The Actin-binding Properties of the Physarum Actin-Fragmin Complex

doi:10.1074/jbc.270.6.2644

The Actin-binding Properties of the Physarum Actin-Fragmin Complex

REGULATION BY CALCIUM, PHOSPHOLIPIDS, AND PHOSPHORYLATION (*)

From the Department of Biochemistry, Laboratory for Physiological Chemistry, Universiteit Gent, B-9000 Gent, Belgium and
the (1)Division of Biochemistry, K.U. Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium

^§ Supported by a Concerted Research Action of the Flemish Community (GOA). To whom correspondence should be addressed:
Universiteit Gent, Laboratory for Physiological Chemistry, Ledeganckstraat 35, B-9000 Gent, Belgium.
Tel.: 32-9-264-52-89; Fax: 32-9-264-53-37.

Abstract

The actin-binding properties of the actin-fragmin complex from Physarum polycephalum microplasmodia were investigated with respect to regulation by Ca, phospholipids, and phosphorylation of the actin subunit by the endogenous actin-fragmin kinase. Fragmin possesses two high affinity actin-binding sites and probably also a third, low affinity site. Its nucleating and F-actin severing activities are inhibited by phosphatidylinositol 4,5-bisphosphate (PIP₂). Actin-fragmin specifically binds PIP₂ which competes with actin for the Ca-sensitive site. However, PIP₂ cannot dissociate the actin-fragmin complex nor the actin₂-fragmin trimer. Efficient F-actin nucleating activity by actin-fragmin is only observed with unphosphorylated actin-fragmin, in the absence of PIP₂ and at high Ca (>μM) concentrations. In the presence of PIP₂, actin-fragmin only caps actin filaments when unphosphorylated. The results suggest that in the cell, hydrolysis of PIP₂, concomitant with the increase of cytosolic Ca, could promote subcortical actin polymerization.

Footnotes

↵^¶ Supported by a Concerted Research Action of the Flemish Community (GOA).
↵** Senior research associate of the National Research Foundation (NFWO).
↵* This work was supported in part by grants from the National Research Foundation and the Concerted Research Action of the Flemish Community (to J. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¹ The abbreviations used are:

PIP₂

phosphatidylinositol 4,5-bisphosphate

A•F

actin-fragmin complex

A₂•F

actin₂-fragmin complex

A^P•F

phosphorylated actin-fragmin complex

F

fragmin

F-actin

filamentous actin

IP₃

inositol 1,4,5-trisphosphate

MOPS

3-(N-morpholino)propanesulfonic acid

PI

phosphatidylinositol

PIP

phosphatidylinositol 4-monophosphate

PC

phosphatidylcholine

PE

phosphatidylethanolamine

PS

phosphatidylserine

14T

N-terminal domain of villin.
↵² J. Gettemans, unpublished observations.
- Received August 10, 1994.
- Revision received November 18, 1994.