Microfilament dynamics: regulation of actin polymerization by actin-fragmin kinase and phosphatases.
Based on the phosphorylation of the purified actin-fragmin complex, an 80 kDa monomeric kinase (AFK) has been isolated from Physarum polycephalum. Protein chemical analysis and studies involving kinase inhibitors and effectors establish that the AFK is a unique kinase that cannot be classified so far in one of the conventional kinase families. The actin-fragmin kinase behaves as an "independent" kinase since its activity towards the actin-fragmin complex is apparently not regulated by the binding of a ligand (e.g., the cyclic-nucleotides, Ca2+, calmodulin, phosphatidylserine and diolein). Rigorous screening of the substrate specificity suggests that the actin-fragmin complex represents the only substrate for this kinase. This kinase phosphorylates the actin moiety of the actin-fragmin complex at two consecutive threonine residues which constitute one of the contact sites for DNase I (37) and which are also located at one of the proposed actin-actin contact sites along the long-pitch helix of F-actin (38, 39). The physiological importance of this phosphorylation was demonstrated by studying the effect of phosphorylation on the nucleation and the capping activity of the actin-fragmin complex using fluorescence enhancement analysis. As could be demonstrated, the nucleation of actin filaments by the actin-fragmin complex is completely abolished upon phosphorylation by the AFK. Phosphorylation of the complex also interferes with its capping activity, which becomes Ca(2+)-dependent. In addition, capping and nucleating activity is regulated in vitro by phosphoinositides, of which PIP2 displays the highest activity and specificity. PIP2 partially inhibits the nucleation and capping activity of the unphosphorylated actin-fragmin. The capping activity of the phosphorylated actin-fragmin complex was inhibited by PIP2 to a much greater extent as compared to the unphosphorylated actin-fragmin complex. Among all phospholipids tested, PIP2 displayed the highest specificity. Initial experiments with purified preparations of the PP-1, PP-2A, PP-2B, alkaline phosphatase and acid phosphatases showed that PP-1 and PP-2A phosphatases were capable of dephosphorylating the phospho actin-fragmin complex. These findings raised the question of whether these or other protein phosphatases were involved in the dephosphorylation of this substrate in vivo. To address this question, Physarum extracts were subjected to fractionation by ion exchange chromatography, and the column fractions were assayed in a variety of conditions, to identify the protein phosphatases involved in the dephosphorylation of this substrate and to identify the elution position of the major Ser/Thr protein phosphatases present in the Physarum extract.(ABSTRACT TRUNCATED AT 400 WORDS)
Waelkens
E
E
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, Belgium.
Gettemans
J
J
De Corte
V
V
De Ville
Y
Y
Goris
J
J
Vandekerckhove
J
J
Merlevede
W
W
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017985:Dalteparin / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010804:Physarum / Q000201:enzymology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D000199:Actins; D004791:Enzyme Inhibitors; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C074377:actin kinase; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D017985:Dalteparin