Publications (33)125.5 Total impact
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Article: Protein tyrosine phosphatases in health and disease.
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ABSTRACT: Protein tyrosine phosphatases (PTPs) represent a super-family of enzymes that play essential roles in normal development and physiology. In this review, we will discuss the PTPs that have a causative role in hereditary diseases in humans. In addition, recent progress in the development and analysis of animal models expressing mutant PTPs will be presented. The impact of PTP signaling on health and disease will be exemplified for the fields of bone development, synaptogenesis and central nervous system diseases. Collectively, research on PTPs since the late 1980's yielded the cogent view that development of PTP-directed therapeutic tools is essential to further combat human disease.FEBS Journal 09/2012; · 3.79 Impact Factor -
Article: Caspase-3 cleavage of DUSP6/MKP3 at the interdomain region generates active MKP3 fragments that regulate ERK1/2 subcellular localization and function.
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ABSTRACT: MAPK (MAP kinase) phosphatase 3 (DUSP6/MKP3) is a cytosolic MKP (MAPK phosphatase) that regulates negatively ERK1/2 downstream to growth factor or apoptotic signaling. Transcription of DUSP6 gene is activated through the ERK1/2 pathway, which constitutes a feedback regulatory loop of ERK1/2 activation. However, the regulation of the function of the DUSP6/MKP3 protein is poorly known. MKP3 possesses a linker region between its N-terminal MAPK-binding domain and its C-terminal catalytic domain, which is conserved in the related MKPs DUSP7/MKPX and DUSP9/MKP4. In MKP3, the interdomain linker region contains a secondary ERK1/2 binding motif and an active nuclear export sequence. Here, we report that MKP3 protein levels are decreased in cells upon apoptotic stimulation in a caspase-dependent manner, and we identify a novel MKP3 regulatory mechanism mediated by the pro-apoptotic protease caspase-3, which involves the MKP3 interdomain linker region. Active caspase-3 targeted the linker region of MKP3 at several residues, rendering N-terminal and C-terminal MKP3 fragments that contain specific arrangements of nuclear export sequence and ERK1/2 interaction motifs. MKP3 caspase-3-generated fragments displayed differential properties to regulate ERK1/2 nuclear/cytosolic localization and activity. Our results indicate that caspase-3 cleavage of MKP3 down-regulates MKP3 full length and renders active MKP3 fragments, which may participate in novel regulatory pathways controlling the subcellular localization and activation of ERK1/2 during apoptosis.Journal of Molecular Biology 04/2012; 420(1-2):128-38. · 4.00 Impact Factor -
Article: A functional network of the tumor suppressors APC, hDlg, and PTEN, that relies on recognition of specific PDZ-domains.
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ABSTRACT: APC and PTEN are tumor suppressor proteins that bind through their C-termini to the PDZ domain containing-hDlg scaffolding protein. We have found that co-expression of PTEN and hDlg enhanced the negative regulation of the PI3K/Akt pathway by PTEN, indicating the physiologic importance of these interactions. APC and PTEN share other PDZ domain containing-interacting partners, including the MAGI scaffolding proteins and the MAST family of protein kinases. Mutational analysis revealed that the C-terminal PDZ-binding motifs from APC and PTEN were differentially recognized by distinct PDZ domains. APC bound to the three PDZ domains from hDlg, whereas PTEN mainly bound to PDZ-2/hDlg. This indicates the existence of overlapping, but distinct PDZ-domain recognition patterns by APC and PTEN. Furthermore, a ternary complex formed by APC, PTEN, and hDlg was detected, suggesting that hDlg may serve as a platform to bring in proximity APC and PTEN tumor suppressor activities. In line with this, tumor-related mutations targeting the PDZ-2/hDlg domain diminished its interaction with APC and PTEN. Our results expand the PDZ-domain counterparts for the tumor suppressor APC, show that APC and PTEN share PDZ-domain partners but have individual molecular determinants for specific recognition of PDZ domains, and suggest the participation of the tumor suppressors APC, PTEN, and hDlg in PDZ-domain interaction networks which may be relevant in oncogenesis.Journal of Cellular Biochemistry 03/2012; 113(8):2661-70. · 2.87 Impact Factor -
Article: Cytoplasmic p27Kip1 counteracts the pro-apoptotic function of the open conformation of PTEN by retention and destabilization of PTEN outside of the nucleus.
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ABSTRACT: The tumor suppressor activity of p27Kip1 takes place in the cell nucleus by inhibitory binding to cyclin/CDK complexes. p27Kip1 can also be localized in the cytoplasm, where it has been proposed to have oncogenic properties. Here, we describe a novel role for cytoplasmic p27Kip1 which could account for its activity as an oncoprotein by negative regulation of the PTEN tumor suppressor. p27Kip1 physically interacted with the open conformation of PTEN, which is competent to enter the nucleus. In mammalian cells, cytoplasmic p27Kip1 retained to nuclear-targeted PTEN in the cytoplasm. This retention was exerted by the C-terminal p27Kip1 region, and was independent of cyclin/CDK-binding. The nuclear accumulation of PTEN triggered by pro-apoptotic TNFα treatment was abolished by cytoplasmic p27Kip1. Furthermore, conformationally-open PTEN displayed diminished protein stability and pro-apoptotic activity in the presence of cytoplasmic p27Kip1. Our results support a conformationally-dependent model of cytoplasmic retention and negative regulation of the activity of nuclear PTEN by oncogenic cytoplasmic p27Kip1, and suggest the existence of reciprocal mechanisms to regulate the levels of both p27Kip1 and PTEN.Cellular signalling 02/2012; 24(2):577-87. · 4.09 Impact Factor -
Article: Epidermal growth factor receptor (EGFR)-mediated positive feedback of protein-tyrosine phosphatase epsilon (PTPepsilon) on ERK1/2 and AKT protein pathways is required for survival of human breast cancer cells.
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ABSTRACT: Increased tyrosine phosphorylation has been correlated with human cancer, including breast cancer. In general, the activation of tyrosine kinases (TKs) can be antagonized by the action of protein-tyrosine phosphatases (PTPs). However, in some cases PTPs can potentiate the activation of TKs. In this study, we have investigated the functional role of PTPε in human breast cancer cell lines. We found the up-regulation and activation of receptor PTPε (RPTPε) in MCF-7 cells and MDA-MB-231 upon PMA, FGF, and serum stimulation, which depended on EGFR and ERK1/2 activity. Diminishing the expression of PTPε in human breast cancer cells abolished ERK1/2 and AKT activation, and decreased the viability and anchorage-independent growth of the cells. Conversely, stable MCF-7 cell lines expressing inducible high levels of ectopic PTPε displayed higher activation of ERK1/2 and anchorage-independent growth. Our results demonstrate that expression of PTPε is up-regulated and activated in breast cancer cell lines, through EGFR, by sustained activation of the ERK1/2 pathway, generating a positive feedback regulatory loop required for survival of human breast cancer cells.Journal of Biological Chemistry 11/2011; 287(5):3433-44. · 4.77 Impact Factor -
Article: Phosphorylation of the kinase interaction motif in mitogen-activated protein (MAP) kinase phosphatase-4 mediates cross-talk between protein kinase A and MAP kinase signaling pathways.
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ABSTRACT: MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.Journal of Biological Chemistry 09/2011; 286(44):38018-26. · 4.77 Impact Factor -
Article: A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes.
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ABSTRACT: The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.Human Molecular Genetics 08/2011; 20(21):4132-42. · 7.64 Impact Factor -
Article: Phylogenetic and genetic linkage between novel atypical dual-specificity phosphatases from non-metazoan organisms.
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ABSTRACT: Dual-specificity phosphatases (DSPs) constitute a large protein tyrosine phosphatase (PTP) family, with examples in distant evolutive phyla. PFA-DSPs (Plant and Fungi Atypical DSPs) are a group of atypical DSPs present in plants, fungi, kinetoplastids, and slime molds, the members of which share structural similarity with atypical- and lipid phosphatase DSPs from mammals. The analysis of the PFA-DSPs from the plant Arabidopsis thaliana (AtPFA-DSPs) showed differential tissue mRNA expression, substrate specificity, and catalytic activity for these proteins, suggesting different functional roles among plant PFA-DSPs. Bioinformatic analysis revealed the existence of novel PFA-DSP-related proteins in fungi (Oca1, Oca2, Oca4 and Oca6 in Saccharomyces cerevisiae) and protozoa, which were segregated from plant PFA-DSPs. The closest yeast homolog for these proteins was the PFA-DSP from S. cerevisiae ScPFA-DSP1/Siw14/Oca3. Oca1, Oca2, Siw14/Oca3, Oca4, and Oca6 were involved in the yeast response to caffeine and rapamycin stresses. Siw14/Oca3 was an active phosphatase in vitro, whereas no phosphatase activity could be detected for Oca1. Remarkably, overexpression of Siw14/Oca3 suppressed the caffeine sensitivity of oca1, oca2, oca4, and oca6 deleted strains, indicating a genetic linkage and suggesting a functional relationship for these proteins. Functional studies on mutations targeting putative catalytic residues from the A. thaliana AtPFA-DSP1/At1g05000 protein indicated the absence of canonical amino acids acting as the general acid/base in the phosphor-ester hydrolysis, which suggests a specific mechanism of reaction for PFA-DSPs and related enzymes. Our studies demonstrate the existence of novel phosphatase protein families in fungi and protozoa, with active and inactive enzymes linked in common signaling pathways. This illustrates the catalytic and functional complexity of the expanding family of atypical dual-specificity phosphatases in non-metazoans, including parasite organisms responsible for infectious human diseases.MGG Molecular & General Genetics 03/2011; 285(4):341-54. · 2.58 Impact Factor -
Article: Dual-specificity MAP kinase phosphatases as targets of cancer treatment.
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ABSTRACT: 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.Anti-cancer agents in medicinal chemistry 02/2011; 11(1):109-32. -
Article: PINK1 displays tissue-specific subcellular location and regulates apoptosis and cell growth in breast cancer cells.
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ABSTRACT: The PINK1 gene is mutated in the germ line of patients with hereditary early-onset Parkinson disease, and PINK1 prosurvival function at neuronal mitochondria has been related with the etiology of this disease. However, the expression and function of PINK1 protein in nonneuronal tissues has not been determined yet. Here, we have analyzed PINK1 protein expression and subcellular distribution in normal and neoplastic human tissues and investigated the function of PINK1 in breast carcinoma cells. PINK1 protein, as stained by a specific anti-PINK1 monoclonal antibody, was widely expressed in human tissues, displaying high expression in epithelial tissues and in the central nervous system and lower expression in tissues of mesenchymal origin. The subcellular distribution of PINK1 was cytoplasmic granular or cytoplasmic diffuse in most tissues. In breast, PINK1 was also associated with the plasma membrane. Human neoplastic tissues ranged from high PINK1 expression in carcinomas to low expression in sarcomas. In neoplastic tissues, PINK1 displayed a diffuse cytoplasmic localization, with an additional membranous localization in breast carcinoma and squamous carcinoma of lung. In the human breast carcinoma Michigan Cancer Foundation-7 cell line, ectopic expression of cytoplasmic or mitochondrial-targeted PINK1 inhibited apoptosis triggered by hydrogen peroxide and suppressed cell growth in soft agar, whereas PINK1 silencing increased hydrogen peroxide-induced apoptosis. Together, our findings indicate that the physiologic functions of PINK1 go beyond its regulatory role of mitochondria-mediated cell survival in neurons.Human pathology 10/2010; 42(1):75-87. · 3.03 Impact Factor -
Article: Differential up-regulation of MAP kinase phosphatases MKP3/DUSP6 and DUSP5 by Ets2 and c-Jun converge in the control of the growth arrest versus proliferation response of MCF-7 breast cancer cells to phorbol ester.
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ABSTRACT: Different levels of regulation account for the inactivation of MAP kinases by MAPK phosphatases (MKPs), in a cell type- and stimuli-dependent manner. MCF-7 human breast carcinoma cells treated with the phorbol 12-myristate 13-acetate (PMA) suffer growth arrest and show morphological alterations, which depend on the activation of the ERK1/2 MAP kinases. MKP3/DUSP6 and DUSP5 MAP kinase phosphatases, two negative regulators of ERK1/2, were specifically up-regulated in MCF-7 and SKBR3 cells in response to PMA. MKP3 and DUSP5 up-regulation required the prolonged activation of the ERK1/2 pathway, and correlated with the shutdown of this route. MKP3 induction relied on the activation of the Ets2 transcription factor, whereas DUSP5 induction depended on the activation of c-Jun. Diminishing the expression of MKP3 and DUSP5 raised the activation of ERK1/2, and accelerated growth arrest of PMA-treated MCF-7 cells. Conversely, MCF-7 cell lines expressing high levels of MKP3 or DUSP5 did not undergo PMA-triggered growth arrest, displayed a migratory phenotype, and formed colonies in soft agar. We propose that the differential up-regulation of MKP3 by Ets2 and of DUSP5 by c-Jun may converge in similar functional roles for these MAP kinase phosphatases in the growth arrest versus proliferation decisions of breast cancer cells.Journal of Biological Chemistry 08/2010; 285(34):26417-30. · 4.77 Impact Factor -
Article: Studying the regulation of MAP Kinase by MAP Kinase phosphatases in vitro and in cell systems.
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ABSTRACT: Signaling through MAPK pathways involves a network of activating kinases and inactivating phosphatases. While single MAPK kinases account for specific activation of the distinct MAPKs, inactivation of MAPKs by phosphatases involves a wider spectrum of enzymes, with phosphatases from distinct families displaying specificity toward MAPKs. The dual-specificity family of MAPK phosphatases, MKPs, constitutes the major group of MAPK inactivating phosphatases. MKPs are widely expressed, in a tissue- and development-regulated manner, and the control of their expression and function is crucial for the regulation of MAPK signaling. Here, we present three methods to analyze the regulation of MAPKs by MKPs, using transient and stable-inducible MKP overexpression cell systems and in vitro phosphatase experiments.Methods in molecular biology (Clifton, N.J.) 01/2010; 661:305-21. -
Article: A one-step method to identify MAP kinase residues involved in inactivation by tyrosine- and dual-specificity protein phosphatases.
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ABSTRACT: 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 p38alpha 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).Analytical Biochemistry 08/2009; 394(1):81-6. · 3.00 Impact Factor -
Article: Phosphatidylinositol 3-kinase-dependent activation of mammalian protein kinase B/Akt in Saccharomyces cerevisiae, an in vivo model for the functional study of Akt mutations.
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ABSTRACT: In animal cells, Akt (also called protein kinase B) is activated by stimuli that elevate the level of phosphatidylinositol 3,4,5-trisphosphate and is a major effector for eliciting responses that support cell growth and survival. We have shown previously that co-expression of Akt1 in budding yeast (Saccharomyces cerevisiae) along with hyperactive p110alpha, the catalytic subunit of mammalian phosphatidylinositol 3-kinase, results in Akt1 relocalization to cellular membranes and activation. In the present study, we show that activation of all three mammalian Akt isoforms by wild-type p110alpha causes deleterious effects on yeast cell growth. Toxicity of Akt in S. cerevisiae required its catalytic activity, its pleckstrin homology domain, and phosphorylation of its activation loop, but not phosphorylation of its hydrophobic motif. We demonstrate that expression in yeast of the only purported oncogenic allele, Akt1(E17K), leads to enhanced phenotypes. Ala-scanning mutagenesis of the VL1 region within the phosphatidylinositol 3,4,5-trisphosphate-interacting pocket of the Akt1 pleckstrin homology domain revealed that most residues in this region are essential for Akt1 activity. We found that active Akt leads to enhanced signaling through the yeast cell wall integrity pathway. This effect requires the upstream Rho1 activator Rom2 and involves both phosphorylation of the MAPK Slt2 and expression of its transcriptional targets, thus providing a quantitative reporter system for heterologous Akt activity in vivo. Collectively, our results disclose a heterologous yeast system that allows the functional assessment in vivo of both loss-of-function and tumorigenic Akt alleles.Journal of Biological Chemistry 04/2009; 284(20):13373-83. · 4.77 Impact Factor -
Article: Protein tyrosine phosphatases: dual-specificity phosphatases in health and disease.
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ABSTRACT: Dual-specificity phosphatases (DSPs) constitute a subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylates phospho-Tyr, phospho-Ser and nonproteinaceous substrates. DSPs are involved in the regulation of both developmental and postnatal essential processes, such as early embryogenesis, placental development and immune responses. Several DSP genes are implicated in familial and sporadic human diseases, including tumor-related, neurological and muscle disorders, and cardiovascular and inflammatory diseases. This association ranges from disease-causative mutations to disease-risk-prone single-nucleotide polymorphisms, promoter methylation or gene duplication (most often in cancer). Deconvolution of the role of DSPs in disease is challenging. The enzymes' activities are regulated at many levels and they form part of extensive, intricate networks with other signaling components. Here, we review current knowledge of the role of cysteine-based PTP-domain DSPs in health and disease, and their suitability as putative therapeutic targets for drugs is discussed.FEBS Journal 04/2008; 275(5):848-66. · 3.79 Impact Factor -
Article: Multimerisation of receptor-type protein tyrosine phosphatases PTPBR7 and PTP-SL attenuates enzymatic activity.
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ABSTRACT: Dimerisation of receptor-type protein tyrosine phosphatases (RPTPs) represents an appealing mechanism to regulate their enzymatic activity. Studies thus far mostly concern the dimerisation behaviour of RPTPs possessing two tandemly oriented catalytic PTP domains. Mouse gene Ptprr encodes four different protein isoforms (i.e. PTPBR7, PTP-SL and PTPPBSgamma-42/37) that contain a single PTP domain. Using selective membrane permeabilisation we here demonstrate that PTP-SL, like PTPBR7, is a single membrane-spanning RPTP. Furthermore, these two receptor-type PTPs constitutively formed homo- and hetero-meric complexes as witnessed in chemical cross-linking and co-immunoprecipitation experiments, in sharp contrast to the cytosolic PTPPBSgamma-42 and PTPPBSgamma-37 PTPRR isoforms. This multimerisation occurs independently of the PTP domain and requires the transmembrane domain and/or the proximal hydrophobic region. Using overexpression of a PTPBR7 mutant that essentially lacks the intracellular PTP domain-containing segment, a monomer-mimicking state was forced upon full-length PTPBR7 immunoprecipitates. This resulted in a significant increase in the enzymatic activity of the PTPRR PTP domain, which strengthens the notion that multimerisation represents a general mechanism to tone down RPTP catalytic activity.Biochimica et Biophysica Acta 03/2008; 1783(2):275-86. · 4.66 Impact Factor -
Article: A novel phosphatase family, structurally related to dual-specificity phosphatases, that displays unique amino acid sequence and substrate specificity.
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ABSTRACT: Members of the superfamily of protein tyrosine phosphatases (PTPs) share the presence of an evolutionarily conserved PTP catalytic domain. Among them, the dual-specificity phosphatases (DSPs) constitute a diverse group of enzymes in terms of substrate specificity, including nonprotein substrates. In recent years, an increasing number of novel DSPs, whose functions and biological substrates are not well defined, have been discovered in a variety of organisms. In this study, we define the structural and functional properties of evolutionarily related atypical DSPs from different phyla. Sets of conserved motifs were defined that (i) uniquely segregated mammalian atypical DSPs from closely related enzymes and (ii) exclusively characterised a novel family of atypical DSPs present in plants, fungi, and kinetoplastids [plant and fungi atypical (PFA)-DSPs]; despite having different sequence "fingerprints," the PTP tertiary structure of PFA-DSPs is conserved. Analysis of the catalytic properties of PFA-DSPs suggests the existence of a unique substrate specificity for these enzymes. Our findings predict characteristic functional motifs for the diverse members of the DSP families of PTPs and provide insights into the functional properties of DSPs of unknown function.Journal of Molecular Biology 01/2008; 374(4):899-909. · 4.00 Impact Factor -
Article: In vivo functional analysis of the counterbalance of hyperactive phosphatidylinositol 3-kinase p110 catalytic oncoproteins by the tumor suppressor PTEN.
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ABSTRACT: The signaling pathways involving class I phosphatidylinositol 3-kinases (PI3K) and the phosphatidylinositol-(3,4,5)-trisphosphate phosphatase PTEN regulate cell proliferation and survival. Thus, mutations in the corresponding genes are associated to a wide variety of human tumors. Heterologous expression of hyperactive forms of mammalian p110alpha and p110beta in Saccharomyces cerevisiae leads to growth arrest, which is counterbalanced by coexpression of mammalian PTEN. Using this in vivo yeast-based system, we have done an extensive functional analysis of germ-line and somatic human PTEN mutations, as well as a directed mutational analysis of discrete PTEN functional domains. A distinctive penetrance of the PTEN rescue phenotype was observed depending on the levels of PTEN expression in yeast and on the combinations of the inactivating PTEN mutations and the activating p110alpha or p110beta mutations analyzed, which may reflect pathologic differences found in tumors with distinct alterations at the p110 and PTEN genes or proteins. We also define the minimum length of the PTEN protein required for stability and function in vivo. In addition, a random mutagenesis screen on PTEN based on this system allowed both the reisolation of known clinically relevant PTEN mutants and the identification of novel PTEN loss-of-function mutations, which were validated in mammalian cells. Our results show that the PI3K/PTEN yeast-based system is a sensitive tool to test in vivo the pathologic properties and the functionality of mutations in the human p110 proto-oncogenes and the PTEN tumor suppressor and provide a framework for comprehensive functional studies of these tumor-related enzymes.Cancer Research 11/2007; 67(20):9731-9. · 7.86 Impact Factor -
Article: Functional assignment of MAPK phosphatase domains.
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ABSTRACT: Mitogen-activated protein kinase (MAPK) pathways are well conserved in most organisms, from yeast to humans. The principal components of these pathways are MAP kinases whose activity is regulated by phosphorylation, implicating various MAPK protein effectors-in particular, protein phosphatases that inactivate MAPKs by dephosphorylation. The molecular basis of binding specificity of such regulatory phosphatases to MAPKs is poorly understood. To try to pinpoint potential functional regions within the sequences and to help identify new family members, we have applied a multimotif pattern-recognition approach to characterize two MAPK phosphatase subfamilies (tyrosine-specific and dual specificity) that are crucial in the regulation of MAPKs. We built "fingerprints" for these two subfamilies that are unique to, and highly discriminatory for, each group of proteins. The fingerprints were used in a genome-wide screen, identifying more than 80 MAPK phosphatase domains, several of which were in partial sequences or unclassified proteins. We confirmed experimentally that one predicted MAPK phosphatase orthologue in Xenopus binds to ERK1/2, suggesting a role in MAPK signaling and thus supporting our functional predictions. Further analysis, mapping the fingerprints on the three-dimensional structure of MAPK phosphatases, revealed that some of the fingerprint motifs reside in the N-terminal noncatalytic regions coinciding with reported MAPK binding sites, while others lie within the catalytic phosphatase domain. These results also suggest the presence of putative allosteric sites in the catalytic region for modulation of protein-protein interactions, and provide a framework for future experimental validation.Proteins Structure Function and Bioinformatics 11/2007; 69(1):19-31. · 3.39 Impact Factor -
Article: Proteolytic processing of the receptor-type protein tyrosine phosphatase PTPBR7.
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ABSTRACT: 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.FEBS Journal 02/2007; 274(1):96-108. · 3.79 Impact Factor
Top co-authors
Top Journals
Institutions
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2005–2012
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Centro de Investigación Príncipe Felipe
Valencia, Valencia, Spain
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2011
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Complutense University of Madrid
- Facultad de Farmacia
Madrid, Madrid, Spain
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2004
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Universidad de Santiago de Compostela
Santiago de Compostela, Galicia, Spain
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