Phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) are lipid second messengers that regulate various cellular processes by recruiting a wide range of downstream effector proteins to membranes. Several pleckstrin homology (PH) domains have been reported to interact with PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 . To understand how these PH domains differentially respond to PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 signals, we quantitatively determined the PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 binding properties of several PH domains, including Akt, ARNO, Btk, DAPP1, Grp1, and C-terminal TAPP1 PH domains by surface plasmon resonance and monolayer penetration analyses. The measurements revealed that these PH domains have significant different phosphoinositide specificities and affinities. Btk-PH and TAPP1-PH showed genuine PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 specificities, respectively, whereas other PH domains exhibited less pronounced specificities. Also, the PH domains showed different degrees of membrane penetration, which greatly affected the kinetics of their membrane dissociation. Mutational studies showed that the presence of two proximal hydrophobic residues on the membrane-binding surface of the PH domain is important for membrane penetration and sustained membrane residence. When NIH 3T3 cells were stimulated with platelet-derived growth factor to generate PtdIns(3,4,5)P 3 , reversible translocation of Btk-PH, Grp1-PH, ARNO-PH, DAPP1-PH, and its L177A mutant to the plasma membrane was consistent with their in vitro membrane binding properties. Collectively, these studies provide new insight into how various PH domains would differentially respond to cellular PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 signals.
Phosphoinositides (PIs)2 are mono-and polyphosphorylated derivatives of phosphatidylinositol (PtdIns) (1-3). AlthoughPIs are minor components of membrane lipids, they regulate a wide range of biological processes, including cell proliferation, cell survival, differentiation, signal transduction, cytoskeleton organization, and membrane trafficking (1-3). PIs regulate these cellular processes primarily by serving as site-specific membrane signals that mediate the membrane recruitment and regulation of effector proteins. The PI-mediated cellular processes allow exceptional spatiotemporal specificity because the spatial and temporal distribution of various PIs is dynamically and tightly regulated by a series of PI-modifying enzymes, such as phospholipases, lipid kinases, and lipid phosphatases, located in different cell membranes (1-3). For instance, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) are mainly found in the plasma membrane, whereas phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 5-phosphate (PtdIns5P), and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2 ) are primarily present in endosomes, Golgi, nucl...