Control of actin filament length by phosphorylation of fragmin-actin complex.

Abstract

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...

The MEK Kinase Ssk2p Promotes Actin Cytoskeleton Recovery After Osmotic Stress

“…It has been shown previously that phosphorylation of Dictyostelium actin plays a role in assembly/disassembly of the actin cytoskeleton (Furuhashi and Hatano, 1990;Howard et al, 1993). In addition, it was recently found that yeast actin is phosphorylated (Futcher et al, 1999).…”

Identification of actin kinase activity in purified fragmin—actin complex

“…In previous studies the purified fragmin-actin complex was not phosphorylated following incubation with ATP alone [6]. In the present work, fragmin-actin complex was purified by a new purification procedure in which ammonium sulfate precipitation for concentrating proteins was omitted.…”

“…Thus, the fragmin-aetin complex purified by the new method was inactivated by incubation with ATP alone. We have demonstrated that the phosphorylation of actin of the fragrnin-actin complex by aetin kinase is responsible for inactivation of the complex [6,8,9]. Because there is a possibility that the inactivation of the complex incubated with ATP alone is due to phosphorylation of actin of the complex by actin kinase, the complex was incubated with [y-32P]ATP and subjected to SDS-PAGE, followed by autoradiography.…”

“…40 btl of 2.5 mg/ml muscle G-actin were added to the incubated sample solution (60/.tl). After incubation at 25°C for 6 rain, the flow birefringenee was measured according to the described method [6].…”

“…Previous studies have shown that the actin :,f t~:e fragmin-actin complex can be phosphorylated by actin kinase purified from Physarum, which abolishes the nucleation and capping activities of the complex [6]. Actin kinase phosphorylates fragmin-Phy.~.arum actin, fragmin 60-Physarum actin, and fragrnin-muscle actin complexes in a highly specific manner [7][8][9].…”