Rho GTPase Control of Protein Kinase C-related Protein Kinase Activation by 3-Phosphoinositide-dependent Protein Kinase*
- From the ‡Imperial Cancer Research Fund, Protein Phosphorylation Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX and the ¶Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom
Abstract
The protein kinase C-related protein kinases (PRKs) have been shown to be under the control of the Rho GTPases and influenced by autophosphorylation. In analyzing the relationship between these inputs, it is shown that activation in vitroand in vivo involves the activation loop phosphorylation of PRK1/2 by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Rho overexpression in cultured cells is shown to increase the activation loop phosphorylation of endogenous PRKs and is demonstrated to influence this process by controlling the ability of PRKs to bind to PDK1. The interaction of PRK1/2 with PDK1 is shown to be dependent upon Rho. Direct demonstration of ternary (Rho·PRK·PDK1) complex formation in situ is provided by the observation that PDK1 is recruited to RhoB-containing endosomes only if PRK is coexpressed. Furthermore, this in vivo complex is maintained after phosphoinositide 3-kinase inhibition. The control of PRKs by PDK1 thus evidences a novel strategy of substrate-directed control involving GTPases.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Present address: University of California Cancer Research Inst., 2340 Sutter St., San Francisco, CA 94115.
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↵‖ To whom correspondence should be addressed.
- Abbreviations:
- PRKs
-
protein kinase C-related protein kinases
- PKC
-
protein kinase C
- GTPγS
-
guanosine 5′-O-(3-thiotriphosphate)
- PDK
-
3-phosphoinositide-dependent protein kinase
- PKB
-
protein kinase B
- PtdIns
-
phosphatidylinositol
- PH
-
pleckstrin homology
- PI3K
-
phosphoinositide 3-kinase
- GST
-
glutathione S-transferase
- DTT
-
dithiothreitol
- MBP
-
myelin basic protein
-
- Received June 24, 1999.
- Revision received December 9, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
