Structural and Membrane Binding Analysis of the Phox Homology Domain of Phosphoinositide 3-Kinase-C2α

Abstract

Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX domains, we determined the crystal structure of the PX domain from phosphoinositide 3-kinase C2␣ (PI3K-C2␣), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ). To delineate the mechanism by which this PX domain interacts with membranes, we measured the membrane binding of the wild type domain and mutants by surface plasmon resonance and monolayer techniques. This PX domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P 2 binding. The membrane binding of the PX domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P 2 . Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P 2 membrane affinity and specificity when compared with the PI3K-C2␣ C2 domain, demonstrating that high affinity PtdIns(4,5)P 2 binding was facilitated by the PX domain in full-length PI3K-C2␣. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2␣ PX domain interacts with PtdIns(4,5)P 2 -containing membranes and thereby mediates the membrane recruitment of PI3K-C2␣.Numerous cellular processes, including cell signaling and membrane trafficking, involve complex arrays of protein-protein and lipid-protein interactions. Research in the past decade has revealed that a large number of cellular proteins reversibly translocate to specific subcellular locations to form lipid-protein interactions (1, 2). These proteins, collectively known as peripheral proteins, typically contain one or more modular domains specialized in lipid binding (3,4). Common targets of many of these lipid-binding domains are phosphoinositides (PIs), 4 which play a crucial role in cell signaling and membrane trafficking by serving as site-specific membrane signals to modulate the intracellular localization and/or biological activity of effector proteins (5-8). PIs are metabolized by kinases and phosphatases, which catalyze the phosphorylation and dephosphorylation of PI at the 3Ј-, 4Ј-, or 5Ј-position (9 -12). PI 3-kinases (PI3Ks) are enzymes that catalyze the phosphorylation of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), or phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) at the 3Ј-position of the inositol ring (13-15). PI3Ks are divided into three classes (class I, II, and III) based upon their structures and in vitro activities (16,17). Class II PI3K includes PI3K-C2␣, PI3K-C2␤, and PI3K-C2␥. These enzymes phosphorylate PtdIns and PtdIns4P at the 3Ј-position in vitro (18 -21) and act downstream of receptors for growth factors (22), chemokines (23), and integr...

Classes of phosphoinositide 3-kinases at a glance

“…Class II PI3Ks also harbor a Ras-binding domain (RBD), although these signaling inputs are not well characterized. All three PI3KC2 subfamily members possess a unique C-terminal extension that carries a C2 domain and a PX domain that preferentially binds to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] (Stahelin et al, 2006).…”

PI3KC2α, a class II PI3K, is required for dynamin-independent internalization pathways

“…The signal that dictates the relocalization of PI3KC2a to specific endomembranes is not known at the moment, but the presence of a PX domain in the C-terminal part of the protein with high affinity for phosphatidylinositol (4,5)-bisphosphate (Stahelin et al, 2006), abundant at the plasma membrane and in some internalizing vesicles, such as the Arf6 compartments (Porat-Shliom et al, 2008), suggests that its intracellular localization could be mediated, at least in some conditions, by protein-lipid interactions.…”

Requirement for Class II Phosphoinositide 3-Kinase C2α in Maintenance of Glomerular Structure and Function

“…In common with the class I enzymes, class II PI3Ks contain Ras-binding, C2-like, and kinase domains (13,33), while also sharing highly conserved Phox (PX) homology and C2 domains that are unique to the class II isoforms in the PI3K family. The latter domains are located within the C-terminal region of the enzyme, and they mediate protein and phospholipid-binding activities and contain a nuclear localization signal (11,45). There is one class III PI3K isoform, vesicular protein-sorting protein Vsp34, a monomer involved in intracellular trafficking (19).…”

“…While the in vivo substrates of PI3KC2␣ have not been identified, in vitro it can generate PI(3)P and PI(3,4)P 2 . PI3KC2␣ is constitutively associated with phospholipid membranes and has been identified in various intracellular locations, including the trans-Golgi network (TGN), endocytic compartments, clathrin-coated vesicles, and nuclear speckles, where it may associate with factors involved in mRNA metabolism (11,39,45). Histological analysis of normal human tissues revealed prominent PI3KC2␣ expression in fully differentiated, nonproliferating cells, including endothelial cells lining capillaries, testicular Leydig cells, and the glomerular tuft, and in spleen and lymph nodes, where expression was prominent in macrophages and polymorphonuclear leukocytes (16).…”