Synergistic Activation of Dynamin GTPase by Grb2 and Phosphoinositides*
- Barbara Barylko,
- Derk Binns,
- Keng-Mean Lin‡,
- Mark A. L. Atkinson§,
- David M. Jameson¶‖,
- Helen L. Yin‡ and
- Joseph P. Albanesi**
- From the Department of Pharmacology and the‡Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, the§University of Texas Health Science Center, Tyler, Texas 75708, and the ¶Department of Biochemistry and Biophysics, University of Hawaii, Honolulu, Hawaii 96822
Abstract
Hydrolysis of GTP by dynamin is essential for budding clathrin-coated vesicles from the plasma membrane. Two distinct domains of dynamin are implicated in the interactions with dynamin GTPase activators. Microtubules and Grb2 bind to the carboxyl-terminal proline/arginine-rich domain (PRD), whereas phosphoinositides bind to the pleckstrin homology (PH) domain. In this study we tested the effect of different phosphoinositides on dynamin GTPase activity and found that the best activator is phosphatidylinositol 4,5-bisphosphate followed by 1-O-(1,2-di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-d-myo-inositol 3,4,5-triphosphate. Phosphatidylinositol 4-phosphate was a weak activator and phosphatidylinositol 3,4-bisphosphate did not activate GTPase at all. We then addressed the question of whether both domains of dynamin, PRD and PH, can be engaged simultaneously, and determined the effects of dual occupancy on dynamin GTPase activity. We found that Grb2 and phosphatidylinositol 4,5-bisphosphate together increased the dynamin GTPase activity up to 4-fold higher than that obtained by these activators tested separately, and also reduced the dynamin concentration required for half-maximal activities by 3-fold. These results indicate that both stimulators can bind to dynamin simultaneously resulting in superactivation of dynamin GTPase activity. We propose that SH3-containing proteins such as Grb2 bind to the dynamin PRD to target it to clathrin-coated pits and prime it for superactivation by phosphoinositides.
Footnotes
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↵* This work was supported in part by American Heart Association Grant-in-Aid 97G-111 (to B. B.), RO1 GM5112 and NS31430 (to H. L. Y.), and a Robert A. Welch Foundation grant (to H. L. Y.).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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↵‖ Supported by the University Research Council of the University of Hawaii.
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↵** To whom correspondence should be addressed: Dept. of Pharmacology, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041. Tel.: 214-648-2995; Fax: 214-648-2971.
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↵1 The abbreviations used are: PRD, proline/arginine-rich domain; PH, pleckstrin homology domain; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4)P2, 1-O-(1,2-di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-d-myo-inositol 3,4-bisphosphate; PI(4)P, phosphatidylinositol 4-phosphate; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; PI(3,4,5)P3, 1-O-(1,2-di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-d-myo-inositol 3,4,5-triphosphate; GST, glutathioneS-transferase.
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- Received October 7, 1997.
- Revision received November 13, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
