Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.
SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism.
Joslin Diabetes Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215.
S E
SE
Shoelson
M
M
Sivaraja
K P
KP
Williams
P
P
Hu
J
J
Schlessinger
M A
MA
Weiss
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D015398:Signal Transduction; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000577:Amides; D010748:Phosphopeptides; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D010450:Endopeptidases