Inositol phospholipid turnover is enhanced during mitogenic stimulation of cells by growth factors and the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) may be important in triggering cell proliferation. PtdInsP2 also binds actin-binding proteins to regulate their activity, but it is not yet understood how this control is achieved. The protein alpha-actinin from striated muscle contains large amounts of endogenous PtdInsP2, whereas that from smooth muscle has only a little but will bind exogenously added PtdInsP2. In vitro alpha-actinin binds to F-actin and will crosslink actin filaments, increasing the viscosity of F-actin solutions. We report here that alpha-actinin from striated muscle is an endogenous PtdInsP2-bound protein and that the specific interaction between alpha-actinin and PtdInsP2 regulates the F-actin-gelating activity of alpha-actinin. Although the F-actin-gelating activity of alpha-actinin from smooth muscle is much reduced compared with that from striated muscle, exogenous PtdInsP2 can enhance the activity of smooth muscle alpha-actinin to the level seen in striated muscles. These results show that PtdInsP2 is present in striated muscle alpha-actinin and that it is necessary for alpha-actinin to realize its maximum gelating activity.

Abstract. Fragmin is a Ca2÷-sensitive F-actin-severing protein purified from a slime mold, Physarum polycephalum (Hasegawa, T., S. Takahashi, H. Hayashi, and S. Hatano. 1980. Biochemistry. 19:2677-2683. It binds to G-actin to form a 1:1 fragmin/actin complex in the presence of micromolar free Ca 2+. The complex nucleates actin polymerization and caps the barbed end of the short F-actin (Sugino, H., and S. Hatano. 1982. Cell Motil. 2:457-470). Subsequent removal of Ca 2÷, however, hardly dissociates the complex. This complex nucleates actin polymerization and caps the F-actin regardless of Ca 2+ concentration. Here we report that this activity of fragmin-actin complex can be abolished by phosphorylation of actin of the complex.When crude extract from Physarum plasmodium was incubated with 5 mM ATP and 1 mM EGTA, the activities of the complex decreased to a great extent. The inactivation of the complex in the crude extract was not observed in the presence of Ca 2+. In addition, the activities of the complex inactivated in the crude extract were restored under conditions suitable for phosphatase reactions. We purified factors that inactivated fragmin-actin complex from the crude extract. These factors phosphorylated actin of the complex, and the activities of the complex decreased with an increased level of phosphorylation of the complex. These factors, termed actin kinase, also inactivated the complex that capped the barbed end of short F-actin, leading to elongation of the short F-actin to long F-actin. Thus the length of F-actin can be controlled by phosphorylation of fragmin-actin complex by actin kinase. IT is considered that actin-binding or actin regulatory proteins play key roles in dynamic behaviors of actin cytoskeletons in nonmuscle cells (9,14,22,29). Actin organizes into bundles of F-actin in plasmodium of a slime mold, Physarum polycephalum. These bundles could be observed as birefringent fibers under a sensitive polarizing microscope. They appear and disappear in accordance with the contraction-relaxation cycle of plasmodium (12). Fragmin (6, 8, 27), profilin (21), and a high molecular weight actinbinding protein (25) , fragmin severs F-actin to produce shorter filaments in the presence of Ca 2÷ of more than 10-6 M (6, 8, 24). It binds to G-actin to form fragmin-actin complex. The fragmin-actin complex becomes a nucleus for actin polymerization and caps the barbed (or fast-growing) end of the short F-actin, so that annealing of the short F-actin is inhibited (24). Because these activities of fragmin are regulated by the physiological concentration of Ca 2÷, fragmin may be responsible for regulating the actin organization. However, reversal of these fragmin activities is not achieved by only removal of Ca 2+. Fragmin bound to actin in the presence of Ca 2÷ hardly dissociated even when Ca 2÷ was removed so as to be <10 -6 M. A prolonged incubation (on the order of days) of fragmin-actin complex with EGTA is required for dissociation of the complex.It has been recently reported that actin or actin re...

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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.