Src-homology region 2 (SH2) domains are stretches of about 100 amino acids which are found to be structurally conserved in a number of signaling molecules. These regions have been shown to bind with high affinity to phosphotyrosine residues within activated receptor tyrosine kinases. Here we report the bacterial expression and purification of individual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase (PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) in amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620–640 nm with Stains-all, a dye known to bind to calcium binding proteins. This effect was not observed for the GAPNSH2 domain. Circular dichroism analysis of the N-terminal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the presence of increasing calcium concentrations. Moreover, the conformational change of p85NSH2 induced by calcium could be replicated by addition of a phosphorylated hexapeptide (DYpMDMK) representing the α-PDGFR binding site for p85. Limited proteolysis studies showed a significant calcium-dependent increase in protection of p85NSH2 but not GAPNSH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown to substitute for calcium, could also protect the p85NSH2 domain from proteolysis even at 10-fold lower concentrations. In vitro binding studies using purified preparations of activated α-PDGFR show that calcium did not affect the binding of GAPNSH2 domains to activated α-PDGFR. In striking contrast, we observed a marked increase in binding of p85NSH2 domains to activated α-PDGFR in the presence of calcium ions. Sequence comparisons and molecular modeling of the p85NSH2 domain based on the v-Src SH2 domain structure show a conserved arrangement of oxygen ligands contributing to two potential calcium binding sites within the p85NSH2 domain. These have been rationalized to be surface loop regions, i.e., loops 2 and 4 and loops 6, 7, and 8, close to the N- and C-terminal α-helical regions, respectively. Together, our findings suggest that the conformation of p85NSH2, but not that of GAPNSH2, is modulated by the presence of calcium ions. This implies that calcium ions may regulate PI-3K (p85α) binding to α-PDGFR in vivo and suggests that the NSH2 domains of p85 and GAP may have distinct functions in receptor-mediated signal transduction.
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