Structure and Phosphatidylinositol-(3,4)- Bisphosphate Binding of the C-Terminal PH Domain of Human Pleckstrin

Abstract

Pleckstrin is the major target of protein kinase C (PKC) in blood platelets. Its phosphorylation triggers responses that ultimately lead to platelet activation and blood clot formation. Pleckstrin consists of three domains: a pleckstrin homology (PH) domain at both termini and a central DEP (Dishevelled, Egl-1, Pleckstrin) domain. Here, we report the solution nuclear magnetic resonance (NMR) structure of the C-terminal PH domain (C-PH) of human pleckstrin-1. We show that this PH domain binds phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) with high specificity in protein lipid overlay assays. Using NMR titration experiments and mutational analysis, residues involved in binding to PtdIns(3,4)P2 are identified. The binding site is formed by a patch of basic residues from the beta1 and beta2 strands and the beta1-beta2 loop. Since PtdIns(3,4)P2 is an important signaling molecule in platelets, our data suggest a C-PH dependent regulation of pleckstrin function in response to PtdIns(3,4)P2.

Loss of pleckstrin defines a novel pathway for PKC-mediated exocytosis

“…In addition, some PH domains may interact with other targets such as the ␤␥ subunits of heterotrimeric G proteins, serine/threonine phosphorylated proteins, and small GTPases. 16,17 The structure of several PH domains complexed to polyphosphoinositides has been solved [18][19][20] (and others), confirming that there is a specific physical interaction between the inositol phosphate headgroup and the positively charged face of the PH domain.…”

Determination of Structural Requirements and Probable Regulatory Effectors for Membrane Association of Maize Sucrose Synthase 1

“…Since the substitution of hydrophobic residues (I367 and W389) and an acidic residue (E387) with Ala decreased binding, whereas the basic residue mutation R373A slightly increased binding, presumably the positionspecific contributions of hydrophobicity and negative charge influence SUS membrane affinity. Two of the residues (I367 and R373) we identified as contributing to SUS membrane binding are conserved (L251 and R257) in pleckstrin and have been identified as important for phosphatidylinositol-(3,4)-bisphosphate interaction (Isakoff et al, 1998;Edlich et al, 2005).…”

“…The alignment of these regions in SUS1 and human pleckstrin required only a single amino acid gap over this 98-amino acid stretch and possessed 34% similarity. The C-terminal PH domain of human pleckstrin binds phosphatidylinositol-(3,4)-bisphosphate (Edlich et al, 2005), making the related region of SUS a possible contributor to membrane binding. This PH-like domain of SUS1 is also highly conserved in the two other known maize SUS isoforms ($74% identity among the SUS isoforms; Fig.…”

“…The SUS b3 segment and the b3/a3 loop sequence are enriched in hydrophobic residues and well conserved among SUS isoforms. Notably, it is amino acids within this region of the C-terminal human pleckstrin PH domain that contact phosphatidylinositol-(3,4)-bisphosphate and confer specificity (Isakoff et al, 1998;Edlich et al, 2005). The presence of a Pro (P371) and different secondary structural elements in this region of SUS may alter this property in its PH-like domain.…”

“…Since PH domains are known to impart polyphosphoinositide and/or membrane localization to about 30% of the proteins that contain them (Lemmon et al, 2002;Yu et al, 2004;Edlich et al, 2005), we investigated the involvement of this region in SUS1 membrane binding. We demonstrate that SUS1 has an intrinsic ability to interact directly with membranes, and that this ability is due, in part, to the identified PH-like domain.…”