Regulation of GRP1-catalyzed ADP Ribosylation Factor Guanine Nucleotide Exchange by Phosphatidylinositol 3,4,5-Trisphosphate*
- Jes K. Klarlund,
- Lucia E. Rameh‡,
- Lewis C. Cantley‡,
- Joanne M. Buxton,
- John J. Holik,
- Christoper Sakelis§,
- Varsha Patki§,
- Silvia Corvera§ and
- Michael P. Czech¶
- From the Program in Molecular Medicine and Department of Biochemistry and Molecular Biology and §Department of Cell Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605 and the ‡Department of Medicine, Division of Signal Transduction, Beth Israel Deaconess Medical Center and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02215
Abstract
Cellular levels of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are rapidly elevated in response to activation of growth factor receptor tyrosine kinases. This polyphosphoinositide binds the pleckstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (Klarlund, J., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997)Science 275, 1927–1930). Here we show that dioctanoyl PtdIns(3,4,5)P3 binds the PH domain of GRP1 with aK d = 0.5 μm, an affinity 2 orders of magnitude greater than dioctanoyl-PtdIns(4,5)P2. Further, the Sec7 domain of GRP1 is found to catalyze guanine nucleotide exchange of ARF1 and -5 but not ARF6. Importantly, PtdIns(3,4,5)P3, but not PtdIns(4,5)P2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphatidylcholine micelles, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and a low concentration of sodium cholate. PtdIns(3,4,5)P3-mediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 μm inositol 1,3,4,5-tetrakisphosphate, which also binds the PH domain of GRP1. Taken together, these data are consistent with the hypothesis that selective recruitment of GRP1 to PtdIns(3,4,5)P3 in membranes activates ARF1 and -5, known regulators of intracellular membrane trafficking.
Footnotes
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↵* This work was supported by Grants KD30898 (to M. P. C.), DK30648 (to M. P. C.), and DK40330 (to S. C.) from the National Institutes of Health.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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↵¶ To whom correspondence should be addressed. Tel.: 508-856-2254; Fax: 508-856-1617; E-mail:Michael.Czech{at}banyan.ummed.edu.
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↵1 The abbreviations used are: PI 3-kinase, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol; PH, pleckstrin homology; ARF, ADP ribosylation factor; GST, glutathioneS-transferase; GTPγS, guanosine 5′-O-(thiotriphosphate); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIP3, phosphatidylinositol trisphosphate.
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↵2 J. K. Klarlund and M. P. Czech, unpublished observations.
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- Received September 15, 1997.
- Revision received November 25, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
