Abstract
3-Phosphoinositide-dependent kinase-1 (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide 3-kinase. Although binding of 3'-phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, to the pleckstrin homology (PH) domain of PDK1 is known to be essential for its interaction with and activation of downstream kinases, the mechanism by which PDK1 is recruited to the plasma membrane remains controversial. Our surface plasmon resonance analysis of the PDK1 PH domain and selected mutants shows that the PH domain specifically binds phosphatidylserine using a site that is separate from the canonical phosphoinositide-binding site. Further cell studies show that this specific phosphatidylserine binding is important for the plasma membrane localization and signaling function of PDK1.
Highlights
The mechanism by which 3-phosphoinositide-dependent kinase-1 (PDK1) is recruited to the plasma membrane is not fully understood
Membrane Binding Properties of the PDK1 pleckstrin homology (PH) Domain—It was reported that PDK1-PH had high affinity for PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and modest affinity for PtdIns(4,5)P2 (7)
To determine whether this PS effect is due to specific binding to PS or nonspecific electrostatic interactions, we measured the binding of PDK1-PH to POPC/1-palmitoyl-2-oleoyl-sn-glycero-3phosphoglycerol (8:2) and POPC/1-palmitoyl-2-oleoyl-snglycero-3-phosphoinositol (8:2) vesicles
Summary
The mechanism by which 3-phosphoinositide-dependent kinase-1 (PDK1) is recruited to the plasma membrane is not fully understood. Binding of 3-phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, to the pleckstrin homology (PH) domain of PDK1 is known to be essential for its interaction with and activation of downstream kinases, the mechanism by which PDK1 is recruited to the plasma membrane remains controversial. Further cell studies show that this specific phosphatidylserine binding is important for the plasma membrane localization and signaling function of PDK1. Akt could be activated in cells expressing a PDK1 mutant, the PH domain of which lacks phosphoinositide binding activity (13) These results suggest the presence of another factor besides 3Ј-phosphoinositides that is important for PM recruitment of PDK1. Earlier in vitro membrane binding and cellular translocation studies of various proteins, including PKC (24) and sphingosine kinase (25), as well as their isolated lipid binding domains (26) have indicated that they are targeted to the PM through direct and specific interactions with PS in the PM. We report that the PH domain of PDK1 binds PS via a well defined site that is separate from its PtdIns(3,4,5)P3-binding pocket, and this specific PS binding is essential for its PM localization and signaling function in response to physiological stimuli
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