Structural and biochemical evaluation of the interaction of the phosphatidylinositol 3-kinase p85 alpha src homology 2 domains with phosphoinositidesand inositol polyphosphates
P. Lo Surdo et al., Structural and biochemical evaluation of the interaction of the phosphatidylinositol 3-kinase p85 alpha src homology 2 domains with phosphoinositidesand inositol polyphosphates, J BIOL CHEM, 274(22), 1999, pp. 15678-15685
Src homology 2 (SH2) domains exist in many intracellular proteins and have
well characterized roles in signal transduction. SH2 domains bind to phosph
otyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation i
s essential for protein-SHE domain interactions, the binding specificity al
so derives from sequences C-terminal to the Tyr(P) residue. The high affini
ty and specificity of this interaction is critical for precluding aberrant
cross-talk between signaling pathways. The p85 alpha subunit of phosphoinos
itide S-kinase (PI 3-kinase) contains two SH2 domains, and it has been prop
osed that in competition with Tyr(P) binding they may also mediate membrane
attachment via interactions with phosphoinositide products of PI 3-kinase.
We used nuclear magnetic resonance spectroscopy and biosensor experiments
to investigate interactions between the p85 alpha SH2 domains and phosphoin
ositides or inositol polyphosphates. We reported previously a similar appro
ach when demonstrating that some pleckstrin homology domains show binding s
pecificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Que
rfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R
., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (199
6) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding sp
ecificity for phosphoinositides in phospholipid bilayers. We show that the
p85 alpha SH2 domain Tyr(P) binding pockets indiscriminately accommodate ph
osphoinositides and inositol polyphosphates. Binding of the SH2 domains to
Tyr(P) peptides was only poorly competed for by phosphoinositides or inosit
ol polyphosphates. We conclude that these ligands do not bind p85 alpha SH2
domains with high affinity or specificity. Moreover, we observed that alth
ough wortmannin blocks PI S-kinase activity in vivo, it does not affect the
ability of tyrosine-phosphorylated proteins to bind to p85 alpha. Conseque
ntly phosphoinositide prodcts of PI 3 kinase are unlikely to regulate signa
ling through p85 alpha SH2 domains.