PSTPIP is a tyrosine-phosphorylated protein involved in the organization of the cytoskeleton. Its ectopic expression induces filipodial-like membrane extensions in NIH 3T3 cells. We previously observed a defect in cytokinesis and an increase in the tyrosine phosphorylation of PSTPIP in PTP-PEST-deficient fibroblasts. In this article, we demonstrate that PTP-PEST and PSTPIP are found in the same complexes in vivo and that they interact directly through the CTH domain of PTP-PEST and the coiled-coil domain of PSTPIP. We tested pathways that could regulate the tyrosine phosphorylation of PSTPIP. We found that the activation of the epidermal growth factor and platelet-derived growth factor receptors can induce PSTPIP phosphorylation. With the use of the PP2 inhibitor, we demonstrate that Src kinases are not involved in the epidermal growth factor-mediated phosphorylation of PSTPIP. Together with previous results, this suggests that c-Abl is the critical tyrosine kinase downstream of growth factor receptors responsible for PSTPIP phosphorylation. We also demonstrate that PTP-PEST dephosphorylates PSTPIP at tyrosine 344. Importantly, we identified tyrosine 344 as the main phosphorylation site of PSTPIP by performing tryptic phosphopeptide maps. This is an important finding since tyrosine 367 of PSTPIP was also proposed as a candidate phosphorylation site involved in the negative regulation of the association between PSTPIP and WASP. In this respect, we observed that the PSTPIP⅐ WASP complex is stable in vivo and is not affected by the phosphorylation of PSTPIP. Furthermore, we demonstrate that PSTPIP serves as a scaffold protein between PTP-PEST and WASP and allows PTP-PEST to dephosphorylate WASP. This finding suggests a possible mechanism for PTP-PEST to directly modulate actin remodeling through the PSTPIP-WASP interaction.

Cotranslational translocation of polypeptides into the ER is controlled by the dynamic interaction of ribosome and translocon components. Analysis of the steps involved in this process by high resolution techniques such as gel electrophoresis is precluded by the high molecular masses of these complexes. We show, here, that modifications to standard native electrophoresis protocols can overcome these problems and lead to an increase in mass range and resolution. Using the modified technique, we show that ER ribosome anchored membrane protein (RAMP) complexes resolve into 3 stable and semistable complexes which range in size between 4 and 8 MDa and are sensitive to relevant concentrations of divalent metals. We demonstrate the molecular composition of the complexes and identify a number of modular components that differentiate them. The components that are common to all three RAMP complexes include the OST translocon subcomplex, Glucosidase I and microtubule tethering protein CLIMP63. The two larger complexes further include the kinesin motor binding protein p180 and Sec61, and the largest complex includes the TRAP translocon component and apoptotic regulator BAP31. On the lumenal side, the BiP cochaperone ERdj3 resides with the three RAMP complexes. Our observations may hint at how subcompartmentalization is achieved in the ER membrane continuum.

The protein tyrosine phosphatase PEST (PTP-PEST) is involved in the regulation of the actin cytoskeleton. Despite the emerging functions attributed to both PTPs and the actin cytoskeleton in apoptosis, the involvement of PTP-PEST in apoptotic cell death remains to be established. Using several cell-based assays, we showed that PTP-PEST participates in the regulation of apoptosis. As apoptosis progressed, a pool of PTP-PEST localized to the edge of retracting lamellipodia. Expression of PTP-PEST also sensitized cells to receptor-mediated apoptosis. Concertedly, specific degradation of PTP-PEST was observed during apoptosis. Pharmacological inhibitors, immunodepletion experiments, and in vitro cleavage assays identified caspase-3 as the primary regulator of PTP-PEST processing during apoptosis. Caspase-3 specifically cleaved PTP-PEST at the 549 DSPD motif and generated fragments, some of which displayed increased catalytic activity. Moreover, caspase-3 regulated PTP-PEST interactions with paxillin, leupaxin, Shc, and PSTPIP. PTP-PEST acted as a scaffolding molecule connecting PSTPIP to additional partners: paxillin, Shc, Csk, and activation of caspase-3 correlated with the modulation of the PTP-PEST adaptor function. In addition, cleavage of PTP-PEST facilitated cellular detachment during apoptosis. Together, our data demonstrate that PTP-PEST actively contributes to the cellular apoptotic response and reveal the importance of caspases as regulators of PTPs in apoptosis.

The mechanisms by which inorganic salts of the trace element vanadium mediate their insulinomimetic effects are not clearly understood and were investigated. We have shown previously that vanadium salts activate mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase activities (PI3-K) via a pathway that does not involve the insulin receptor (IR) tyrosine kinase function [Pandey, S. K., Anand-Srivastava, M. B., and Srivastava, A. K. (1998) Biochemistry 37, 7006-7014]. Herein, we have examined a possible role of PI3-K in the vanadyl sulfate (VS)-mediated increase in the level of ras-MAPK activation as well as the contribution of signaling components upstream to MAPK in this VS response. Treatment of IR-overexpressing cells with VS resulted in an increased level of tyrosine phosphorylation of p44(mapk) (ERK-1) and p42(mapk) (ERK-2) along with stimulation of MAPK, MAPK kinase (MEK), and C-raf-1 activities, and ras activation. Preincubation with wortmannin and LY294002, two structurally and mechanistically different inhibitors of PI3-K, blocked the VS-mediated increase in MAPK activity and phosphorylation of ERK-1 and ERK-2. Furthermore, wortmannin inhibited activation of ras, C-raf-1, and MEK in response to VS. The addition of a farnesyltransferase inhibitor, B581, to cells reduced the level of MAPK activation as well as ERK-1 and ERK-2 phosphorylation stimulated by VS. Finally, VS increased PI3-K activity in ras immunoprecipitates. A VS-mediated increase in p70(s6k) activity was also found to be inhibited by wortmannin. Taken together, these results demonstrate that the insulinomimetic effects of VS may be mediated, in part, by PI3-K-dependent stimulation of the ras-MAPK and p70(s6k) pathways.