The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr131 or Tyr132 by Src kinase

Bucciantini, Monica; Chiarugi, Paola; Cirri, Paolo; Taddei, Letizia; Stefani, Massimo; Raugei, Giovanni; Nordlund, P.; Ramponi, Giampietro

doi:10.1016/s0014-5793(99)00828-5

Cited by 70 publications

(77 citation statements)

References 29 publications

(43 reference statements)

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“…One possible mechanism is through Src kinase. Although Src can phosphorylate both LMW-PTP and p190 RhoGAP (Bucciantini et al, 1999) (Haskell et al, 2001;Roof et al, 2000;Roof et al, 1998), the phenotype of MCF-10A-EPHA2 cells suggest that p190 RhoGAP is most likely not a candidate substrate for Src. As EPHA2 can physically associate with both Src kinase and LMW-PTP, Src could possibly regulate cell-cell adhesion through modulating LMW-PTP activity.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of EPHA2 receptor destabilizes adherens junctions via a RhoA-dependent mechanism

Fang

Ireton

Zhuang

et al. 2008

Journal of Cell Science

113

103

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EPHA2 receptor tyrosine kinase is overexpressed in several human cancer types and promotes malignancy. However, the mechanisms by which EPHA2 promotes tumor progression are not completely understood. Here we report that overexpression of a wild-type EPHA2, but not a signaling-defective cytoplasmic truncation mutant (ΔC), in human mammary epithelial cells weakens E-cadherin-mediated cell-cell adhesion. Interestingly, the total level of cadherins and the composition of the adherens junction complexes were not affected, nor was the tyrosine phosphorylation of the cadherin complex components changed. By contrast, RhoA GTPase activity was significantly affected by modulating the EPHA2 activity in MCF-10A cells. Treatment with a ROCK kinase inhibitor rescued cell-cell adhesion defects in EPHA2-overexpressing cells, whereas expression of constitutively activated Rho disrupted adherens junctions in ΔC-expressing cells. EPHA2-dependent Rho activation and destabilization of adherens junctions appeared to be regulated via a signaling pathway involving Src kinase, low molecular weight phosphotyrosine phosphatase (LMW-PTP) and p190 RhoGAP. EPHA2 interacted with both Src and LMW-PTP, and the interactions increased in EPHA2-overexpressing cells. In addition, LMW-PTP phosphatase activity was elevated, and this elevation was accompanied by a decrease in tyrosine phosphorylation of p190 RhoGAP and destabilization of cell-cell adhesion. Expression of either a dominant negative LMW-PTP mutant, C12S, or a wild-type p190 RhoGAP rescued adhesion defects in EPHA2-overexpressing cells. Together, these data suggest that EPHA2 promotes tumor malignancy through a mechanism involving RhoA-dependent destabilization of adherens junctions.

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Section: Discussionmentioning

confidence: 99%

Overexpression of EPHA2 receptor destabilizes adherens junctions via a RhoA-dependent mechanism

Fang

Ireton

Zhuang

et al. 2008

Journal of Cell Science

113

103

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“…PTP Activity Assay-The PTP activity was measured as previously reported (7). Briefly, 1.5 ϫ 10 6 cells were collected in 300 l of 0.1 M sodium acetate, pH 5.5, 10 mM EDTA, 1 mM ␤-mercaptoethanol and sonicated for 10 s. The lysates were clarified by centrifugation, and 50 l were used in the PTP activity assay with 50 l of 10 mM paranitrophenylphosphate, at 37°C for 30 min.…”

Section: Site-specific Mutagenesis and Cloning Of Lmw-ptp Mutants In mentioning

confidence: 99%

“…These two LMW-PTP pools are differentially regulated because only the cytoskeleton-associated LMW-PTP fraction is specifically phosphorylated by c-Src after PDGF stimulation (6). As a consequence of its phosphorylation, LMW-PTP shows an average 20-fold increase in its in vitro catalytic activity (7,8,9). Cytoskeleton-associated LMW-PTP influences cell adhesion, spreading, and migration, controlling the phosphorylation level of p190Rho-GAP, a protein that is able to regulate Rho activity and, consequently, cytoskeleton rearrangement in response to PDGF stimulation.…”

mentioning

confidence: 99%

Two Vicinal Cysteines Confer a Peculiar Redox Regulation to Low Molecular Weight Protein Tyrosine Phosphatase in Response to Platelet-derived Growth Factor Receptor Stimulation

Chiarugi

Fiaschi

Taddei

et al. 2001

Journal of Biological Chemistry

Self Cite

175

154

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Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H 2 O 2 or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H 2 O 2 , evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H 2 O 2 production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.Protein tyrosine phosphorylation plays a key role in the regulation of many cellular processes in eukaryotes such as cellular metabolism, proliferation, differentiation, and oncogenic transformation (1). Accumulating evidence indicates that the contribution of phosphotyrosine protein phosphatases (PTPs) 1 to the control of the cell phosphorylation state is as relevant as that of phosphotyrosine protein kinases. The PTP superfamily is composed of over 70 enzymes that, despite very limited sequence similarity, share a common CX 5 R active site motif and an identical catalytic mechanism. On the basis of their function, structure, and sequence, PTPs can be classified in four main families: 1) tyrosine-specific phosphatases, 2) VH1-like dual specificity PTPs, 3) the cdc25 phosphatases, and 4) the low molecular weight phosphatase (2).The low molecular weight protein tyrosine phosphatase (LMW-PTP) is an 18-kDa enzyme that is expressed in many mammalian tissues (3). Our previous studies on the molecular biology of LMW-PTP in NIH3T3 cells demonstrated a well defined role of this enzyme in platelet-derived growth factor (PDGF)-induced mitogenesis. The most relevant phenotypic effect of LMW-PTP overexpression is a strong reduction of the cell growth rate in response to PDGF stimulation. We ...

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“…lysine, arginine, and glycine) in class II LMW-PTPs. The phosphorylation state of Tyr 132 plays an important physiological role in regulating the activity of the protein BPTP and controlling the strength of NIH-3T3 cell substrate adhesion (46,47). The absence of a tyrosine at this position in class II LMW-PTPs suggests that their enzyme activities might be regulated by other processes (48).…”

mentioning

confidence: 99%

The Solution Structure of Escherichia coli Wzb Reveals a Novel Substrate Recognition Mechanism of Prokaryotic Low Molecular Weight Protein-tyrosine Phosphatases

Lescop

Hu²,

Xu³

et al. 2006

Journal of Biological Chemistry

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Low molecular weight protein-tyrosine phosphatases (LMWPTPs) are small enzymes that ubiquitously exist in various organisms and play important roles in many biological processes. In Escherichia coli, the LMW-PTP Wzb dephosphorylates the autokinase Wzc, and the Wzc/Wzb pair regulates colanic acid production. However, the substrate recognition mechanism of Wzb is still poorly understood thus far. To elucidate the molecular basis of the catalytic mechanism, we have determined the solution structure of Wzb at high resolution by NMR spectroscopy. The Wzb structure highly resembles that of the typical LMW-PTP fold, suggesting that Wzb may adopt a similar catalytic mechanism with other LMW-PTPs. Nevertheless, in comparison with eukaryotic LMW-PTPs, the absence of an aromatic amino acid at the bottom of the active site significantly alters the molecular surface and implicates Wzb may adopt a novel substrate recognition mechanism. Furthermore, a structure-based multiple sequence alignment suggests that a class of the prokaryotic LMW-PTPs may share a similar substrate recognition mechanism with Wzb. The current studies provide the structural basis for rational drug design against the pathogenic bacteria.Low molecular weight protein-tyrosine phosphatases (LMW-PTPs) 3 are small cytoplasmic enzymes (ϳ18 kDa) that are widely distributed in prokaryotes and eukaryotes (1, 2). In eukaryotes, LMW-PTPs specifically dephosphorylate and down-regulate many tyrosine kinase receptors, such as platelet-derived growth factor receptor (3, 4), insulin receptor (5), or ephrin receptor (6). The reaction mechanism of eukaryotic LMW-PTPs has been structurally, thermodynamically, and kinetically characterized (7). In contrast, very limited knowledge is available for prokaryotic LMW-PTPs thus far. All LMW-PTPs share a highly conserved C(X) 5 RS motif, where X can be any amino acid. This motif adopts a loop structure, where the phosphate group of the substrate binds to, and is known as the P-loop (1). In addition, some amino acids required for the catalysis are also highly conserved, such as an aspartate residue that acts as a general acid (8). Based on the structures of mammalian LMW-PTPs in complex with exogenous substrates or serendipitous ligands, determinants for substrate specificity were proposed, including the ring stacking around the targeted phosphotyrosine provided by two aromatic side chains of the LMW-PTPs (9, 10).Many bacterial species harbor a layer of capsular polysaccharides and surface-associated exopolysaccharides (EPS). In Escherichia coli, the group 1 capsular polysaccharides and the colanic acid EPS are assembled in a Wzy-dependent polymerization system (11). The ca gene cluster contains one tyrosine autokinase (Wzc) and one LMW-PTP (Wzb) that function as a pair of kinase/phosphatase in the regulation of colanic acid production (12, 13). As a consequence, colanic acid is only produced after the dephosphorylation of the phosphorylated Wzc by Wzb (14). Wzc was identified to regulate the activity of UDPglucose dehydrogenase b...

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The low M_r phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr¹³¹ or Tyr¹³² by Src kinase

Cited by 70 publications

References 29 publications

Overexpression of EPHA2 receptor destabilizes adherens junctions via a RhoA-dependent mechanism

Overexpression of EPHA2 receptor destabilizes adherens junctions via a RhoA-dependent mechanism

Two Vicinal Cysteines Confer a Peculiar Redox Regulation to Low Molecular Weight Protein Tyrosine Phosphatase in Response to Platelet-derived Growth Factor Receptor Stimulation

The Solution Structure of Escherichia coli Wzb Reveals a Novel Substrate Recognition Mechanism of Prokaryotic Low Molecular Weight Protein-tyrosine Phosphatases

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