KSR-1 Binds to G-protein βγ Subunits and Inhibits βγ-induced Mitogen-activated Protein Kinase Activation

doi:10.1074/jbc.274.12.7982

KSR-1 Binds to G-protein βγ Subunits and Inhibits βγ-induced Mitogen-activated Protein Kinase Activation*

From the ^‡Center for Cardiovascular Research, Department of Medicine, ^§Department of Cell Biology and Physiology, and ^¶Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110

Abstract

The protein kinase KSR-1 is a recently identified participant in the Ras signaling pathway. The subcellular localization of KSR-1 is variable. In serum-deprived cultured cells, KSR-1 is primarily found in the cytoplasm; in serum-stimulated cells, a significant portion of KSR-1 is found at the plasma membrane. To identify the mechanism that mediates KSR-1 translocation, we performed a yeast two-hybrid screen. Three clones that interacted with KSR-1 were found to encode the full-length γ₁₀ subunit of heterotrimeric G-proteins. KSR-1 also interacted with γ₂and γ₃ in a two-hybrid assay. Deletion analysis demonstrated that the isolated CA3 domain of KSR-1, which contains a cysteine-rich zinc finger-like domain, interacted with γ subunits. Coimmunoprecipitation experiments demonstrated that KSR-1 bound to β₁γ₃ subunits when all three were transfected into cultured cells. Lysophosphatidic acid treatment of cells induced KSR-1 translocation to the plasma membrane from the cytoplasm that was blocked by administration of pertussis toxin but not by dominant-negative Ras. Finally, transfection of wild-type KSR-1 inhibited β₁γ₃-induced mitogen-activated protein kinase activation in cultured cells. These results demonstrate that KSR-1 translocation to the plasma membrane is mediated, at least in part, by an interaction with βγ and that this interaction may modulate mitogen-activated protein kinase signaling.

Footnotes

↵* 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.
↵‖ Supported by grants from the Barnes-Jewish Hospital Foundation, the National Institutes of Health, and the American Heart Association. To whom correspondence should be addressed: Center for Cardiovascular Research, Box 8086, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-747-3525; Fax: 314-362-0186; E-mail: amuslin{at}imgate.wustl.edu.
↵2 Heming Xing and Anthony J. Muslin, unpublished observations.
Abbreviations:

KSR-1

kinase suppressor of Ras

mKSR-1

murine KSR-1

FCS

fetal calf serum

LPA

lysophosphatidic acid

PTX

pertussis toxin

PAGE

polyacrylamide gel electrophoresis

GST

glutathione S-transferase

X-gal

5-bromo-4-chloro-3-indolyl β-d-galactopyranoside

ERK

extracellular signal-regulated kinase

MAP

mitogen-activated protein

MEK

MAP kinase or extracellular signal-regulated kinase kinase
- Received October 20, 1998.
- Revision received December 23, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.