Polyphosphoinositides Inhibit the Interaction of Vinculin with Actin Filaments*
- From the ‡Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and ¶Myriad Genetics, Salt Lake City, Utah 84108
Abstract
Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin’s head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P2) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P2 on the actin and talin-binding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P2.
Footnotes
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↵* This work was supported by research Grant GM41605 from the National Institutes of Health and by a grant from the Muscular Dystrophy Association.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. Present address: Dept. of Physiology and Biophysics, Case Western Reserve School of Medicine, Cleveland, OH 44106-4970. Tel.: 216-368-8947; Fax: 216-368-1693; E-mail: pas5{at}po.cwru.edu.
- Abbreviations:
- PI4,5P2
-
phosphatidylinositol 4,5-bisphosphate
- BSA
-
bovine serum albumin
- GST
-
glutathione S-transferase
- βME
-
β-mercaptoethanol
- PI
-
phosphatidylinositol
- PI3P
-
phosphatidylinositol 3-phosphate
- PI4P
-
phosphatidylinositol 4-phosphate
- PI3,4P2
-
phosphatidylinositol 3,4-bisphosphate
- PI3,4,5P3
-
phosphatidylinositol 3,4,5-triphosphate
- Sa
-
streptavidin
- SaAP
-
SA-conjugated alkaline phosphatase
- PAGE
-
polyacrylamide gel electrophoresis
- Vh
-
head domain of vinculin
- Vt
-
tail domain of vinculin
- PBS
-
phosphate-buffered saline
-
- Received February 23, 1999.
- Revision received April 7, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
