Abstract
Cofilin is a widely distributed actin-modulating protein that has the ability to bind along the side of F-actin and to depolymerize F-actin in a pH-dependent manner. We found that phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) inhibited both actions of cofilin in a dose-dependent manner, while inositol 1,4,5-triphosphate (IP3), 1-oleoyl-2-acetylglycerol (OAG), phosphatidylserine (PS), or phosphatidylcholine (PC) had little or no effect on them. Gel filtration analyses showed that PIP2 bound to cofilin and thereby inhibited the binding of cofilin to G-actin. Destrin is a mammalian, pH-independent actin-depolymerizing protein. The actin-depolymerizing activity of destrin was also inhibited by PI, PIP, and PIP2, but not by IP3, OAG, PS, or PC. In addition, we found further that an actin-depolymerizing activity of bovine pancreas deoxyribonuclease I, a G-actin-sequestering protein, was inhibited by PIP and PIP2, but not by PI, IP3, OAG, PS, or PC. These results together with previous findings (Lassing, I., and Lindberg, U. (1985) Nature 314, 472-474; Janmey, P. A., and Stossel, T. P. (1987) Nature 325, 362-364) suggest that the sensitivity to polyphosphoinositides may be a common feature in vitro among actin-binding proteins that can bind to G-actin and regulate the state of actin polymerization.
Highlights
From the Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan and the SDepartment of Cell Biology, the Tokyo Metropolitan Institute of Medical Science, Hon-Komagome, Cofilin is a widely distributed actin-modulating protein that has the ability to bind along the side of F
It has already been reported that polyphosphoinositides affect the functions of a G-actin-sequestering protein, profilin
We have found that besides these actin-regulatory proteins, an F-actinside-binding and -depolymerizing protein, cofilin, and actindepolymerizing/severing protein, destrin, and a G-actin sequestering protein, DNase I, are sensitive to polyphosphoinositides
Summary
Proteins-Actin was prepared from rabbit skeletal muscle by the method of Spudich and Watt [21] and further purified by Sephadex. Porcine brain cofilin was purified by the previously described method [22]. Molecular mass on SDS-polyacrylamide gel electrophoresis is a little larger than that of porcine brain cofilin. Destrin was purified from porcine brains by using DNase I affinity column chromatography as previously described [9, 10]. Assays for the Binding of Cofilin to F-actin and the Actin-depolymerizing Activities of Cofilin, Destrin, and DNase I-Recombinant cofilin, porcine brain cofilin, destrin, or DNase I was first incubated with lipids or IP3 for 10 min at 25 “C, the aliquot of polymerized actin was added, and the solution (final 100 ~1) was incubated at. Since the staining intensities of proteins were linear, in the range of 0.3-3 pg/lane, in our SDS-polyacrylamide gel electrophoretic experiments, we adjusted the sample volume of pellet or supernatant fractions electrophoresed in one lane within this range.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
