IN-VIVO PHOSPHORYLATION OF ACTIN IN PHYSARUM-POLYCEPHALUM - STUDY OF THE SUBSTRATE-SPECIFICITY OF THE ACTIN-FRAGMIN KINASE

Actin-fragmin is a heterodimeric protein complex from Physarum polycep halum microplasmodia that is phosphorylated in vitro at residues Thr20 3 and Thr202 of the actin subunit by the endogenous actin-fragmin kina se. Following phosphorylation, the F-actin capping activity of the com plex becomes Ca2+-dependent, suggesting a fundamental regulatory role in controlling F-actin growth [Gettemans, J., De Ville, Y., Waelkens E . and Vandekerckhove, J. (1995) J. Biol. Chem. 270, 2644-2651]. In thi s study we analysed actin phosphorylation in vivo. We demonstrate that the actin-fragmin complex constitutes the only substrate of the actin -fragmin kinase in plasmodia. Monomeric actin is not phosphorylated. I mmunoprecipitation of actin-fragmin reveals that approximately 40% of the actin subunit of the complex is phosphorylated in vivo. However, u sing purified substrate and kinase, the complex can be quantitatively phosphorylated as judged by two-dimensional gel electrophoresis. Throu gh comparative phosphopeptide fingerprinting, we show that the phospho rylation sites in vivo are identical to those identified in vitro. We additionally characterized a complex of actin and the NH2-terminal hal f of fragmin (residues 1-168) that is also phosphorylated by the same kinase. In contrast to actin-fragmin, phosphorylation of the complex b etween actin and residues 1-168 of fragmin is independent of Ca2+ beca use the second Ca2+-dependent regulatory actin-binding domain is missi ng. By artificially varying the actin-fragmin concentration or the act in-fragmin kinase activity present in microplasmodia cytosolic extract s, we attempted to detect alternative protein substrates for the actin -fragmin kinase. The fact that none could be identified suggests that the control and properties of actin-fragmin phosphorylation observed i n vitro may stand as a model for F-actin growth control in Physarum ce lls.