PLD2 is a newly identified mammalian PLD isoform with novel regulatory properties. Our findings suggest that regulated secretion and morphological reorganization, the two most frequently proposed biological roles for PLD, are likely to be effected separately by PLD1 and PLD2.

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...

Ribozymes are potential therapeutic agents which suppress specific genes in disease-affected cells. Ribozymes have high substrate cleavage efficiency, yet their medical application has been hindered by RNA degradation, aberrant cell trafficking, or misfolding when fused to a carrier. In this study, we constructed a chimeric ribozyme escorted by the motor pRNA of bacteriophage phi29 to achieve proper folding and enhanced stability. A pRNA molecule contains an interlocking loop domain and a 5'/3' helical domain, which fold independently of one another. When a ribozyme is connected to the helical domain, the chimeric pRNA/ribozyme reorganizes into a circularly permuted form, and the 5'/3' ends are relocated and buried in the original 71'/75' positions. Effective silencing of the anti-apoptotic gene survivin by an appropriately designed chimeric ribozyme, as demonstrated at mRNA and protein levels, led to programmed cell death in various human cancer cell lines, including breast, prostate, cervical, nasopharyngeal, and lung, without causing significant non-specific cytotoxicity. Through the interlocking interaction of right and left loops, monomer pRNA/ribozyme chimeras can be incorporated into multi-functional dimer, trimer and hexamer complexes for specific gene delivery. Using the phi29 motor pRNA as an escort may revive the ribozyme's strength in medical application.

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.