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Role of Phosphatidic Acid in the Coupling of the ERK Cascade

J. Biol. Chem., 26 December 2008
Vol. 283, Issue 52, p. 36636-36645
DOI: 10.1074/jbc.M804633200

Role of Phosphatidic Acid in the Coupling of the ERK Cascade

  1. Catherine A. Kraft1,
  2. José Luis Garrido1,
  3. Eric Fluharty,
  4. Luis Leiva-Vega and
  5. Guillermo Romero2
  1. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
  1. 2 To whom correspondence should be addressed. Tel.: 412-648-9408; E-mail: ggr{at}pitt.edu.

Abstract

The production of phosphatidic acid plays a crucial role in the activation of the ERK cascade. This role was linked to the binding of phosphatidate to a specific polybasic site within the kinase domain of Raf-1. Here we show that phosphatidate promotes ERK phosphorylation in intact cells but does not activate Raf in vitro. The kinase suppressor of Ras (KSR) contains a sequence homologous to the phosphatidate binding site of Raf-1. Direct binding of phosphatidate to synthetic peptides derived from the sequences of the binding domains of Raf-1 and KSR was demonstrated by spectroscopic techniques. The specificity of these interactions was confirmed using synthetic lipids and mutated peptides in which the core of the phosphatidic acid binding domain was disrupted. Insulin and exogenous dioleoyl phosphatidate induced a rapid translocation of a mouse KSR1-EGFP construct to the plasma membrane of HIRcB cells. Mutation of two arginines located in the core of the putative phosphatidate binding site abolished dioleoyl phosphatidate- and insulin-induced translocation of KSR1. Overexpression of the mutant KSR1 in HIRcB cells inhibited insulin-dependent MEK and ERK phosphorylation. The addition of dioleoyl phosphatidate or insulin increased the co-localization of KSR1 and H-Ras and promoted the formation of plasma membrane patches enriched in both proteins and phosphatidic acid. These results, in conjunction with our previous work, suggest the formation of phosphatidate-enriched membrane microdomains that contain all components of the ERK cascade. We propose that these domains act as molecular scaffolds in the coupling of signaling events.

Footnotes

  • * This work was supported, in whole or in part, by National Institutes of Health Grants R01-DK-54782 and T32-GM-54813. This work was also supported by an internal grant from the Office of the Senior Vice Chancellor for the Health Sciences of the University of Pittsburgh. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Embedded Image The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Movie S1.

  • 1 Both of these authors contributed equally to this work.

  • Received June 17, 2008.
  • Revision received October 22, 2008.

Citation:

C. A. Kraft, J. L. Garrido, E. Fluharty, L. Leiva-Vega, and G. Romero, Role of Phosphatidic Acid in the Coupling of the ERK Cascade. J. Biol. Chem. 283, 36636-36645 (2008).

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