IntroductionA remarkable event that takes place during platelet activation is the reorganization that occurs when platelets adhere and spread on exposed collagen fibrils or become activated in the circulation by agonists, such as thrombin or adenosine diphosphate (ADP). In response to either stimulus, the shape of the platelet changes from a smooth disc to an irregular form with multiple, finger-like projections. This transformation is due to cytoskeletal rearrangements within the platelet. The platelet cytoskeleton is an intricately woven network1 arranged in three major structures: a cytoplasmic actin network, a rim of membrane-associated cytoskeleton, and a marginal band consisting of a microtubule coil. Together, these lend support to the platelet plasma membrane and give shape to both resting and activated platelets.At several levels, phosphoinositides are involved in the regulation of the platelet cytoskeleton. Actin binding, capping, and severing proteins are regulated by binding to phosphatidylinositol 4,5-bisphosphate (PIP2). The action of specific phosphoinositide kinases and phosphatases, leading to the regulation of levels of D3- and D4-containing phosphoinositides, has a profound impact on actin assembly. For example, synthesis of D3-containing phosphoinositides by phosphoinositide 3-kinases (PI3Ks) can lead to cortical actin assembly and the formation of lamellipodia downstream of stimulation by growth factor receptors, insulin receptors, and G protein-coupled receptors.2-5
There is increasing evidence that other lipid kinases also regulate cytoskeletal reorganization. Phosphatidylinositol 4-P 5-kinase enzymes, acting downstream of Rho family GTPases, have been shown to stimulate actin assembly.6 Because these areas have been covered in other articles,7,8 this review will, instead, concentrate on the role of pleckstrin and pleckstrin homology (PH) domains in the regulation of the actin cytoskeleton.Pleckstrin homology (PH) domains are the most wellrecognized phosphoinositide-binding protein motifs, comprising “modules” within more than 100 signaling proteins, and are used to mediate intermolecular interactions. The threedimensional structures of all PH domains studied to date are virtually superimposable, despite divergence in their amino acid sequence.9-17 The basic PH domain structure is composed of a β “sandwich,” capped at one end by a carboxyl-terminal α-helix, and all PH domains exhibit a striking polarity in their distribution of surface charge (Fig. 1). Based on the similarity of the structure of the NH2-terminal PH domain of pleckstrin to that of the retinol-binding protein, which was known to bind lipids, Harlan and coworkers tested PH domains and demonstrated that they bind to phosphoinositides.18 Since then, a number of laboratories, including our own, have published reports showing that the binding of PH domains to phosphoinositides can regulate protein function.4,19-22 It is now accepted that PH domains serve to localize their molecules into membrane structures by binding to phosphoinositides;18,23 though some PH domains may interact with other targets, such as the βγ subunits of heterotrimeric G proteins (Gβγ)24-27 or protein kinase C (PKC).28-30
The structure of several PH domains complexed to inositol trisphosphate (IP3) has been solved,11,13 confirming a physical interaction between the inositol phosphate headgroup and the positively charged face of the PH domain. For example, the association of the PH domain of PLCδ with IP3 is shown in Figure 1. Pleckstrin is a 43-kDa hematopoietic protein that contains the amino- and carboxyl- termini of the two prototypic PH domains (Fig. 2). Pleckstrin was first described as a major substrate for PKC in platelets and leukocytes, and its phosphorylation has long been used as a marker for platelet activation. Though its function in vivo remains unclear, expressed pleckstrin can affect PIP2-based signaling mediated by phospholipase C, PI3K, and inositol phosphatases.31-33 Ser113, Thr114, and Ser117, the three residues phosphorylated by PKC, lie adjacent to, but not within, the amino-terminal PH domain. Phosphorylation at these sites has been shown to regulate the function of this PH domain.34 Recently, a third functional motif has been described within pleckstrin.35 This motif is termed the DEP domain after the first three proteins known to possess this sequence (disheveled, Egl-10, and pleckstrin).
Localization of a high-affinity inositol 1,4,5-trisphosphate/inositol 1,4,5,6-tetrakisphosphate binding domain to the pleckstrin homology module of a new 130 kDa protein: characterization of the determinants of structural specificity