Mechanistic Studies on the Membrane Recruitment and Function of 3’-Phosphoinositide Dependent Kinase-1

Lipid binding domains rank among the most common modular domains found in the eukaryotic proteome; consequently their structure and function have been the focus of intense investigation over the years. 3’-Phosphoinositide dependent kinase-1 (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)P3) and phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) is thought to recruit PDK1 and its substrates to the 3’-phosphoinositide-rich regions of the plasma membrane for subsequent phosphorylation. PDK1 contains an N-terminal catalytic domain and a C-terminal pleckstrin homology (PH) domain that was reported to have high affinity for PtdIns(3,4,5)P3 and PtdIns(3,4)P2. 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)P3, PtdIns(3,4)P2, but also for phosphatidylserine (PS). We identified the separate binding sites for phosphoinositides (PtdInsP) and PS through molecular modeling and mutagenesis and analyzed the differential effects of PtdInsP and PS in membrane recruitment of PDK1. Furthermore, mutation of the putative PS-binding site disrupts subcellular localization properties of PDK1-PH and also affects the physiological function of the full length protein. Collectively, results establish that PS plays a critical role in plasma membrane localization and signaling function of PDK1.