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Ca2+-dependent actin-binding phosphoprotein in Physarum polycephalum. I. Ca2+/actin-dependent inhibition of its
Abstract:
When crude extracts of the slime mold Physarum polycephalum were incubated with ATP and Mg2+ at 35 degrees C, a peptide of approximately 42,000 Da was predominantly phosphorylated. The kinase, separated from the phosphorylatable peptide, phosphorylated neither actin nor fragmin, both proteins of 42,000 Da, the latter known to cap and shorten actin filaments in a Ca2+-dependent manner. The phosphorylatable peptide was phosphorylated only at threonine residue(s), and its phosphorylation was almost completely inhibited by micromolar concentrations of Ca2+ in the extracts. The Ca2+-dependent inhibition of the phosphorylation was reversed by the subsequent addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid but not by trifluoperazine. The Ca2+-dependent inhibition of the phosphorylation required either actin or another, so far unidentified, protein(s) which is distinct from calmodulin. Fragmin reversed the Ca2+/actin-dependent inhibition of the phosphorylation. The Ca2+-dependent actin-binding phosphorylatable protein named Cap 42 (a + b), consisting of two distinct 42,000-Da peptides a and b, was purified to near homogeneity. Peptide b was identified as the phosphorylatable subunit. Substoichiometric amounts of Cap 42 (a + b) reduced the low shear viscosity of F-actin solutions.
Insights
A slime mold protein, Cap 42, is phosphorylated on threonine residues. Calcium ions inhibit this phosphorylation, which is reversed by actin and fragmin, revealing a novel Ca2+-regulated actin-binding protein.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Slime mold Physarum polycephalum contains various proteins involved in cellular processes.
- Actin dynamics are crucial for cell motility and shape, often regulated by Ca2+.
- Protein phosphorylation plays a key role in signal transduction and cellular regulation.
Purpose of the Study:
- To identify and characterize a novel phosphorylatable protein in Physarum polycephalum extracts.
- To investigate the regulatory mechanisms, particularly Ca2+ dependence, of this protein's phosphorylation.
- To determine the protein's interaction with actin and its functional role.
Main Methods:
- Incubation of crude extracts with ATP and Mg2+.
- Separation and purification of phosphorylated peptides.
- Kinase assays using purified proteins.
- Calcium ion concentration manipulation and use of chelators (EGTA) and inhibitors (trifluoperazine).
- Viscosity measurements of F-actin solutions with purified protein.
Main Results:
- A 42,000 Da peptide was predominantly phosphorylated.
- Phosphorylation occurred at threonine residues and was inhibited by Ca2+.
- The Ca2+-dependent inhibition required actin and was reversed by fragmin.
- A Ca2+-dependent actin-binding protein, Cap 42 (a + b), was purified, with peptide b being the phosphorylatable subunit.
- Cap 42 (a + b) reduced F-actin viscosity.
Conclusions:
- Cap 42 (a + b) is a novel Ca2+-regulated actin-binding protein from Physarum polycephalum.
- Peptide b of Cap 42 is the phosphorylatable subunit, with phosphorylation occurring on threonine.
- The protein's function involves regulating actin filament dynamics, potentially through Ca2+-dependent mechanisms involving actin and fragmin.