A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment.
Insulin drives the formation of a complex between tyrosine-phosphorylated IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR cells) in a cell-free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co-immunoprecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitated the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinase in vitro. Together, these results suggest that in CHO-IR cells, insulin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dynamin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin-Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Ando
A
A
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Yonezawa
K
K
Gout
I
I
Nakata
T
T
Ueda
H
H
Hara
K
K
Kitamura
Y
Y
Noda
Y
Y
Takenawa
T
T
Hirokawa
N
N
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D034281:Dynamins; D051380:GRB2 Adaptor Protein; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020664:Son of Sevenless Proteins; D014443:Tyrosine / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D008565:Membrane Proteins; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020664:Son of Sevenless Proteins; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011972:Receptor, Insulin; D020558:GTP Phosphohydrolases; D034281:Dynamins