Characterization of fodrin phosphorylation by spleen protein tyrosine kinase.
Fodrin, an actin and calmodulin binding and spectrin-like protein present in many nonerythrocyte tissues, could be phosphorylated up to more than 1.5 mol of phosphate/mol of protein by a highly purified non-receptor-associated protein tyrosine kinase from bovine spleen. The protein phosphorylation was not affected by Ca2+/calmodulin or by F-actin. Km and Vmax values of the reaction were 91 nM and 0.35 nmol of P2 min-1 (mg of kinase)-1, respectively. Both subunits A and B of fodrin were phosphorylated, with the rate of subunit A phosphorylation much greater than that of subunit B phosphorylation. Tryptic phosphopeptide mapping of the phosphorylated subunits suggested that there were three major phosphorylation sites in subunit A and one in subunit B. Phosphotyrosylfodrin could be dephosphorylated by the calmodulin-stimulated phosphatase (calcineurin) in the presence of activating metal ions; Ni2+ was a much more effective activator than Mn2+ for this reaction. Fodrin phosphorylation by the spleen protein tyrosine kinase did not appear to alter the actin and calmodulin binding properties of the protein. On the other hand, the calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin was enhanced by 101% +/- 3% (n = 3) upon fodrin phosphorylation. Ni2+-calcineurin, which was shown to effectively dephosphorylate the phosphotyrosyl residues on fodrin, could reverse the phosphorylation-enhanced Mg2+-ATPase stimulatory activity of fodrin.
Wang
C Y
CY
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Kong
S K
SK
Wang
J H
JH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity; D013049:Spectrin
D000255:Adenosine Triphosphate; D000596:Amino Acids; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D010748:Phosphopeptides; D011505:Protein-Tyrosine Kinases; D013049:Spectrin