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Control of actin filament length by phosphorylation of fragmin-actin complex
1Department of Molecular Biology, School of Science, Nagoya University, Japan.
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 Ca2+. 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 Ca2+, however, hardly dissociates the complex. This complex nucleates actin polymerization and caps the F-actin regardless of Ca2+ 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 Ca2+. 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.
Insights
Phosphorylation by actin kinase inactivates the fragmin-actin complex, controlling F-actin length. This inactivation, observed without calcium, can be reversed by phosphatases, revealing a novel regulatory mechanism for actin dynamics.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Fragmin is a calcium-sensitive protein that severs F-actin and forms a complex with G-actin.
- The fragmin/actin complex nucleates actin polymerization and caps F-actin barbed ends, regulating actin filament length.
- Calcium removal does not significantly dissociate the fragmin-actin complex, suggesting calcium-independent functions.
Purpose of the Study:
- To investigate the regulation of the fragmin-actin complex activity.
- To identify factors that modulate the fragmin-actin complex's ability to control actin filament length.
- To elucidate the role of phosphorylation in regulating actin dynamics mediated by fragmin.
Main Methods:
- Purification of fragmin from Physarum polycephalum.
- Formation and characterization of the fragmin/actin complex.
- Incubation of the complex with crude Physarum extract, ATP, and EGTA to assess inactivation.
- Purification of actin kinase from crude extract.
- Phosphorylation assays to determine the effect of actin kinase on the fragmin-actin complex activity.
Main Results:
- The activity of the fragmin-actin complex was significantly decreased upon incubation with crude extract, ATP, and EGTA (low calcium).
- This inactivation was not observed in the presence of calcium.
- Inactivated complex activity was restored under conditions favoring phosphatase activity.
- Purified factors, termed actin kinase, were identified that phosphorylate actin within the fragmin-actin complex.
- Increased phosphorylation correlated with decreased activity of the fragmin-actin complex, including its ability to cap F-actin barbed ends, leading to F-actin elongation.
Conclusions:
- Actin phosphorylation by actin kinase is a mechanism to inactivate the fragmin-actin complex.
- This phosphorylation-dependent inactivation regulates F-actin length by affecting nucleation and capping.
- The study reveals a novel pathway for controlling actin dynamics through post-translational modification of actin within regulatory complexes.