Antigen-related Protein Tyrosine Phosphatase Research Articles

Liprin-α1 contributes to oncogenic MAPK signaling by counteracting ERK activity.

PTPRF interacting protein alpha 1 (PPFIA1) encodes for liprin-α1, a member of the leukocyte common antigen-related protein tyrosine phosphatase (LAR-RPTPs)-interacting protein family. Liprin-α1 localizes to adhesive and invasive structures in the periphery of cancer cells, where it modulates migration and invasion in head and neck squamous cell carcinoma (HNSCC) and breast cancer. To study the possible role of liprin-α1 in anticancer drug responses, we screened a library of oncology compounds in cell lines with high endogenous PPFIA1 expression. The compounds with the highest differential responses between high PPFIA1-expressing and silenced cells across cell lines were inhibitors targeting mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinases (ERK) signaling. KRAS proto-oncogene, GTPase (KRAS)-mutated MDA-MB-231 cells were more resistant to trametinib upon PPFIA1 knockdown compared with control cells. In contrast, liprin-α1-depleted HNSCC cells with low RAS activity showed a context-dependent response to MEK/ERK inhibitors. Importantly, we showed that liprin-α1 depletion leads to increased p-ERK1/2 levels in all our studied cell lines independent of KRAS mutational status, suggesting a role of liprin-α1 in the regulation of MAPK oncogenic signaling. Furthermore, liprin-α1 depletion led to more pronounced redistribution of RAS proteins to the cell membrane. Our data suggest that liprin-α1 is an important contributor to oncogenic RAS/MAPK signaling, and the status of liprin-α1 may assist in predicting drug responses in cancer cells in a context-dependent manner.

LAR Downregulation Protects the Astrocytic U251 and Cocultured SH-SY5Y Cells in a Rotenone-Induced Parkinson's Disease Cell Model.

Leukocyte common antigen-related protein tyrosine phosphatase (LAR) is a member of the protein tyrosine phosphatase family that serves as a key regulator of cellular survival. It is also involved in neurodevelopment and brain disorders. This study was designed to investigate the role of LAR in a cell-based model of Parkinson's disease (PD) in which U251 and SH-SY5Y cells were used as models of astrocytes and dopaminergic neurons, respectively. Cell viability, cell death, cell morphology, protein phosphorylation and expression, ATP levels, reactive oxygen species (ROS) generation, and mitochondrial membrane potential were analyzed in the wild-type (WT) and heterozygous LAR-knockout astrocytoma U251 cells to assess the cell state, signal transduction, and mitochondrial function. LAR downregulation showed a protective effect in rotenone-exposed U251 cells by increasing cell viability, reducing cell mortality, and restoring appropriate cellular morphology. LAR downregulation enhanced IGF-1R phosphorylation and downstream signal transduction as evidenced by increases in the Akt and GSK-3β phosphorylation, as well as the upregulation of NRF2 and HO-1. The downregulation of LAR also augmented DJ-1 levels in these cells. The enhanced Akt and GSK-3β phosphorylation contributed to a reduced Bax/Bcl2 ratio and suppressed apoptosis after rotenone exposure. Heterozygous LAR-knockout U251 cells exhibited higher mitochondrial function evidenced by increased mitochondrial membrane potential, ATP contents, and reduced ROS production compared to the WT cells following rotenone exposure. Further studies showed that the astrocytic protection mediated by the heterozygous knockout of LAR was associated with the activation of Akt. A specific Akt inhibitor, MK2206, reduced the cell viability, Akt and GSK3β phosphorylation, and HO-1 and NRF2 expression in U251 cells exposed to rotenone. Astrocytes provide structural and metabolic support to maintain neuronal health. Astrocytic glial cell-derived neurotrophic factor (GDNF) production is vital for dopaminergic neuron survival. Heterozygous LAR-knockout U251 cells produced higher amounts of GDNF than the WT cells. The SH-SY5Y cells cocultured with heterozygous LAR-knockout U251 cells exhibited greater viability than that of cells cocultured with WT U251 cells in response to rotenone. Together, these findings demonstrate that the heterozygous knockout of LAR in astrocytes can play a key role in protecting both astrocytic cells and cocultured neurons in a rotenone-induced cell-based model of PD. This neuroprotective effect is attributable to the augmentation of IGF1R-Akt-GDNF signaling and the maintenance of astrocytic mitochondrial function.

LAR-RPTPs Directly Interact with Neurexins to Coordinate Bidirectional Assembly of Molecular Machineries.

Neurexins (Nrxns) and LAR-RPTPs (leukocyte common antigen-related protein tyrosine phosphatases) are presynaptic adhesion proteins responsible for organizing presynaptic machineries through interactions with nonoverlapping extracellular ligands. Here, we report that two members of the LAR-RPTP family, PTPσ and PTPδ, are required for the presynaptogenic activity of Nrxns. Intriguingly, Nrxn1 and PTPσ require distinct sets of intracellular proteins for the assembly of specific presynaptic terminals. In addition, Nrxn1α showed robust heparan sulfate (HS)-dependent, high-affinity interactions with Ig domains of PTPσ that were regulated by the splicing status of PTPσ. Furthermore, Nrxn1α WT, but not a Nrxn1α mutant lacking HS moieties (Nrxn1α ΔHS), inhibited postsynapse-inducing activity of PTPσ at excitatory, but not inhibitory, synapses. Similarly, cis expression of Nrxn1α WT, but not Nrxn1α ΔHS, suppressed the PTPσ-mediated maintenance of excitatory postsynaptic specializations in mouse cultured hippocampal neurons. Lastly, genetics analyses using male or female Drosophila Dlar and Dnrx mutant larvae identified epistatic interactions that control synapse formation and synaptic transmission at neuromuscular junctions. Our results suggest a novel synaptogenesis model whereby different presynaptic adhesion molecules combine with distinct regulatory codes to orchestrate specific synaptic adhesion pathways.SIGNIFICANCE STATEMENT We provide evidence supporting the physical interactions of neurexins with leukocyte common-antigen related receptor tyrosine phosphatases (LAR-RPTPs). The availability of heparan sulfates and alternative splicing of LAR-RPTPs regulate the binding affinity of these interactions. A set of intracellular presynaptic proteins is involved in common for Nrxn- and LAR-RPTP-mediated presynaptic assembly. PTPσ triggers glutamatergic and GABAergic postsynaptic differentiation in an alternative splicing-dependent manner, whereas Nrxn1α induces GABAergic postsynaptic differentiation in an alternative splicing-independent manner. Strikingly, Nrxn1α inhibits the glutamatergic postsynapse-inducing activity of PTPσ, suggesting that PTPσ and Nrxn1α might control recruitment of a different pool of postsynaptic machinery. Drosophila orthologs of Nrxns and LAR-RPTPs mediate epistatic interactions in controlling synapse structure and strength at neuromuscular junctions, underscoring the physiological significance in vivo.

Structural Basis for LAR-RPTP-Mediated Synaptogenesis.

Leukocyte common antigen-related protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that regulate neurite outgrowth and neuronal regeneration. LAR-RPTPs have also received particular attention as the major presynaptic hubs for synapse organization through selective binding to numerous postsynaptic adhesion partners. Recent structural studies on LAR-RPTP–mediated trans-synaptic adhesion complexes have provided significant insight into the molecular basis of their specific interactions, the key codes for their selective binding, as well as the higher-order clustering of LAR-RPTPs necessary for synaptogenic activity. In this review, we summarize the structures of LAR-RPTPs in complex with various postsynaptic adhesion partners and discuss the molecular mechanisms underlying LAR-RPTP–mediated synaptogenesis.

Activity measurement of protein kinase and protein phosphatase by microchip phosphate-affinity electrophoresis

We previously proposed microchip-based phosphate-affinity electrophoresis (μPAE) and demonstrated its application to activity measurement of a tyrosine kinase, c-Src. In this study, we extended the μPAE application to a serine/threonine kinase, protein kinase A (PKA), and to a tyrosine phosphatase, leukocyte antigen-related protein tyrosine phosphatase (LAR PTPase). For standard peptide samples, we obtained linear calibration plots, and the limits of detection were 1.2% (PKA) and 1.5% (LAR PTPase) product peptides in the total peptides. The μPAE was also proven to be effective for unpurified enzyme reaction products.

Pharmacological profiles of a novel protein tyrosine phosphatase 1B inhibitor, JTT‐551

Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signalling, is a novel therapeutic target for type 2 diabetes mellitus. We evaluated in vitro and in vivo the pharmacological profiles of a new PTP1B inhibitor, JTT-551: monosodium ({[5-(1,1-dimethylethyl)thiazol-2-yl]methyl} {[(4-{4-[4-(1-propylbutyl)phenoxy]methyl}phenyl)thiazol-2-yl]methyl}amino)acetate. PTP1B inhibitory activity and the inhibition mode were assayed with p-nitrophenyl phosphate as a substrate, and the selectivity of JTT-551 against other PTPs, including T-cell protein tyrosine phosphatase (TCPTP), CD45 protein tyrosine phosphatase (CD45) and leucocyte common antigen-related protein tyrosine phosphatase (LAR), was evaluated. Glucose uptake with JTT-551 treatment was evaluated in L6 rat skeletal myoblasts (L6 cells). In the in vivo study, we investigated the effects on insulin receptor (IR) phosphorylation and blood chemical parameters with JTT-551 administration in ob/ob mice and db/db mice. JTT-551 showed an inhibitory effect on PTP1B with a Ki value of 0.22 microM, and a mixed-type inhibition mode. Ki values of TCPTP, CD45 and LAR were 9.3, 30 or higher and 30 or higher microM, respectively, and JTT-551 exhibited clear selectivity against the other PTPs. Moreover, JTT-551 increased the insulin-stimulated glucose uptake in L6 cells. A single administration of JTT-551 in ob/ob mice enhanced the IR phosphorylation of liver and reduced the glucose level. In db/db mice, chronic administration showed a hypoglycaemic effect without an acceleration of body weight gain. JTT-551, a newly developed PTP1B inhibitor, improves glucose metabolism by enhancement of insulin signalling and could be useful in the treatment of type 2 diabetes mellitus.

Leukocyte Antigen-related Protein Tyrosine Phosphatase Negatively Regulates Hydrogen Peroxide-induced Vascular Smooth Muscle Cell Apoptosis

Reactive oxygen species (ROS) have been implicated in vascular smooth muscle cell (VSMC) apoptosis, a hallmark of advanced atherosclerotic lesions. Transient oxidation and inactivation of protein-tyrosine phosphatases play a critical role in cellular response to ROS production. However, the function of leukocyte antigen-related (LAR) protein-tyrosine phosphatase in ROS signaling is not known. To determine the expression of LAR in ROS-induced apoptosis, we investigated hydrogen peroxide-induced cell death and signaling in aortic VSMCs from wild-type and LAR(-/-) mice. Histone-associated DNA fragmentation and caspase-3/7 activity were significantly enhanced, mitochondrial membrane integrity was compromised, and cell viability was significantly decreased following H(2)O(2) treatment in LAR(-/-) VSMCs compared with wild-type cells. Stronger and sustained increase in autophosphorylation and activity of Fyn, an Src family tyrosine kinase, was observed in LAR(-/-) cells compared with wild-type cells following H(2)O(2) treatment. LAR binds to activated Fyn in H(2)O(2)-treated VSMCs, and recombinant LAR dephosphorylates phosphorylated-Fyn in vitro. In addition, LAR deficiency enhanced H(2)O(2)-induced phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). PP2, a Fyn-specific inhibitor, blocked JAK2, STAT3, and p38 MAPK activation and significantly attenuated apoptosis induced by H(2)O(2). AG490, a JAK2-specific inhibitor, significantly attenuated H(2)O(2)-induced apoptosis, and blocked H(2)O(2)-induced activation of STAT3, but not p38 MAPK in both wild-type and LAR(-/-) VSMCs. Attenuation of Fyn expression by short hairpin RNA significantly decreased H(2)O(2)-induced downstream signaling and apoptosis in VSMCs. Together, these data indicate that LAR regulates Fyn/JAK2/STAT3 and Fyn/p38 MAPK pathways involved in ROS-induced apoptosis.

Leukocyte Antigen-related Protein Tyrosine Phosphatase Negatively Regulates Hydrogen Peroxide-induced Vascular Smooth Muscle Cell Apoptosis

Leukocyte Antigen-related Protein Tyrosine Phosphatase Negatively Regulates Hydrogen Peroxide-induced Vascular Smooth Muscle Cell Apoptosis

Knock-down of LAR protein tyrosine phosphatase induces insulin resistance

To test the role of the leukocyte common antigen-related protein tyrosine phosphatase (LAR) as a regulator of insulin receptor (IR) signalling, an siRNA probe against LAR was developed. Knock-down of LAR induced post-receptor insulin resistance with the insulin-induced activation of PKB/Akt and MAP kinases markedly inhibited. The phosphorylation and dephosphorylation of the IR and insulin receptor substrate (IRS) proteins were unaffected by LAR knock-down. These results identify LAR as a crucial regulator of the sensitivity of two key insulin signalling pathways to insulin. Moreover, the siRNA probe provides a molecular tool of general applicability for further dissecting the precise targets and roles of LAR.

Targets of fibroblast growth factor 1 (FGF-1) and FGF-2 signaling involved in the invasive and tumorigenic behavior of carcinoma cells.

Fibroblast growth factor (FGF)-1 and -2 have potent biological activities implicated in malignant tumor development. Their autocrine and nonautocrine activity in tumor progression of carcinoma was investigated in the NBT-II cell system. Cells were manipulated to either produce and be autocrine for FGF-1 or -2 or to only produce but not respond to these factors. The autocrine cells are highly invasive and tumorigenic and the determination of specific targets of FGF/fibroblast growth factor receptor (FGFR) signaling was assessed. In vitro studies showed that nonautocrine cells behave like epithelial parental cells, whereas autocrine cells have a mesenchymal phenotype correlated with the overexpression of urokinase plasminogen activator receptor (uPAR), the internalization of E-cadherin, and the redistribution of beta-catenin from the cell surface to the cytoplasm and nucleus. uPAR was defined as an early target, whereas E-cadherin and the leukocyte common antigen-related protein-tyrosine phosphatase (LAR-PTP) were later targets of FGF signaling, with FGFR1 activation more efficient than FGFR2 at modulating these targets. Behavior of autocrine cells was consistent with a decrease of tumor-suppressive activities of both E-cadherin and LAR-PTP. These molecular analyses show that the potential of these two growth factors in tumor progression is highly dependent on specific FGFR signaling and highlights its importance as a target for antitumor therapy.

Identification of hepatic transcriptional changes in insulin-resistant rats treated with peroxisome proliferator activated receptor-alpha agonists.

Peroxisome proliferator activated receptor (PPAR)-alpha controls the expression of multiple genes involved in lipid metabolism, and activators of PPAR-alpha, such as fibrates, are commonly used drugs in the treatment of hypertriglyceridemia and other dyslipidemic states. Recent data have also suggested a role for PPAR-alpha in insulin resistance and glucose homeostasis. In the present study, we have assessed the transcriptional and physiological responses to PPAR-alpha activation in a diet-induced rat model of insulin resistance. The two PPAR-alpha activators, fenofibrate and Wy-14643, were dosed at different concentrations in high-fat fed Sprague-Dawley rats, and the transcriptional responses were examined in liver using cDNA microarrays. In these analyses, 98 genes were identified as being regulated by both compounds. From this pool of genes, 27 correlated to the observed effect on plasma insulin, including PPAR-alpha itself and the leukocyte antigen-related protein tyrosine phosphatase (PTP-LAR). PTP-LAR was downregulated by both compounds, and showed upregulation as a result of the high-fat feeding. This regulation was also observed at the protein level. Furthermore, downregulation of PTP-LAR by fenofibric acid was demonstrated in rat FaO hepatoma cells in vitro, indicating that the observed regulation of PTP-LAR by fenofibrate and Wy-14643 in vivo is mediated as a direct effect of the PPAR agonists on the hepatocytes. PTP-LAR is one of the first genes involved in insulin receptor signaling to be shown to be regulated by PPAR-alpha agonists. These data suggest that factors apart from skeletal muscle lipid supply may influence PPAR-alpha-mediated amelioration of insulin resistance.

Leukocyte Antigen-Related Protein Tyrosine Phosphatase Receptor: A Small Ectodomain Isoform Functions as a Homophilic Ligand and Promotes Neurite Outgrowth

The identities of ligands interacting with protein tyrosine phosphatase (PTP) receptors to regulate neurite outgrowth remain mainly unknown. Analysis of cDNA and genomic clones encoding the rat leukocyte common antigen-related (LAR) PTP receptor predicted a small, approximately 11 kDa ectodomain isoform, designated LARFN5C, containing a novel N terminal followed by a C-terminal segment of the LAR fifth fibronectin type III domain. RT-PCR and Northern blot analysis confirmed the presence of LARFN5C transcripts in brain. Transfection of COS cells with LARFN5C-Fc cDNA resulted in expression of the predicted protein, and Western blot analysis verified expression of approximately 11 kDa LARFN5C protein in vivo and its developmental regulation. Beads coated with rLARFN5C demonstrated aggregation consistent with homophilic binding, and pull-down and immunoprecipitation assays demonstrated that rLARFN5C associates with the LAR receptor. rLARFN5C binding to COS cells was dependent on LAR expression, and rLARFN5C binding to LAR +/+ hippocampal neurons was fivefold greater than that found by using LAR-deficient (-/-) neurons. Substratum-bound rLARFN5C had potent neurite-promoting effects on LAR +/+ neurons, with a fivefold loss in potency with the use of LAR -/- neurons. rLARFN5C in solution at low nanomolar concentrations inhibited neurite outgrowth induced by substratum-bound rLARFN5C, consistent with receptor-based function. These studies suggest that a small ectodomain isoform of a PTP receptor can function as a ligand for the same receptor to promote neurite outgrowth.

Leukocyte Antigen-Related Protein Tyrosine Phosphatase Receptor: A Small Ectodomain Isoform Functions as a Homophilic Ligand and Promotes Neurite Outgrowth

Leukocyte Antigen-Related Protein Tyrosine Phosphatase Receptor: A Small Ectodomain Isoform Functions as a Homophilic Ligand and Promotes Neurite Outgrowth

A decrease in size and number of basal forebrain cholinergic neurons is paralleled by diminished hippocampal cholinergic innervation in mice lacking leukocyte common antigen-related protein tyrosine phosphatase activity

The leukocyte common antigen-related (LAR) receptor, composed of an extracellular region with three immunoglobulin-like and eight fibronectin type III-like domains, and a cytoplasmic region containing two protein tyrosine phosphatase domains, is thought to play a role in axonal outgrowth and guidance during neural development. LAR mutant mice were generated completely lacking the two cytoplasmic protein tyrosine phosphatase domains, resulting in the loss of ability to bind intracellular associating proteins, but (may be) still containing the ability to perform extracellular functions. A reduction in size of basal forebrain cholinergic neurons and diminished hippocampal innervation reported for knockout mice that contain a leaky gene trap inserted into the 5′ part of the LAR gene [Yeo T. T. et al. (1997) J. Neurosci. Res. 47, 348–360] warranted a computer-assisted quantitative image analysis throughout the basal forebrain and hippocampus of our LAR mutant mice. The total number, longest diameter and cell body area were calculated for the choline acetyltransferase-positive neurons in the medial septum and vertical diagonal band, and optical density measurements were performed to determine the extent of acetyl cholinesterase-positive fibre innervation of the different layers in the dentate gyrus. In LAR mutant mice, the number of cholinergic cells was significantly reduced (∼25%) in the vertical diagonal band. Also, the cross-sectional area of the cholinergic neurons in the medial septum and vertical diagonal band was reduced (5%). These findings were paralleled by a diminished cholinergic innervation of the supragranular (18%) and molecular (4%) layers of the dentate gyrus. Thus, LAR protein tyrosine phosphatase activity appears crucial for size, number and target projection of basal forebrain cholinergic neurons, further strengthening a role for LAR in CNS development.

Extinction of insulin-like growth factor-I mitogenic signaling by antiestrogen-stimulated Fas-associated protein tyrosine phosphatase-1 in human breast cancer cells.

Steroidal (ICI 182, 780) and nonsteroidal hydroxytamoxifen (OH-Tam) antiestrogens inhibit growth factor-mitogenic activity in MCF 7 estrogen receptor-positive human breast cancer cells. Cell inhibition is correlated with an increase in membrane protein tyrosine phosphatase (PTP) activity, and the addition of orthovanadate prevents OH-Tam inhibition. After RT-PCR cloning of PTPs expressed in MCF 7 cells with primers to their catalytic domains, we have shown, by differential screening, that the expression of two enzymes, leukocyte common antigen-related PTP (LAR) and Fas-associated PTP-1 (FAP-1), was modulated by antiestrogens. By comparative RT-PCR, in situ hybridization, and Northern blot, LAR and FAP-1 mRNAs accumulation was found to be dose- and time-dependently increased by antiestrogens. To further demonstrate that PTPs were key mediators of antiestrogen-inhibitory action on the growth factor pathway, a panel of stable FAP-1 transfectants expressing low to high levels of antisense mRNAs was established. In these clones, the level of antisense RNA expression was correlated with a reduction in basal levels and a complete inhibition of antiestrogen-stimulated values of PTP activity. When FAP-1 expression was abolished, OH-Tam was no longer able to block insulin-like growth factor I mitogenic activity even though it remained strongly antiestrogenic. However, ICI 182,780 was still inhibitory, indicating that its effect was not exclusively mediated by PTP. Our data first demonstrate that a specifically regulated phosphatase (FAP-1) is implicated in the triggering of negative proliferation signals in breast cancer cells.

Novel alternative splicing predicts a secreted extracellular isoform of the human receptor-like protein tyrosine phosphatase LAR

RT-PCR was used to examine the expression of LAR (encoding the leukocyte-common antigen-related protein tyrosine phosphatase) in normal human colon mucosa, and colon polyps and tumors. Although the LAR protein was not detected in the colon in a previous immunohistochemical study, amplification of a region of LAR between the most membrane proximal (eighth) fibronectin type-III (FN-III) repeat and the transmembrane domain demonstrated LAR expression in all samples, but showed no difference in expression within matched samples from each patient examined. An additional minor fragment amplified in all reactions was consistently observed in colon and various cell line samples using this and two other LAR-specific sets of primers. Cloning and sequencing of the fragment identified it as deriving from a novel alternatively spliced form of LAR containing a retained intron of 85 bp. This intron encodes an additional 13 amino acids followed by an in-frame stop codon, thus its retention is predicted to give rise to a secreted LAR extracellular region isoform(s). LAR transcripts containing the intron were detected by RNase protection assay of colon samples and were present in most human tissues examined by Northern analysis. A protein in colon tumor extract was recognized by antiserum raised to the intron-encoded sequence. Soluble isoforms of the LAR extracellular immunoglobulin (Ig)-like/FN-III repeat-containing region could have a biological function distinct from those isoforms localized at the cell surface and/or coupled to intracellular phosphatase activity.

Cloning and Characterization of Islet Cell Antigen-related Protein-tyrosine Phosphatase (PTP), a Novel Receptor-like PTP and Autoantigen in Insulin-dependent Diabetes

Cloning of the cDNA encoding a novel human protein- tyrosine phosphatase (PTP) called islet cell antigen-related PTP (IAR) predicts a receptor-like molecule with an extracellular domain of 614 amino acids containing a hydrophobic signal peptide, one potential N-glycosylation site, and an RGDS peptide which is a possible adhesive recognition sequence. The 376-amino acid intracellular region contains a single catalytic domain. Recombinant IAR polypeptide has phosphatase activity. Northern blot analysis shows tissue-specific expression of two IAR transcripts of 5.5 and 3. 7 kilobases, which are most abundant in brain and pancreas. The IAR PTP is homologous in its intracellular region to IA-2, a putative PTP that is an insulin-dependent diabetes mellitus (IDDM) autoantigen. IAR is also reactive with IDDM patient sera. IAR and IA-2 may distinguish different populations of IDDM autoantibodies since they identify overlapping but nonidentical sets of IDDM patients. Thus IAR is likely to be an islet cell antigen useful in the preclinical screening of individuals for risk of IDDM.

Identification of a Protein-Tyrosine Phosphatase from Human Platelet Membranes by an Immobilon-Based Solid Phase Assay

The present investigations show that a 53-kDa platelet-membrane protein is a protein tyrosine phosphatase. Identification involved a novel methodology in which membrane proteins are resolved by polyacrylamide gel electrophoresis and then transblotted to a polyvinylidene difluoride membrane surface-labeled with [32P-Tyr](Glu4 · Tyr1)n. Phosphatase activity appears as clear areas in the autoradiograph of the renatured 32P Western blot. Studies using this new solid-phase system indicate that a 53-kDa platelet-membrane protein dephosphorylates [32P-Tyr](Glu4 · Tyr1)n. Dephosphorylation is both time- and dose-dependent. The protein-tyrosine phosphatase antagonists orthovanadate and molybdate block dephosphorylation in a concentration-dependent manner. Inhibitors of protein serine/threonine phosphatases and of acid and alkaline phosphatases do not significantly affect enzymatic activity. The enzyme is active toward phosphotyrosyl proteins but not phosphoseryl proteins. Results from the solid-phase phosphatase assays correlate with data from standard liquid-phase studies. A Lineweaver-Burk plot gives an apparent Km of 4.3 μM and an apparent Vmax = 168 nmol Pi transferred/min/mg for the membrane enzyme towards the 32P-labeled polymer in a liquid system. Platelet lysate was shown by the solid-phase assay to possess a protein-tyrosine phosphatase of ≅50 kDa. The tyrosine phosphatase activities associated with a placental preparation, pure recombinantprotein-tyrosine phosphatase 1B. and YOPR, the product of the YOP51 gene of Yersinia enterocolitica, which contains the C235R mutation, were also visualized by this technique. However, the catalytic activity of domain 1 of the transmembrane leukocyte antigen-related protein-tyrosine phosphatase was not detected using the solid-phase assay.