title: Phosphylation and protein kinase activity of platelet tubulin.
abstract: Platelet tubulin isolated by two successive cycles of polymerization-depolymerization was shown to contain protein kinase activity. The phosphorylating activity measured by incorporation of [32P]phosphate from [gamma-32P]ATP was cAMP-independent and behaved with respect to substrate specificity, cation requirement, and maximum incorporation of phosphate similarly to tubulin of brain. Contrary to tubulin from that source, however, platelet tubulin itself, not one of its co-purifying proteins appeared to be the source of the protein kinase activity. This was suggested by assays of tubulin freed from its associated proteins by chromatography on DEAE-Sephadex and on immunosorbent columns containing monospecific antibody to human platelet tubulin. Further corroboration was obtained from experiments in which tubulin was applied to casein affinity columns. No separation of protein kinase from colchicine binding activity could be obtained. Gel filtration showed that all of the in vitro phosphorylated tubulin was aggregated. Tryptic peptide patterns of 32P-labeled alpha- and beta-tubulin subunits were analyzed by ion exchange chromatography. Multiple peptides in both tubulin subunits were identified as acceptors of [32P]phosphate. In vivo phosphorylated tubulin was demonstrated to contain an average of 5 phosphoserine residues/monomer.
mesh_terms: D000469:Alkaline Phosphatase; D000596:Amino Acids / Q000032:analysis; D001792:Blood Platelets / Q000201:enzymology*; D003078:Colchicine / Q000378:metabolism; D006023:Glycoproteins / Q000097:blood*; D006801:Humans; D008870:Microtubules / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000097:blood*; D014404:Tubulin / Q000097:blood* / Q000302:isolation & purification
chemical_list: D000596:Amino Acids; D006023:Glycoproteins; D010446:Peptide Fragments; D014404:Tubulin; D011494:Protein Kinases; D000469:Alkaline Phosphatase; D003078:Colchicine