Specific binding of the C-terminal Src homology 2 domain of the p85alpha subunit of phosphoinositide 3-kinase to phosphatidylinositol 3,4,5-trisphosphate. Localization and engineering of the phosphoinositide-binding motif.
Phosphoinositide second messengers, generated from the action of phosphoinositide 3-kinase (PI3K), mediate an array of signaling pathways through the membrane recruitment and activation of downstream effector proteins. Although pleckstrin domains of many target proteins have been shown to bind phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and/or phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) with high affinity, published data concerning the phosphoinositide binding specificity of Src homology 2 (SH2) domains remain conflicting. Using three independent assays, we demonstrated that the C-terminal (CT-)SH2 domain, but not the N-terminal SH2 domain, on the PI3K p85alpha subunit displayed discriminative affinity for PIP(3). However, the binding affinity diminished precipitously when the acyl chain of PIP(3) was shortened. In addition, evidence suggests that the charge density on the phosphoinositol ring represents a key factor in determining the phosphoinositide binding specificity of the CT-SH2 domain. In light of the largely shared structural features between PIP(3) and PI(4,5)P(2), we hypothesized that the PIP(3)-binding site on the CT-SH2 domain encompassed a sequence that recognized PI(4,5)P(2). Based on a consensus PI(4,5)P(2)-binding sequence (KXXXXXKXKK; K denotes Arg, Lys, and His), we proposed the sequence (18)RNKAENLLRGKR(29) as the PIP(3)-binding site. This binding motif was verified by using a synthetic peptide and site-directed mutagenesis. More importantly, neutral substitution of flanking Arg(18) and Arg(29) resulted in a switch of ligand specificity of the CT-SH2 domain to PI(4,5)P(2) and PI(3,4)P(2), respectively. Together with computer modeling, these mutagenesis data suggest a pseudosymmetrical relationship in the recognition of the phosphoinositol head group at the binding motif.
Ching
T T
TT
Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, USA.
Lin
H P
HP
Yang
C C
CC
Oliveira
M
M
Lu
P J
PJ
Chen
C S
CS
D000595:Amino Acid Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D018909:src Homology Domains
D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D019869:Phosphatidylinositol 3-Kinases