Potential involvement of FRS2 in insulin signaling.
Shp-2 is implicated in several tyrosine kinase receptor signaling pathways. This phosphotyrosine phosphatase is composed of a catalytic domain in its C-terminus and two SH2 domains in its N-terminus. Shp-2 becomes activated upon binding through one or both SH2 domains to tyrosine phosphorylated molecules such as Shc or insulin receptor substrates. We were interested in finding a new molecule(s), tyrosine phosphorylated by the insulin receptor (IR), that could interact with Shp-2. To do so, we screened a human placenta complementary DNA (cDNA) library with the SH2 domain-containing part of Shp-2 using a modified yeast two-hybrid system. In this system we induce or repress the expression of a constitutive active IR beta-subunit. When expressed, IR phosphorylates proteins produced from the library that can then associate with Shp-2. Using this approach, we isolated FRS2 as a potential target for tyrosine phosphorylation by the IR. After cloning the entire cDNA, we found that 1) in the yeast two-hybrid system, FRS2 interacts with Shp-2 in a fashion dependent on the presence of the IR; and 2) in the PC12/IR cell-line, insulin leads to an increase in FRS2 association with the phosphatase. We next wanted to determine whether FRS2 could be a direct substrate for IR. In an in vitro kinase assay we found that wheat-germ agglutinin-purified IR phosphorylates glutathione-S-transferase-FRS2 fusion protein. Finally, in intact cells we show that insulin stimulates tyrosine phosphorylation of endogenous FRS2. In summary, by screening a two-hybrid cDNA library, we have isolated FRS2 as a possible substrate for IR. We found that IR can directly phosphorylate FRS2. Moreover, in intact cells insulin stimulates tyrosine phosphorylation of FRS2 and its subsequent association with Shp-2. Taken together these results suggest that FRS2 could participate in insulin signaling by recruiting Shp-2 and, hence, could function as a docking molecule similar to insulin receptor substrate proteins.
Delahaye
L
L
INSERM, U-145 and IFR 50, Nice, France. delahaye@unice.fr
Rocchi
S
S
Van Obberghen
E
E
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D005260:Female; D015723:Gene Library; D017930:Genes, Reporter; D006801:Humans; D007328:Insulin / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction; D014162:Transfection; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C106534:FRS2 protein, human; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D011972:Receptor, Insulin; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C516727:Ptpn11 protein, rat; C509250:Ptpn6 protein, rat; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases