No abstract

Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs).Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTP␣-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTP␣-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTP␣-D1 and RPTP␣-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTP-D1, with a similar conformation as RPTP␣-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H 2 O 2 concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.

Human intestine 407 cells respond to hypo-osmotic stress by the rapid release of ATP into the extracellular medium. A difference in the time course of activation as well as in the sensitivity to cytochalasin B treatment and BAPTA-AM [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester] loading suggests that ATP leaves the cell through a pathway distinct from volume-regulated anion channels. To evaluate a putative role for nucleotides as autocrinic/paracrinic factors in osmotic signalling, the effects of extracellular ATP on the regulation of volume-sensitive anion channels as well as on the hypotonicity-induced activation of extracellular signal-regulated protein kinases (Erk-1/2) were investigated. Micromolar concentrations of ATP were unable to elicit an isotope efflux from (125)I(-)-loaded cells by itself, but strongly potentiated the hypotonicity-provoked anion efflux through a Ca(2+)-dependent mechanism. The order of potency of nucleotides (ATP = UTP = ATP[S] > ADP = AMP >> adenosine = cAMP) indicated the involvement of P2Y(2) receptors. In contrast, millimolar concentrations of ATP markedly inhibited both the osmotically induced isotope efflux and whole-cell Cl(-) currents. Inhibition of whole-cell Cl(-) currents, not only by millimolar ATP but also by the purinoceptor antagonists suramin and reactive blue, was observed most prominently at depolarizing holding potentials, suggesting a direct interaction with volume-sensitive Cl(-) channels rather than interaction with purinoceptors. Both ATP and UTP, at submicromolar levels, were found to act as potent activators of Erk-1/2 in intestine 407 cells. Addition of the ATP hydrolase apyrase to the bath greatly reduced the hypotonicity-induced Erk-1/2 activation, but did not affect the swelling-induced isotope efflux or whole-cell Cl(-) currents. Furthermore, pre-treatment with suramin or reactive blue almost completely prevented the hypo-osmotic activation of Erk-1/2. The results indicate that extracellularly released ATP functions as an autocrinic/paracrinic factor that mediates hypotonicity-induced Erk-1/2 activation but does not serve as an activator of volume-sensitive compensatory Cl(-) currents.

Receptor protein-tyrosine phosphatase ␣ (RPTP␣) constitutively forms dimers in the membrane, and activity studies with forced dimer mutants of RPTP␣ revealed that rotational coupling of the dimer defines its activity. The hemagglutinin (HA) tag of wild type RPTP␣ and of constitutively dimeric, active RPTP␣-F135C with a disulfide bond in the extracellular domain was not accessible for antibody, whereas the HA tag of constitutively dimeric, inactive RPTP␣-P137C was. All three proteins were expressed on the plasma membrane to a similar extent, and the accessibility of their extracellular domains did not differ as determined by biotinylation studies. Dimerization was required for masking the HA tag, and we identified a region in the N terminus of RPTP␣ that was essential for the effect. Oxidative stress has been shown to induce a conformational change of the membrane distal PTP domain (RPTP␣-D2). Here we report that H 2 O 2 treatment of cells induced a change in rotational coupling in RPTP␣ dimers as detected using accessibility of an HA tag in the extracellular domain as a read-out. The catalytic site Cys 723 in RPTP␣-D2, which was required for the conformational change of RPTP␣-D2 upon H 2 O 2 treatment, was essential for the H 2 O 2 -induced increase in accessibility. These results show for the first time that a conformational change in the intracellular domain of RPTP␣ led to a change in conformation of the extracellular domains, indicating that RPTPs have the capacity for inside-out signaling.Two antagonistically acting families of enzymes regulate phosphorylation of protein tyrosine residues, an important determinant for many cellular functions: protein-tyrosine kinases (PTKs) 1 and protein-tyrosine phosphatases (PTPs). PTKs catalyze the phosphorylation of tyrosine residues, and PTPs catalyze the dephosphorylation. The classical PTPs can be subdivided into the cytoplasmic PTPs and the transmembrane PTPs (reviewed in Ref. 1). Transmembrane PTPs, tentatively called receptor-like PTPs (RPTPs), are interesting because of their potential to signal across the membrane. Besides one transmembrane domain, most RPTPs possess two catalytic domains of which the first contains most of the catalytic activity. The RPTP extracellular domains vary greatly (2). Clear evidence for ligands that regulate RPTP activity remains elusive. In some cases, as for the interaction between RPTP␤, leukocyte common antigen-related (LAR), and PTP and their ligands contactin, laminin/nidogen, and heparan sulfate proteoglycans, respectively, the binding compound is known, but the effect of the ligand on PTP activity needs to be established (3-5). Sorby et al. (6) reported an effect of Matrigel on DEP1 activity; however, the exact responsible compound still needs to be established. Only pleiotrophin, a heparin-binding cytokine, was reported to reduce RPTP␤/ activity, in that ␤-catenin phosphorylation was enhanced in cells expressing RPTP␤/ in response to pleiotrophin (7).Dimerization induced by ligand binding is a well known regulatory mechanis...

Receptor protein-tyrosine phosphatase ␣ (RPTP␣) belongs to the subfamily of receptor-like protein-tyrosine phosphatases that are characterized by two catalytic domains of which only the membrane-proximal one (D1) exhibits appreciable catalytic activity. The C-terminal catalytic domain (D2) regulates RPTP␣ catalytic activity by controlling rotational coupling within RPTP␣ dimers. RPTP␣-D2 changes conformation and thereby rotational coupling within RPTP␣ dimers in response to changes in the cellular redox state. Here we report a decrease in motility of RPTP␣ from cells treated with H 2 O 2 on non-reducing SDS-polyacrylamide gels to a position that corresponds to RPTP␣ dimers, indicating intermolecular disulfide bond formation. Using mutants of all individual cysteines in RPTP␣ and constructs encoding the individual protein-tyrosine phosphatase domains, we located the intermolecular disulfide bond to the catalytic Cys-723 in D2. Disulfide bond formation and dimer stabilization showed similar levels of concentration and time dependence. However, treatment of lysates with dithiothreitol abolished intermolecular disulfide bonds but not stable dimer formation. Intermolecular disulfide bond formation and rotational coupling were also found using a chimera of the extracellular domain of RPTP␣ fused to the transmembrane and intracellular domain of the leukocyte common antigen-related protein (LAR). These results suggest that H 2 O 2 treatment leads to oxidation of the catalytic Cys in D2, which then rapidly forms a disulfide bond with the D2 catalytic Cys of the dyad-related monomer, rendering an inactive RPTP dimer. Recovery from oxidative stress first leads to the reduction of the disulfide bond followed by a slower refolding of the protein to the active conformation. Protein-tyrosine phosphatases (PTPs)1 form a family of enzymes that catalyze the dephosphorylation of tyrosine residues in proteins. They are characterized by one or two catalytic domains containing a signature sequence (I/V)HCXAGXXR(S/ T/G) including a catalytic cysteine (for review, see Refs. 1 and 2). This cysteine forms a thiol-phosphate intermediate in the dephosphorylation reaction and is therefore essential for enzyme activity (3). Because of the low pK a of the catalytic cysteine, PTPs are very susceptible to oxidation (for review, see Ref. 4). Reactive oxygen species induce oxidation of catalytic cysteines, thereby inactivating these PTPs (5-8). Extracellular stimuli like growth factors and UV irradiation result in an increase in intracellular reactive oxygen species and oxidation of PTPs (5, 9 -12). Inhibition of enzyme activity by oxidative stress is increasingly recognized as an important mechanism of regulation of the PTP family. Therefore, PTPs may serve as sensors of the cellular redox state.RPTP␣ belongs to the receptor-like PTPs that are characterized by a single transmembrane domain. RPTP␣ has two catalytic domains of which the N-terminal one (D1) contains almost all of the catalytic activity of the enzyme. RPTP␣ was found to constitutive...