Expression and function of IRS-1 in insulin signal transmission.
IRS-1 is a major insulin receptor substrate which may play an important role in insulin signal transmission. The mRNA for IRS-1 in rat cells and tissues is about 9.5 kilobases (kb). Rat liver IRS-1 was stably expressed in Chinese hamster ovary (CHO) cells (CHO/IRS-1). Although its calculated molecular mass is 131 kDa, IRS-1 from quiescent cells migrated between 165 and 170 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRS-1 was phosphorylated strongly on serine residues and weakly on threonine residues before insulin stimulation. Insulin immediately stimulated tyrosine phosphorylation of IRS-1, and after 10-30 min with insulin its apparent molecular mass increased to 175-180 kDa. Expression of the human insulin receptor and rat IRS-1 together in CHO/IR/IRS-1 cells increased the basal serine phosphorylation of IRS-1 and strongly increased tyrosine phosphorylation during insulin stimulation. Purified insulin receptors directly phosphorylated baculovirus-produced IRS-1 exclusively on tyrosine residues. By immunofluorescence, IRS-1 was absent from the nucleus, but otherwise distributed uniformly before and after insulin stimulation. Some IRS-1 associated with the insulin receptor during insulin stimulation. In addition, a phosphatidylinositol 3'-kinase associated with IRS-1 during insulin stimulation, and this association was more sensitive to insulin in CHO cells overexpressing the insulin receptor (CHO/IR cells), more responsive to insulin to CHO/IRS-1 cells, and both sensitive and responsive in CHO/IR/IRS-1 cells. Similarly, insulin-stimulated DNA synthesis was more sensitive to insulin in CHO/IR cells, and more responsive in CHO/IRS-1 cells; however, insulin-stimulated DNA synthesis was sensitive but poorly responsive to insulin in CHO/IR/IRS-1 cells. Together, these results suggest that IRS-1 is a direct physiologic substrate of the insulin receptor and may play an important role in insulin signal transmission.
Sun
X J
XJ
Research Division, Joslin Diabetes Center, Boston, Massachusetts.
Miralpeix
M
M
Myers
M G
MG
Glasheen
E M
EM
Backer
J M
JM
Kahn
C R
CR
White
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D015870:Gene Expression; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D004247:DNA; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin