Cooperation of Src homology domains in the regulated binding of phosphatidylinositol 3-kinase. A role for the Src homology 2 domain.
Fibroblasts transformed by the v-Src oncoprotein exhibit elevated activity of the enzyme phosphatidylinositol 3'-kinase (PI 3-kinase), which binds to, and is activated by, a wide range of receptor tyrosine kinases as well as v-Src and transforming polyoma middle T/c-Src complexes. Here we consider the role of the v-Src homology (SH) domains, SH3 and SH2, and the tyrosine kinase catalytic domain, in the stimulation of v-Src-associated PI 3-kinase activity in response to rapid activation of the oncoprotein. As shown by others, we find that the v-Src SH3 domain tightly binds the PI 3-kinase p85 regulatory subunit in normal growing chicken embryo fibroblasts. However, we also find that in transformed cells there is additional efficient binding of PI 3-kinase to the v-Src SH2 domain in a catalytically active form. Furthermore, the binding of p85 to the SH2 domain, which is almost undetectable in quiescent cells, is rapidly stimulated upon activation of temperature-sensitive v-Src and consequent cell cycle entry, demonstrating that binding is a target for regulation. We also show that v-Src-associated PI 3-kinase differs considerably from PDGF receptor-associated enzyme by a different mode of binding, a lack of substantial allosteric activation, and a dependence on the tyrosine kinase activity of v-Src. The rapidly induced binding and activation of PI 3-kinase thus provides sensitive regulation of recruitment of PI 3-kinase to its substrates and into other signaling complexes at the cell membrane, which involves all the Src homology domains.
Haefner
B
B
Beaston Institute for Cancer Research, Cancer Research Campaign Beatson Laboratories, Bearsden, Glasgow United Kingdom.
Baxter
R
R
Fincham
V J
VJ
Downes
C P
CP
Frame
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D002642:Chick Embryo; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011485:Protein Binding; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D010748:Phosphopeptides; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D017479:Receptors, Platelet-Derived Growth Factor; D015688:Oncogene Protein pp60(v-src)