Phosphorylated pleckstrin induces cell spreading via an integrin-dependent pathway.

Pleckstrin is a 40-kD phosphoprotein containing NH(2)- 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, alphaIIbbeta3. 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 alphaIIbbeta3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant alphaIIbbeta3 Ser753Pro, or beta3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that alphaIIbbeta3 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 beta3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of beta3 Ser753Pro or alphaIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin beta-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.

Pleckstrin 2, a widely expressed paralog of pleckstrin involved in actin rearrangement.

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.