Caspase-3 Regulates Catalytic Activity and Scaffolding Functions of the Protein Tyrosine Phosphatase PEST, a Novel Modulator of the Apoptotic Response

Abstract

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 ⁵⁴⁹DSPD 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.

SUPPLEMENTAL MATERIAL

We are grateful for support from the National Science Council of Taiwan (grants NSC 94-2311-B-011-032 and NSC 94-2311-B-001-020 to T.-C.M.). M.H. is a recipient of a Rolande and Marcel Gosselin Graduate Studentship; S.H. was supported by a postdoctoral fellowship from the Canadian Institute of Health Research (CIHR); C.B. is a recipient of a Human Frontier Science Program postdoctoral fellowship; and A.B. is a recipient of a fellowship from the Lymphoma Research Foundation. M.L.T. is the recipient of the Jeanne et Jean-Louis Lévesque Chair in Cancer Research, a chercheur-Nationaux of the Fonds de la Rercherche en Santé du Québec, and recipient of a James McGill Professor fellowship. This work was supported by a CIHR operating grant (MT-12466) to M.L.T.

We thank Ryan Petrie and Jacynthe Laliberté for their advice on live imaging and John W. Hanrahan for access to the confocal microscope. PTP-PEST polyclonal antibodies were provided by Jean-François Côté. We are grateful to Serge Morin and Martin Calille for their assistance with computer software and to Josée N. Lavoie for helpful discussions. We thank Yves Boiclair and Matthew Stuible for critical reviews of the manuscript.