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The 80-kDa actin-fragmin kinase (AFK) was purified from Physarum polycephalum microplasmodia to apparent homogeneity through a procedure involving six chromatographic steps. Taking the activity at the first purification step as 100%, the kinase was purified more than 1500-fold, with an overall yield of 8%. The specific activity of the purified enzyme was 700 U/mg. The total amount of AFK present could be estimated as 34 ng/mg extracted protein. One of the polyclonal antibodies raised against four tryptic peptides of the purified 80-kDa AFK recognized the 80-kDa band in an immunoblot and could inhibit the AFK activity up to 100%. A minor 78-kDa protein, also displaying AFK activity, appeared to be derived from the 80-kDa AFK. A partial amino acid sequence analysis, covering up to 20% of the protein (150 amino acids), was performed and confirmed the lack of similarity with any of the sequenced kinases. These data are in agreement with the unique physical and enzymatic properties of the AFK.

Actin kinase phosphorylates actin of fragmin-actin complex, resulting in the inactivation of the nucleation and capping activities of the complex. Fragmin-actin complex was prepared by a new purification procedure. Incubation with ATP caused inactivation of the purified complex and phosphorylation of actin of fragmin-actin complex. The detailed analysis of the complex by SDS-gel electrophoresis showed that actin kinase was co-purified with the fragmin-actin complex. Formation of such an association between actin kinase and substrate suggests that the kinase is localized on the fragmin-actin complex to efficiently regulate actin cytoskeletons.

The Physarum EGTA-resistant actin-fragmin complex, previously named cap 42(a+b), is phosphorylated in the actin subunit by an endogenous kinase [Maruta and Isenberg (1983) J. Biol. Chem., 258, 10151-10158]. This kinase has been purified and characterized. It is an 80 kDa monomeric enzyme, unaffected by known kinase regulators. Staurosporine acts as a potent inhibitor. The actin-fragmin complex is the preferred substrate. The phosphorylation is inhibited by micromolar Ca2+ concentrations, but only in the presence of additional actin. Polymerized actin (vertebrate muscle and non-muscle isoforms) and actin complexes with various actin-binding proteins are poorly phosphorylated. The heterotrimer consisting of two actins and one fragmin, which is formed from cap 42(a+b) and actin in the presence of micromolar concentrations of Ca2+, is also a poor substrate. From the other substrates tested, only histones were significantly phosphorylated, in particular histone H1. In the same manner, casein kinase I could also phosphorylate the actin-fragmin complex. The major phosphorylation site in actin is Thr203. A second minor site is Thr202. These residues constitute one of the contact sites for DNase I [Kabsch et al. (1990) Nature, 347, 37-44] and are also part of one of the predicted actin-actin contact sites in the F-actin model [Holmes et al. (1990) Nature, 347, 44-49].

Fragmin from plasmodium of Physarum polycephalum binds G-actin and severs F-actin in the presence of Ca2+ over 10(-6) M. The fragmin-actin complex consisting of fragmin and G-actin nucleates actin polymerization and caps the barbed (fast growing) end of F-actin, regardless of the concentrations of Ca2+, and the actin filaments are shortened. Actin kinase purified from plasmodium abolishes the nucleation and capping activities of the complex by phosphorylating actin of the fragmin-actin complex (Furuhashi, K., and Hatano, S. (1990) J. Cell. Biol. 111, 1081-1087). This inactivation of the complex leads to production of long actin filaments. We obtained evidence that Physarum actin is phosphorylated by actin kinase at Thr-201, and probably at Thr-202 and/or Thr-203, with 1 mol of phosphate distributed among them. This finding raises the possibility that the site of phosphorylation, Thr-201 to Thr-203, is positioned on the pointed (slow growing) end domain of the actin molecule, because growth of actin filaments from the fragmin-actin complex occurs only from the pointed end. These observations are consistent with a model of the three-dimensional structure of G-actin. Inactivation of the fragmen-actin complex may follow phosphorylation of the pointed end domain of actin.

Many protein factors regulating actin polymerization can be extracted from plasmodia of Physarum polycephalum in the presence of a high EGTA concentration (30 mM). A protein factor with the molecular weight of 60,000 (60 kDa protein) was especially interesting because of its fragmin-like properties. We purified and characterized this 60 kDa protein in the present study. The purified 60 kDa protein enhanced the initial rate of G-actin polymerization, severed F-actin, and capped the barbed end of F-actin in a Ca2+-dependent way. The threshold concentration for Ca2+ was around 10(-6) M. The flow birefringence measurement showed that the length of F-actin decreased from 2.8 to 1.0 microns depending on the concentration of 60 kDa protein added to F-actin. These properties were identical to those of fragmin (Mr 42,000) isolated from plasmodia (Hasegawa et al. (1980) Biochemistry 19, 2677-2683). However, the molecular weight, the tryptic peptide map, and the cross-reactivities with polyclonal anti-fragmin antibodies were different from those of fragmin. We concluded from these results that 60 kDa protein is a new Ca2+-sensitive F-actin-severing protein. Considering its similarity to fragmin, we termed the 60 kDa protein fragmin 60.