Identification of the amino acids 300-600 of IRS-2 as 14-3-3 binding region with the importance of IGF-1/insulin-regulated phosphorylation of Ser-573.
Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300-600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300-600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573.
Division of Clinical Chemistry and Pathobiochemistry, Department of Internal Medicine IV, University Hospital Tuebingen, Tuebingen, Germany.
Sabine S
SS
Neukamm
Rachel
R
Toth
Nick
N
Morrice
David G
DG
Campbell
Carol
C
Mackintosh
Rainer
R
Lehmann
Hans-Ulrich
HU
Haering
Erwin D
ED
Schleicher
Cora
C
Weigert
D048948:14-3-3 Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics* / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*
D048948:14-3-3 Proteins; C526297:IRS2 protein, human; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I