Mechanistic Basis of Differential Cellular Responses of Phosphatidylinositol 3,4-Bisphosphate- and Phosphatidylinositol 3,4,5-Trisphosphate-binding Pleckstrin Homology Domains

Abstract

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...

Phosphoinositides Differentially Regulate Protrudin Localization through the FYVE Domain

“…As summarized in Table 1, the protrudin-FYVE domain has high affinity for vesicles containing PtdIns(3,4)P 2 , PtdIns(4,5)P 2 , and PtdIns(3,4,5)P 3 , respectively. However, it shows very low affinity for vesicles containing PtdIns(3)P, PtdIns(4)P, or PtdIns(5)P. The K d values for binding of the protrudin-FYVE domain to vesicles containing PtdIns(3,4)P 2 , PtdIns(4,5)P 2 , and PtdIns(3,4,5)P 3 range from 100 to 200 nM, which are comparable with that for PtdIns(3,4,5)P 3 -specific PH domains determined under similar conditions (30). These values also compare well with the K d value for binding of other prototypical FYVE domains to vesicles containing PtdIns(3)P (49).…”

Lipid binding domains: more than simple lipid effectors

“…The membrane binding of PH domains is initially driven by nonspecific electrostatic interactions, which is followed by specific PI binding to increase the membrane residence time. A recent report demonstrated some PH domains anchor to the membrane through aliphatic residues adjacent to the PI-binding site (38).…”

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains

“…Our previous studies on Fab1, YOTB, Vac1, and EEA1 domain (FYVE) (50), phox homology (PX) (51), ENTH (52), and pleckstrin homology (PH) domains (53) showed that PtdInsP binding triggers membrane penetration of the domains by inducing an electrostatic potential switch and/or local conformational changes. To see whether PtdIns(4,5)P 2 exerts a similar effect on membrane binding of the two N-BAR domains, we measured the interaction of the domains with lipid monolayers in the presence and absence of PtdIns(4,5)P 2 .…”