We have identified a cDNA for pleckstrin 2 that is 39% identical and 65% homologous to the original pleckstrin. Like the original pleckstrin 1, this protein contains a pleckstrin homology (PH) domain at each end of the molecule as well as a DEP (Dishevelled, Egl-10, and pleckstrin) domain in the intervening sequence. A Northern blot probed with the full-length cDNA reveals that this homolog is ubiquitously expressed and is most abundant in the thymus, large bowel, small bowel, stomach, and prostate. Unlike pleckstrin 1, this newly discovered protein does not contain obvious sites of PKC phosphorylation, and in transfected Cos-7 cells, it is a poor substrate for phosphorylation, even after PMA stimulation. Cells expressing pleckstrin 2 undergo a dramatic shape change associated with actin rearrangement, including a loss of central F-actin and a redistribution of actin toward the cell cortex. Overexpression of pleckstrin 2 causes large lamellipodia and peripheral ruffle formation. A variant of pleckstrin 2 lacking both PH domains still had some membrane binding but did not efficiently induce lamellipodia, suggesting that the PH domains of pleckstrin 2 contribute to lamellipodia formation. This work describes a novel, widely expressed, membrane-associating protein and suggests that pleckstrin 2 may help orchestrate cytoskeletal arrangement.Pleckstrin homology, or PH, 1 domains are amino acid motifs that are capable of binding polyphosphoinositides and regulating protein function (1-6). Frequently, the binding of polyphosphoinositides to the PH domains within a protein localizes the molecules to the cell membrane (7). In addition, some PH domains may interact with other targets such as the ␤/␥ subunits of heterotrimeric G proteins (8 -11) or protein kinase C (12-14). The structure of several PH domains complexed to inositol trisphosphate has been solved (15, 16), confirming a physical interaction between the inositol phosphate headgroup and the positively charged face of the PH domain.Pleckstrin 1 is a 40-kDa protein containing the prototypic PH domains at its amino and carboxyl termini. It was first described as a major substrate for protein kinase C (PKC) in platelets and leukocytes, and its phosphorylation has long been used as a marker for platelet activation. Although its function in vivo is unclear, heterologously expressed pleckstrin can affect second messenger-based signaling events mediated by phospholipase C, PI3K␥, and 5Ј-inositol phosphatases (17-19). Overexpression and microinjection studies suggest that pleckstrin 1 is membrane-localized, induces a shift of F-actin toward the cell cortex, and participates in the production of lamellipodia (3). These functions are tightly regulated by PKC-mediated phosphorylation of three residues (Ser 113 , Thr 114 , and Ser 117 ) located near, but not within, the amino-terminal PH domain (24). Recently, a DEP (Dishevelled, Egl-10, and pleckstrin) domain has been described in pleckstrin 1, but the function of this motif is unknown (20).Regulation of pleckstrin 1 is uni...

Pleckstrin-2 is composed of 2 pleckstrin homology (PH) domains and a disheveled-Egl-10-pleckstrin (DEP) domain. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to D3 and D5 phosphoinositides. Pleckstrin-2 expressed in Jurkat T cells bound to the cellular membrane and enhanced actindependent spreading only after stimulation of the T-cell antigen receptor or the integrin ␣4␤1. A pleckstrin-2 variant containing point mutations in both PH domains failed to associate with the Jurkat membrane and had no effect on spreading under the same conditions. Although still membrane bound, a pleckstrin-2 variant containing point mutations in the DEP domain demonstrated a decreased ability to induce membrane ruffles and spread. Pleckstrin-2 also colocalized with actin at the immune synapse and integrin clusters via its PH domains. Although pleckstrin-2 can bind to purified D3 and D5 phosphoinositides, the intracellular membrane association of pleckstrin-2 and cell spreading are dependent on D3 phosphoinositides, because these effects were disrupted by pharmacologic inhibition of phosphatidylinositol 3-kinase (PI3K). Our results indicate that pleckstrin-2 uses its modular domains to bind to membraneassociated phosphatidylinositols generated by PI3K, whereby it coordinates with the actin cytoskeleton in lymphocyte spreading and immune synapse formation. IntroductionPleckstrin-1, the platelet and leukocyte C kinase substrate, is the major substrate of protein kinase C (PKC) in platelets, monocytes, macrophages, lymphocytes, and granulocytes. 1 Its 350 amino acid sequence can be divided into 3 motifs: PH domains at the aminoand carboxy-termini of the molecule and an intervening DEP domain. 2-4 A short stretch of amino acids between the aminoterminal PH domain and the DEP domain contains 3 sites of phosphorylation (Ser113, Thr114, and Ser117) by PKC, which are essential for the function of pleckstrin-1. [5][6][7] PH domains have been identified in approximately 252 other human proteins (Simple Modular Architecture Research Tool [SMART] database) and are found in many molecules involved in cellular signaling, cytoskeletal organization, membrane trafficking, and phospholipid modification. 8 Previous results have suggested that PH domains mediate binding of their host proteins to certain phosphoinositides. [9][10][11] Consistent with this hypothesis, nearly all PH domain-containing proteins require membrane association for their function in signal transduction, including pathways that contribute to cytoskeletal assembly, membrane budding, and fusion. 4,7,[12][13][14][15] DEP homology domains are present in numerous signaling proteins, 3,16 including 67 found in the human genome (SMART database). The DEP domain is a protein module of approximately 100 amino acids, first found in the signaling proteins disheveled, Egl-10, and pleckstrin. 3 The domain is also present in certain kinases, regulators of G-protein signaling proteins (RGSs), and Epac, the cyclic adenosine monophosphateregulated guanine nucleotide ...

Pleckstrin is a 40-kD phosphoprotein containing NH2- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, αIIbβ3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with αIIbβ3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant αIIbβ3 Ser753Pro, or β3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that αIIbβ3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of β3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of β3 Ser753Pro or αIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin β-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.