Selective recognition of phosphatidylinositol 3,4,5-trisphosphate by a synthetic peptide.
The present study takes a novel approach to explore the mode of action of phosphoinositide 3-kinase lipid products by identifying a synthetic peptide W-NG(28-43) (WAAKIQASFRGHMARKK) that displays discriminative affinity with phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). This PtdIns(3,4,5)P3-binding peptide was discovered by a gel filtration-based binding assay and exhibits a high degree of stereochemical selectivity in phosphoinositide recognition. It forms a 1:1 complex with PtdIns(3,4,5)P3 with Kd of 2 microM, but binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) with substantially lower affinity (5- and 40-fold, respectively) despite the largely shared structural motifs with PtdIns(3,4,5)P3. Other phospholipids examined, including phosphatidylserine, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, show low or negligible affinity with the peptide. Several lines of evidence indicate that this phosphoinositide-peptide interaction is not due to nonspecific electrostatic interactions or phospholipid aggregation, and requires a cooperative action among the hydrophobic and basic residues to exert the selective recognition. CD data suggest that the peptide acquires an ordered structure upon binding to PtdIns(3,4,5)P3. Further, we demonstrate that PtdIns(3,4,5)P3 enhances the phosphorylation rate of this binding peptide by protein kinase C (PKC)-alpha in a dose-dependent manner. In view of the findings that this stimulatory effect is not noted with other PKC peptide substrates lacking affinity with PtdIns(3,4,5)P3 and that PKC-alpha is not susceptible to PtdIns(3,4,5)P3 activation, the activity enhancement is thought to result from the substrate-concentrating effect of the D-3 phosphoinositide, i.e. the presence of PtdIns(3,4,5)P3 allows the peptide to bind to the same vesicles/micelles to which PKC is bound. Moreover, it is noteworthy that neurogranin, the full-length protein of W-NG(28-43) and a relevant PKC substrate in the forebrain, binds PtdIns(3,4,5)P3 with high affinity. Taken together, it is plausible that, in addition to PKC activation, PtdIns(3,4,5)P3 provides an alternative mechanism to regulate PKC activity in vivo by recruiting and concentrating its target proteins at the interface to facilitate the subsequent PKC phosphorylation.
Lu
P J
PJ
Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082, USA.
Chen
C S
CS
D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D051579:Neurogranin; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence
D002148:Calmodulin-Binding Proteins; D008823:Micelles; D009419:Nerve Tissue Proteins; C496651:Nrgn protein, rat; D010455:Peptides; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; C060974:phosphatidylinositol 3,4,5-triphosphate; D051579:Neurogranin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C