Identification of a NPXY motif in growth factor receptor-bound protein 14 (Grb14) and its interaction with the phosphotyrosine-binding (PTB) domain of IRS-1.
Recently we have shown that insulin fails to induce the phosphorylation of IRS-1 in the retina [Rajala et al. (2004) Biochemistry 43, 5637-5650], even though there is widespread expression of IRS-1 throughout the retina. These results suggest the expression of tissue-specific regulators in the retina. Yeast two-hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of retinal insulin receptor identified a novel member of the Grb7 gene family, Grb14. Phosphorylation prediction software indicated 6 out of 18 tyrosine residues were most likely to be phosphorylated. Out of six tyrosine phosphorylation sites, one of the tyrosine residues in Grb14 is present in a conserved sequence motif, FXNPXY. The NPXY motifs are recognized by proteins containing a domain known as phosphotyrosine-binding (PTB) or phosphotyrosine-interacting domain (PID). The biological function of the PTB domain is to drive recruitment of signaling adapters such as IRS-1 or Shc to NPXpY (pY stands for phosphotyrosine) on activated receptor tyrosine kinases. We have made a novel finding that the PTB domain of IRS-1 binds to the NPXY motif of Grb14 in a phosphorylation-independent manner. In addition, Grb14-IRS-1 complexes are detected in lysates prepared from retina tissues. We suggest that the Grb14 NPXY motif could be acting as a dominant negative for IRS-1 functions in the retina, and this hypothesis is consistent with the recent study that Grb14-deficient mice exhibit enhanced IRS-1 phosphorylation and activation of protein kinase B. This is the first report describing the presence of the NPXY motif in Grb14 and binding of the PTB domain of IRS-1 in a phosphorylation-independent manner.
Rajala
Raju V S
RV
Department of Cell Biology, University of Oklahoma Health Sciences Center, and Dean A. McGee Eye Institute, Oklahoma City, Oklahoma 73104, USA. raju-rajala@ouhsc.edu
Chan
Michael D
MD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012160:Retina / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics
D002352:Carrier Proteins; C526292:IRS1 protein, human; C526297:IRS2 protein, human; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526298:Irs2 protein, mouse; D010750:Phosphoproteins; D021122:Protein Subunits; D019000:Phosphotyrosine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin