Identification of insulin-stimulated phosphorylation sites on calmodulin.
Insulin enhances calmodulin phosphorylation in vivo. To determine the insulin-sensitive phosphorylation sites, phosphocalmodulin was immunoprecipitated from Chinese hamster ovary cells expressing human insulin receptors (CHO/IR). Calmodulin was constitutively phosphorylated on serine, threonine, and tyrosine residues, and insulin enhanced phosphate incorporation on serine and tyrosine residues. Phosphocalmodulin immunoprecipitated from control and insulin-treated CHO/IR cells, and calmodulin phosphorylated in vitro by the insulin receptor kinase and casein kinase II were resolved by two-dimensional phosphopeptide mapping. Several common phosphopeptides were detected. The phosphopeptides from the in vitro maps were eluted and phosphoamino acid analysis, manual sequencing, strong cation exchange chromatography, and additional proteolysis were performed. This strategy demonstrated that Tyr-99 and Tyr-138 were phosphorylated in vitro by the insulin receptor kinase and Thr-79, Ser-81, Ser-101 and Thr-117 were phosphorylated by casein kinase II. In vivo phosphorylation sites were identified by comigration of phosphopeptides on two-dimensional maps with phosphopeptides derived from calmodulin phosphorylated in vitro and by phosphoamino acid analysis. This approach revealed that Tyr-99 and Tyr-138 of calmodulin were phosphorylated in CHO/IR cells in response to insulin. Additional sites remain to be identified. The identification of the insulin-stimulated in vivo tyrosine phosphorylation sites should facilitate the elucidation of the physiological role of phosphocal-modulin.
Joyal
J L
JL
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Crimmins
D L
DL
Thoma
R S
RS
Sacks
D B
DB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D047390:Casein Kinase II; D006224:Cricetinae; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D002147:Calmodulin; D007328:Insulin; D011994:Recombinant Proteins; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D011972:Receptor, Insulin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases