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The single-copy mouse gene Ptprr gives rise to different protein tyrosine phosphatase (PTP) isoforms in neuronal cells through the use of distinct promoters, alternative splicing, and multiple translation initiation sites. Here, we examined the array of post-translational modifications imposed on the PTPRR protein isoforms PTPBR7, PTP-SL, PTPPBSgamma42 and PTPPBSgamma37, which have distinct N-terminal segments and localize to different parts of the cell. All isoforms were found to be short-lived, constitutively phosphorylated proteins. In addition, the transmembrane isoform, PTPBR7, was subject to N-terminal proteolytic processing, in between amino acid position 136 and 137, resulting in an additional, 65-kDa transmembrane PTPRR isoform. Unlike for some other receptor-type PTPs, the proteolytically produced N-terminal ectodomain does not remain associated with this PTPRR-65. Shedding of PTPBR7-derived polypeptides at the cell surface further adds to the molecular complexity of PTPRR biology.

Contains fulltext : 48764.pdf (Publisher’s version ) (Open Access) The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors.

The protein tyrosine phosphatases (PTPs) PTP-SL, STEP and HePTP are mitogen-activated protein kinase (MAPK) substrates and regulators that bind to MAPKs through a kinase-interaction motif (KIM) located in their non-catalytic regulatory domains. We have found that the binding of these PTPs to the MAPKs extracellular-signal-regulated kinase 1 and 2 (ERK1/2), and p38alpha is differentially determined by the KIM-adjacent C-terminal regions of the PTPs, which have been termed kinase-specificity sequences, and is influenced by reducing agents. Under control conditions, PTP-SL bound preferentially to ERK1/2, whereas STEP and HePTP bound preferentially to p38alpha. Under reducing conditions, the association of p38alpha with STEP or HePTP was impaired, whereas the association with PTP-SL was unaffected. On the other hand, the association of ERK1/2 with HePTP was increased under reducing conditions, whereas the association with STEP or PTP-SL was unaffected. In intact cells, PTP-SL and STEP distinctively regulated the kinase activity and the nuclear translocation of ERK1/2 and p38alpha. Our results suggest that intracellular redox conditions could modulate the activity and subcellular location of ERK1/2 and p38alpha by controlling their association with their regulatory PTPs.

MAP kinases (MAPKs) are enzymes directly involved in the control of cellular homeostasis in response to external cues, from differentiation and developmental processes to cell transformation. The activation status of MAPKs, both in magnitude and in duration, reflects the balance of phosphorylation at their Thr and Tyr regulatory residues by specific MAPK kinases and their dephosphorylation by inactivating protein serine/threonine phosphatases (PPs) and protein tyrosine phosphatases (PTPs). The dephosphorylation of MAPKs by PTPs relies on molecular docking between the two enzymes at specific interaction sites. Here we outline a one-step method to identify ERK1/2 and p38 alpha mutations that prevent binding and inactivation by PTPs (tyrosine-or dual-specificity phosphatases) based on the use of anti-pTyr antibodies and cell lysis buffers lacking or containing the broad PTP inhibitor sodium orthovanadate (Na3VO4). (C) 2009 Elsevier Inc. All rights reserved.

Regulated function of mitogen-activated protein (MAP) kinases involves their selective association through docking sites with both activating MAP kinase kinases and inactivating phosphatases, including dual specificity and protein-tyrosine phosphatases (PTP). Site-directed mutagenesis on the mammalian MAP kinases ERK2 and p38alpha identified within their C-terminal docking grooves two clusters of residues important for association with their regulatory PTPs, PTP-SL and STEP. ERK2 and p38alpha mutations that resembled the sevenmaker gain-of-function mutation in the Rolled D. melanogaster ERK2 homologue failed to associate with PTP-SL, were not retained in the cytosol, and were poorly inactivated by this PTP. Additional ERK2 mutations at the docking groove showed deficient association and dephosphorylation by PTP-SL, although their cytosolic retention was unaffected. Other ERK2 mutations, resembling gain-of-function mutations in the FUS3 yeast ERK2 homologue, associated to PTP-SL and were inactivated normally by this PTP. Our results demonstrate that mutations at distinct regions of the docking groove of ERK2 and p38alpha differentially affect their association and regulation by the PTP-SL and STEP PTPs.

The protein tyrosine phosphatase family (PTP) contains a group of dual-specificity phosphatases (DUSPs) that regulate the activivity of MAP kinases (MAPKs), which are key effectors in the control of cell growth and survival in physiological and pathological processes, including cancer. These phosphatases, named as MKP-DUSPs, include the MAPK phosphatases (MKPs) as well as a group of small-size atypical DUSPs structurally and functionally related to the MKPs. MKP-DUSPs, in most of the cases, are direct inactivators of MAPKs by dephosphorylation of both the Thr and the Tyr regulatory residues at the MAPKs catalytic loop. In some other cases, MKP-DUSPs regulate the activity of MAPKs indirectly, acting through upstream MAPK pathways components. The active involvement of MKP-DUSPs in oncogenesis or resistance to cancer therapies is now well documented, making the search and validation of MKP-DUSPs inhibitors a prominent area in clinical cancer research. Here, we review the current knowledge on the role of MKP-DUSPs in human cancer, the status of the preclinical development and validation of specific MKP-DUSP inhibitors, and the potential of MKP-DUSPs as targets for anti-cancer drugs.