Two phosphorylation-independent sites on the p85 SH2 domains bind A-Raf kinase.
Src homology 2 (SH2) domains mediate phosphotyrosine (pY)-dependent protein:protein interactions involved in signal transduction pathways. We have found that the SH2 domains of the 85-kDa alpha subunit (p85) of phosphatidylinositol 3-kinase (PI3 kinase) bind directly to the serine/threonine kinase A-Raf. In this report we show that the p85 SH2:A-Raf interaction is phosphorylation-independent. The affinity of the p85 C-SH2 domain for A-Raf and phosphopeptide pY751 was similar, raising the possibility that a p85:A-Raf complex may play a role in the coordinated regulation of the PI3 kinase and Raf-MAP kinase pathways. We further show that the p85 C-SH2 domain contains two distinct binding sites for A-Raf; one overlapping the phosphotyrosine-dependent binding site and the other a separate phosphorylation-independent site. This is the first evidence for a second binding site on an SH2 domain, distinct from the phosphotyrosine-binding pocket.
Fang
Yun
Y
Department of Biochemistry, University of Saskatchewan, 107 Wiggins Avenue, Saskatoon, Saskatchewan, S7N 5E5, Canada.
Johnson
Lindsey M
LM
Mahon
Elizabeth S
ES
Anderson
Deborah H
DH
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D048491:Proto-Oncogene Proteins A-raf; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018909:src Homology Domains
D011993:Recombinant Fusion Proteins; D019869:Phosphatidylinositol 3-Kinases; D048491:Proto-Oncogene Proteins A-raf; D019908:Proto-Oncogene Proteins c-raf