositol 4,5-bisphosphate, consistent with recent evidence that the inositide can activate, localize, and orient PKCα in membranes. Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with fluorescence resonance energy transfer microscopy sensing GTP-RhoA levels, the data reveal a common pathway in cell adhesion linking two essential mediators, conventional PKC and RhoA.

Highlights

Protein kinase C (PKC)6 isoforms have a wide array of functions in cell adhesion, cytoskeleton, trafficking, and polarity [1,2,3] to transcription, survival, and differentiation [4]
Conventional PKC Phosphorylates RhoGDI␣ in Vitro on Serine 34—The recombinant RhoGDI␣ fusion protein was first cleaved with recombinant tobacco etch virus [23] to remove the hexahistidine tag, because the two threonine residues in the tag linker region are PKC substrates
Recombinant RhoGDI␣ was phosphorylated by PKC in the presence of PtdIns[4,5]P2 but much less in the presence of the more commonly used phosphatidylserine/diacylglycerol/calcium (PtdSer/DL/Ca2ϩ)

Summary

Introduction

Protein kinase C (PKC)6 isoforms have a wide array of functions in cell adhesion, cytoskeleton, trafficking, and polarity [1,2,3] to transcription, survival, and differentiation [4]. Of several serine residue mutations within the N-terminal domain of full-length, wild-type RhoGDI␣, that of Ser-34 to Ala had the most marked effect, reducing phosphorylation by recombinant PKC␣ or PKC␣␤␥ by around 70%, relative to the wild-type protein (Fig. 1D). The data show that Ser-34 is a major site in RhoGDI␣ that can be phosphorylated by PKC␣ and, as shown below, regardless of whether the GDI is bound to GTPase or not.

Results

Conclusion