Identification of Insulin-Stimulated Phosphorylation Sites on Calmodulin

Abstract

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.

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

“…Although the CK2 inhibitor DRB has been used to evaluate the role of CK2 in other endothelial signaling pathways (22), to our knowledge no CK2 inhibitor has previously been used to evaluate pathways controlling CaM phosphorylation in any cell type. Indeed, the only existing evidence supporting a role for CK2 in mediating CaM phosphorylation in vivo is indirect: phosphopeptides generated from CaM isolated from insulin-treated rat hepatocytes overlap significantly with those isolated from in vitro CK2-phosphorylated CaM (18).…”

“…The precleared supernatant was incubated with anti-CaM, anti-FLAG, or isotype control IgG1 mAb for 2 h and then with protein G for 1 h at 4°C. Immune complexes were then processed as described (18).…”

“…CaM was immunoprecipitated from [ 32 P i ] biosynthetically labeled BAEC with a specific antiCaM mAb (18). As shown in Fig.…”

“…Furthermore, CaM itself has been shown to be a phosphoprotein (reviewed in ref. 14 (15)(16)(17) and has been implicated in insulin-induced CaM phosphorylation in hepatocytes (18).…”

Hypertonicity Activates Na+/H+ Exchange through Janus Kinase 2 and Calmodulin

“…Casein kinase II phosphorylates CaM on multiple serine and threonine residues (Thr 79 , Ser 81 , Ser 101 , and Thr 117 ) (35,36). Tyr 99 is phosphorylated after activation of both the insulin receptor (37,38) and the epidermal growth factor receptor (39,40) and Tyr 138 after activation of the insulin receptor (37,38). There is also significant precedent for the idea that tyrosine phosphorylation of CaM can alter its ability to interact with and activate its downstream targets.…”