Role of the Src homology 2 (SH2) domain and C-terminus tyrosine phosphorylation sites of SH2-containing inositol phosphatase (SHIP) in the regulation of insulin-induced mitogenesis.
To examine the role of SHIP in insulin-induced mitogenic signaling, we used a truncated SHIP lacking the SH2 domain (deltaSH2-SHIP) and a Y917/1020F-SHIP (2F-SHIP) in which two tyrosines contributing to Shc binding were mutated to phenylalanine. Wild-type (WT)-, deltaSH2-, and 2F-SHIP were transiently transfected into Rat1 fibroblasts overexpressing insulin receptors (HIRc). Insulin-stimulated tyrosine phosphorylation of WT-SHIP and deltaSH2-SHIP, whereas tyrosine phosphorylation of 2F-SHIP was not detectable, indicating that 917/1020-Tyr are key phosphorylation sites on SHIP. Although SHIP can bind via its 917/1020-Tyr residues and SH2 domain to Shc PTB domain and 317-Tyr residue, respectively, insulin-induced SHIP association with Shc was more greatly decreased in 2F-SHIP cells than that in deltaSH2-SHIP cells. Insulin stimulation of Shc association with Grb2, which is important for p21ras-MAP kinase activation, was decreased by overexpression of WT- and 2F-SHIP. Importantly, insulin-induced Shc x Grb2 association was not detectably reduced in deltaSH2-SHIP cells. In accordance with the extent of Shc association with Grb2, insulin-induced MAP kinase activation was relatively decreased in both WT-SHIP and 2F-SHIP cells, but not in deltaSH2-SHIP cells. To examine the functional role of SHIP in insulin's biological action, insulin-induced mitogenesis was compared among these transfected cells. Insulin stimulation of thymidine incorporation and bromodeoxyuridine incorporation was decreased in WT-SHIP cells compared with that of control HIRc cells. Expression of 2F-SHIP also significantly reduced insulin-induced mitogenesis, whereas it was only slightly affected by overexpression of deltaSH2-SHIP. Furthermore, the reduction of insulin-induced mitogenesis in WT-SHIP cells was partly compensated by coexpression of Shc. These results indicate that SHIP plays a negative regulatory role in insulin-induced mitogenesis and that the SH2 domain of SHIP is important for its negative regulatory function.
Wada
T
T
First Department of Medicine, Toyama Medical and Pharmaceutical University, Japan.
Sasaoka
T
T
Ishiki
M
M
Hori
H
H
Haruta
T
T
Ishihara
H
H
Kobayashi
M
M
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008938:Mitosis / Q000502:physiology*; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D051381:Rats; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495950:Grb2 protein, rat; D007328:Insulin; D011506:Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D001973:Bromodeoxyuridine; D013936:Thymidine