Phosphotyrosine Protein Phosphatase Activity Research Articles

Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa.

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.

Low Mr phosphotyrosine protein phosphatase activity on fibroblast growth factor receptor is not associated with enzyme translocation

Fibroblast growth factor receptor (class IV) shares a certain degree of similarity with class III members like platelet-derived growth factor and macrophage-colony-stimulating factor receptors, which, once activated, are substrates of low M r phosphotyrosine protein phosphatase. Up until now no phosphotyrosine phosphatase has been shown to act on this receptor in vivo. Here we demonstrate that low M r phosphotyrosine protein phosphatase is able to reduce receptor tyrosine phosphorylation and cell proliferation in response to basic fibroblast growth factor. Contrary to what was previously observed for platelet-derived growth factor, during cell stimulation with basic fibroblast growth factor, no enzyme redistribution among cellular compartments is observed.

Phosphotyrosine protein phosphatases activation by acth in rat adrenal gland

The steady state level of most cellular phosphoproteins is dependent on the relative catalytic activities of intracellular protein kinases and phosphatases. In adrenal cortex, ACTH acts through PKA activation and Ser/Tre phosphorylation. Phosphatases involved in this pathway are not completely described, particularly the role of phosphotyrosine protein phosphatase (PTP) activity on ACTH action. We investigated potential changes in PTPs activity in adrenal gland upon in vivo and in vitro PKA activation. In vivo ACTH stimulates cytosolic PTP activity (2-fold). Similar effect is detected by in vitro stimulation. In accordance with the effects of ACTH on PTP activity, cell permeable PTP inhibitors block ACTH stimulation on adrenal zona fasciculata (ZF) cells: ACTH (1 nM) = 108.2 ± 3.5 ng corticosterone/105 cells vs. ACTH + phenylarsine oxide (2 nM) = 60 ± 4 (P < 0,001) and ACTH + pervanadate (10 mM) = 68 ± 2 (P < 0,01). These results are reproduced when cells are stimulated with cAMP. The inhibition is not observed when steroidogenesis is supported by 22(R)OH cholesterol. We describe, for the first time, a hormonal regulation of PTP activity. According to the effect of PTP inhibitors on steroid production activated by ACTH we propose that PTP activation is a crucial event in hormone action in the steroidogenic pathway. We also propose that PTP activity is located after PKA activation and prior to cholesterol transport to the inner mitochondrial membrane.

The endogenous substrate of low molecular weight acid phosphatase in the brain is an epidermal growth factor receptor

We have recently reported that low molecular weight (LMW) acid phosphatase, which is supposed to possess phosphotyrosine protein phosphatase activity, showed a significant decrease of activity in Alzheimer brains compared to control brains [ Ann. Neurol., 33 (1993) 616–621]. In the present study, we investigated the endogenous substrate of LMW acid phosphatase in the brain. LMW acid phosphatase was purified from bovine brain, and the enzyme was obtained with both a high specific activity and a good yield. The bovine brain enzyme was a monomer with a molecular mass of 17 kDa. We used a specific monoclonal anti-phosphotyrosine antibody to detect phosphotyrosine protein in rat brain extracts. The LMW acid phosphatase from bovine brain dephosphorylated a M r 170 kDa phosphotyrosine protein in rat brain extracts. This M r 170 kDa protein was considered to be the epidermal growth factor (EGF) receptor using a specific antibody. These results suggest that LMW acid phosphatase in the brain may regulate EGF receptor-dependent transmembrane signalling by dephosphorylating the phosphorylated receptor.

P-509 - Inhibition study and partial purification of phosphotyrosine protein phosphatase activity of a clonal osteoblastic cell line (MC3T3-E1 cell).

P-509 - Inhibition study and partial purification of phosphotyrosine protein phosphatase activity of a clonal osteoblastic cell line (MC3T3-E1 cell).

Placental alkaline phosphatase activity is inversely related to cell growth rate in HeLaS3 cervical cancer cells

Placental alkaline phosphatase is an inducible enzyme, expressed in HeLaS3 cells, which has been shown to possess protein phosphotyrosine phosphatase activity. Since phosphotyrosine levels are known to increase in actively dividing cells we sought an inverse correlation between PLAP activity and growth rate in HeLaS3 cells. We found that PLAP inducers, Na-butyrate, dexamethasone, bromodeoxyuridine and dibutyryl cAMP caused a dose-dependent reduction in growth rate. Mimosine, an agent that blocks the cell cycle in Gl, caused an increase in PLAP activity whilst the mitogen EGF caused a corresponding decrease in PLAP activity. PLAP activity may therefore be related to cell proliferation rate.

Reduction of low‐molecular‐ weight acid phosphatase activity in Alzheimer brains

Recent studies in Alzheimer brains have shown aberrant protein phosphorylation, suggesting an alteration in protein kinases and/or phosphoprotein phosphatases. In the present study, the activity of acid phosphatase was investigated in samples prepared from postmortem normal human and Alzheimer brains. p-Nitrophenyl phosphate, a nonprotein phosphoester, was used as a substrate for acid phosphatase. The separation profile on Sephadex G-100 gel filtration chromatography revealed that two major forms of high-molecular-weight and low-molecular-weight acid phosphatase were present in the crude extracts of both rat and human brains. Another class of zinc ion (Zn2+)-dependent acid p-nitrophenyl phosphatase was also detected in rat and human brains. In Alzheimer brains, the low-molecular-weight acid phosphatase activity was significantly decreased compared to that in control brains; however, the high-molecular-weight and Zn(2+)-dependent acid phosphatase activity in control and Alzheimer brains was not different. These results suggest that reduced activity of the low-molecular-weight acid phosphatase, which possesses phosphotyrosine protein phosphatase activity, might be linked to aberrant protein tyrosine phosphorylation found in Alzheimer brains.

Insulin differentially regulates protein phosphotyrosine phosphatase activity in rat hepatoma cells.

We have studied the effect of insulin stimulation on phosphotyrosine phosphatase (PTPase) activity in the well-differentiated rat hepatoma cell line Fao. PTPase activity was measured using a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation. Of the PTPase activity in Fao cells, 14% was in the cytosolic fraction, whereas 86% was in the particulate fraction; this latter fraction also had a 4-fold higher specific activity. Purification of the particulate fraction by lectin chromatography resulted in a 50% increase in specific activity, although this glycoprotein-rich fraction contained only 1.5% of the total activity. Both the cytosolic and particulate PTPase fractions were active toward the tyrosyl-phosphorylated insulin receptor in vitro. The activity of the particulate fraction but not the cytosolic fraction was inhibited by addition of a micromolar concentration of a phosphorylated peptide corresponding to residues 1142-1153 of the human insulin receptor sequence. By contrast, addition of the nonphosphorylated peptide even at millimolar concentration was without effect. Both PTPase fractions were inhibited by Zn+ at similar concentrations, whereas the cytosolic PTPase activity was 10-fold more sensitive to vanadate inhibition. Treatment of cells with 100 nM insulin increased PTPase activity in the particulate fraction by 40% and decreased activity in the cytosolic fraction by 35%. These effects occurred within 15 min and were half-maximal at 3-4 nM insulin. When assessed as total activity, the magnitude of the changes in PTPase activity in the particulate and cytosolic fractions could not be explained on the basis of a translocation of PTPases between the two pools.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat liver low M(r) phosphotyrosine protein phosphatase isoenzymes: purification and amino acid sequences.

Two low M(r) phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as low M(r) acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH2-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40-73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.

Differential activation of phosphotyrosine protein phosphatase activity in a murine T cell hybridoma by monoclonal antibodies to CD45.

The effect of two anti-CD45 (T200, LCA, Ly5) antibodies on the activation of the murine T-cell hybridoma 13.13 has been evaluated. These studies have been carried out in a system that did not require cross-linking or coclustering of antibodies. Activation of 13.13 cells with the anti-CD3 monoclonal antibody, 145.2C11, gave rise to rapid increases in intracellular calcium and interleukin-2 production. Additionally, within 1 min, phosphorylation on tyrosine of four major proteins of about 130,000, 110,000, 80,000, and 37,000 daltons could be seen. Pretreatment of the cells with the anti-CD45 mAb M1/89.18.7.HK markedly inhibited all three biological responses, while an alternate anti-CD45 antibody, M1/9.3.4.HL.2, had little effect. The two antibodies bound to CD45 with similar affinities, and no differences in the lateral mobility of antibody-CD45 complexes in the cell membrane were observed. The inhibition of activation of the cells by M1/89.18.7.HK was abrogated significantly both by the phosphotyrosine protein phosphatase inhibitor orthovanadate and by excess M1/9.3.4.HL.2. If M1/89.18.7.HK was added to the 13.13 cells after they had already been activated with anti-CD3, it very effectively stimulated dephosphorylation of substrates that had been phosphorylated on tyrosines prior to adding the anti-CD45 antibody. These results indicate that the phosphotyrosine protein phosphatase activity of CD45 is critical to its biological function and that bivalent (i.e. uncross-linked) anti-CD45 antibodies can give rise to markedly different responses. One of the antibodies, M1/89.18.7.HK, appears to behave much like a receptor ligand and is able to activate the enzymatic activity associated with the CD45 transmembrane protein.

Endogenous protein substrates for prostatic acid phosphatase in human prostate

In order to identify the endogenous phosphoprotein substrates for human prostatic acid phosphatase (PAP), cellular proteins of human normal, benign, and malignant prostatic tissues as well as carcinoma cell lines were phosphorylated by the cellular kinases in the presence of (gamma-32P)-ATP and then were subjected to dephosphorylation reaction by PAP. Of several endogenous phosphoproteins, PAP preferentially dephosphorylated a cytosolic protein of Mr 83 kDa. The dephosphorylation of the 83 kDa phosphoprotein (designated pp83) by PAP was uniformly observed in all cells/tissues of prostate origin, and was completely inhibited by L(+)-tartrate, the classic inhibitor of PAP. Phosphoamino acid analysis revealed that pp83 was a tyrosine-poor phosphoprotein and was mostly dephosphorylated by PAP at serine/threonine residues rather than tyrosine residues. Further comparison of dephosphorylation rate with that of an endogenous phosphotyrosine-containing phosphoprotein (pp53) revealed that PAP possessed both phosphoserine/threonine protein phosphatase and phosphotyrosine protein phosphatase activity. These results demonstrate that pp83 apparently is an endogenous substrate of PAP in human prostate, and that, instead of a phosphotyrosine protein specific phosphatase, PAP is a universal protein phosphatase hydrolyzing equally well the phosphotyrosine, serine, and threonine residues.

Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines.

CD45 is a family of high molecular weight leukocyte cell surface glycoproteins. Recently, two related subregions of the cytoplasmic domain of CD45 have been shown to have 30-40% amino acid identity with a human placental protein phosphotyrosine phosphatase, and CD45 isolated from human spleen was found to exhibit intrinsic protein phosphotyrosine phosphatase (EC 3.1.3.48) activity. In the present studies, we demonstrate that each of the known isoforms of murine CD45 has an equivalent basal level of protein phosphotyrosine phosphatase activity and establish that this enzymatic activity is associated with the cytoplasmic domain of the glycoprotein. Studies with three independent sets of well-characterized parental CD45+, mutant CD45-, and revertant CD45+ lymphoma cell lines indicate that loss of CD45 increases the phosphorylation of the src-related leukocyte-specific tyrosine protein kinase p56lck on tyrosine-505, a putative negative regulatory site. This suggests that CD45 may play a role in leukocyte growth regulation by altering the kinase activity of p56lck.

Purification and characterization of a protein-phosphotyrosine phosphatase from rat spleen which dephosphorylates and inactivates a tyrosine-specific protein kinase

A particulate form of protein-phosphotyrosine phosphatase was solubilized and purified over 2,000-fold from the particulate fraction of rat spleen. Phosphorylated poly(Glu, Tyr), a random copolymer of glutamic acid and tyrosine, was used as substrate for measuring protein-phosphotyrosine phosphatase activity. Nonionic detergents like Triton X-100 increased the protein-phosphotyrosine phosphatase activity of the particulate fraction (but not of the soluble fraction) by 4-8-fold. Chromatography of the Triton extract of the particulate fraction on DEAE-Sephacel gave three peaks of protein-phosphotyrosine phosphatase activity. The major peak of activity was further purified on Bio-Gel HTP, Sephadex G-75, and phosphocellulose columns. On polyacrylamide gel electrophoresis in the presence of Na-dodecyl-SO4 the purified enzyme showed a major protein band of Mr 36,000 which comigrated with enzyme activity on the phosphocellulose column. The apparent Vmax and Km for phosphorylated poly(Glu,Tyr) were 6,150 nmol min-1 mg-1 and 1.6 microM, respectively. This enzyme was strongly inhibited by microM concentrations of orthovanadate and zinc acetate. Fluoride (50 mM) inhibited this enzyme only by 30-40%. Divalent metal ions Ca2+, Mg2+, and Mn2+ were inhibitory at 1-10 mM concentration. EDTA had no effect on the activity of the purified enzyme. This phosphatase could dephosphorylate and inactivate the phosphorylated form of a tyrosine-specific protein kinase (TK-I) previously purified from rat spleen. Dephosphorylation and inactivation of TK-I by purified phosphatase were inhibited by orthovanadate. After dephosphorylation and inactivation by phosphatase, TK-I could be rephosphorylated and reactivated on incubation with ATP. These results suggest that this protein-phosphotyrosine phosphatase may be involved in the regulation of the kinase activity of TK-I.

Purification and characterization of a protein tyrosine kinase from bovine spleen.

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.

Vanadate stimulation of phosphotyrosine protein levels in quiescent nakano mouse lens cells

Incubation of quiescent Nakano mouse lens epithelial cells with sodium orthovanadate resulted in time- and concentration-dependent stimulation of protein tyrosine phosphorylation levels in the cells. Protein tyrosine phosphorylation in the 27,000 g pellet showed a 100% stimulation by vanadate. However, upon detergent solubilization, 30% activation of basal endogenous tyrosine phosphorylation was observed but no additional increase was obtained with vanadate. Protein phosphotyrosine phosphatase activity was found in both pellet and cytosolic fractions of the cell. Vanadate inhibited these activities with an IC50 of 57 microM in the cytosolic fraction and 3 microM in the pellet. These data suggest that vanadate increases phosphotyrosine protein levels in these cells by inhibition of a membrane-associated tyrosine-specific phosphatase rather than by activation of protein tyrosine kinases. These data correlate well with the vanadate stimulation of DNA synthesis in these cells, thus indicating a role for phosphotyrosine proteins in regulation of cell division in these cells.

Regulation of protein phosphotyrosine content by changes in tyrosine kinase and protein phosphotyrosine phosphatase activities during induced granulocytic and monocytic differentiation of HL-60 leukemia cells

About 1.5% of phosphorylated amino acid residues of HL-60 promyelocytic leukemia cells are phosphotyrosine. Induction of granulocytic differentiation by exposure to dimethylsulfoxide decreased tyrosine phosphorylation to 0.2%. A maximum 3-fold increase in tyrosine kinase activity and a 7-fold increase in protein phosphotyrosine phosphatase activity accompanied this change. Monocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate, caused a decrease in phosphotyrosine levels to 0.1%; tyrosine kinase activity maximally increased 2-fold, and protein phosphotyrosine phosphatase activity increased 11-fold in these differentiated cells. Thus, although total tyrosine kinase activity markedly increased during differentiation, this was counteracted by an even greater elevation in protein phosphotyrosine phosphatase activity. The findings support the concept that tyrosine phosphorylation is important in the regulation of growth and differentiation of leukemia cells.

The human red cell acid phosphatase is a phosphotyrosine protein phosphatase which dephosphorylates the membrane protein band 3

Human red cell cytosol acid phosphatase activity is supported by a main enzyme which can be extracted by DEAE and phosphocellulose chromatography. It uses pNPP as a substrate and is a protein phosphatase specific to phosphotyrosine. It dephosphorylates the tyrosine-phosphorylated cytosolic fragment of membrane protein 3. When taken together, these results suggest that the physiological role of red cell acid phosphatase is the FB3 phosphotyrosine dephosphorylation. Whatever it may be phosphotyrosine protein phosphatase activity is the first role of red cell acid phosphatase to be demonstrated.

Specificity of protein phosphotyrosine phosphatases. Comparison with mammalian alkaline phosphatase using polypeptide substrates.

The specificity of cytosolic protein phosphotyrosine (PPT) phosphatases was investigated using different peptides and proteins that were phosphorylated on tyrosine residues by the EGF receptor kinase. The acidic phosphoproteins, serum albumin, casein, and myosin light chains, were dephosphorylated by the PPT phosphatases with apparent Km values of 1.2 to 12.5 microM and apparent velocities of 0.2 to 18 mumol/min/mg. In contrast, [Tyr(32P)]histone and the phosphotyrosine peptides [Val5]angiotensin and RR-src, a peptide with sequence Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, were unreactive with the PPT phosphatases. However, each of these unreactive phosphopolypeptides was dephosphorylated under the same conditions by calf-intestine alkaline phosphatase. The data reveal how PPT phosphatase activity has been ascribed to different cellular enzymes. When acidic phosphotyrosine proteins were used as substrates in assays for PPT phosphatase activity the cytosolic enzymes were isolated, whereas when phosphotyrosine histones were used as substrates only the membrane-bound alkaline phosphatase was detected. Apparently the protein tyrosine kinase and the protein tyrosine phosphatases do not have the same specificity, so substrates such as histone, angiotensin, or RR-src are phosphorylated but not hydrolyzed. Therefore, these polypeptides would be ideal for the characterization of protein tyrosine kinases in cellular extracts.

An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.