Phosphatidylserine binding is important for plasma membrane targeting of 3′‐phosphoinositide‐dependent kinase‐1 pleckstrin homology domain

3′‐Phosphoinositide dependent kinase (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide‐3‐kinase activation. An increase in phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns(3,4,5)P 3 ) and phosphatidylinositol‐3,4‐bisphosphate (PtdIns(3,4)P 2 ) is thought to recruit PDK1 and its substrates to the 3′‐phosphoinositide‐rich regions of the plasma membrane for subsequent phosphorylation. PDK1 contains a C‐terminal catalytic domain and an N‐terminal pleckstrin homology (PH) domain that was reported to have high affinity for PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 . To better understand how lipids regulate the subcellular localization and activation of PDK1, we rigorously investigated the membrane binding properties of PDK1 PH domain by surface plasmon resonance analysis. The results show that the PDK1 PH domain has high affinity for not only PtdIns(3,4,5)P 3 , PtdIns(3,4)P 2 and phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P 2 ) but also for phosphatidylserine (PS). We identified the separate binding sites for phosphoinositides and PS through molecular modeling and mutagenesis and analyzed the differential effects of phosphoinositides and PS in membrane recruitment of PDK1. Supported by a NIH grant GM68849.