The use of the yeast two hybrid system to evaluate ErbB-3 interactions with SH2 domain containing proteins.
Several mutations in the tyrosine kinase domain of ErbB-3 have been postulated to render this enzyme catalytically inactive. To test which amino acid mutations in ErbB-3 might be critical for kinase inactivation, we used a yeast two hybrid assay of protein-protein interaction. We monitored restoration of ErbB-3 kinase activity by investigating the ability of wild type or mutant ErbB-3 to associate with the SH2 containing proteins Syp and Phosphatidyl-inositol-3-kinase (PI3K). Our results demonstrate that changing individual amino acids to tyrosine kinase consensus sequences did not increase the interaction of ErbB-3 with Syp or PI3K. Mutation of the consensus Asp832 of rat ErbB-3 to Asn observed in human and bovine ErbB-3 significantly increased the interaction of ErbB-3 and Syp and PI3K 11 or 26 fold respectively. A double mutant (Asp832Asn, Asp757 His) exhibited a 96 or 350 fold increase in the ability to bind PI3K and Syp.
Yoo
J Y
JY
Molecular and Cellular Biology Program, University of Maryland, Baltimore, Maryland, 21201, USA.
Hamburger
A W
AW
D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D016296:Mutagenesis; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020893:Receptor, ErbB-3; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013045:Species Specificity; D018909:src Homology Domains
D066246:ErbB Receptors; D047908:Intracellular Signaling Peptides and Proteins; D019869:Phosphatidylinositol 3-Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D011518:Proto-Oncogene Proteins; D020893:Receptor, ErbB-3; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; C516727:Ptpn11 protein, rat; C509250:Ptpn6 protein, rat