Use of the two hybrid system to detect the association of the protein-tyrosine-phosphatase, SHPTP2, with another SH2-containing protein, Grb7.
SHPTP2 is a ubiquitously-expressed SH2-containing tyrosine phosphatase that is tyrosine phosphorylated in response to activation of various receptor and nonreceptor tyrosine kinases. SHPTP2 associates with the platelet-derived growth factor (PDGF) receptor after ligand stimulation, and binding of SHPTP2 to this receptor promotes tyrosine phosphorylation of SHPTP2. The yeast two-hybrid system was modified to identify partners of tyrosine-phosphorylated proteins. Using SHPTP2 as bait and supplying an exogenous tyrosine kinase gene to the yeast cells, we have found that SHPTP2 interacts with another signaling protein, Grb7. We have localized the region of interaction to tyrosine 580 in the carboxyl end of SHPTP2 and to the SH2 domain in the carboxy-terminus of Grb7. We demonstrate that Grb7 binds to SHPTP2 in vitro under conditions where the latter is tyrosine-phosphorylated. These experiments show that this modified two hybrid technique may be useful for the identification of proteins involved in tyrosine kinase signal transduction cascades.
Keegan
K
K
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.
Cooper
J A
JA
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D051382:GRB7 Adaptor Protein; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism
D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D051382:GRB7 Adaptor Protein; D014443:Tyrosine; D066246:ErbB Receptors; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases