Human prostatic acid phosphatase has phosphotyrosyl protein phosphatase activity.
ABSTRACT: The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 188.8.131.52), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.
Project description:We describe the isolation and characterization of a gene (ptpA) from Streptomyces coelicolor A3(2) that codes for a protein with a deduced M(r) of 17,690 containing significant amino acid sequence identity with mammalian and prokaryotic small, acidic phosphotyrosine protein phosphatases (PTPases). After expression of S. coelicolor ptpA in Escherichia coli with a pT7-7-based vector system, PtpA was purified to homogeneity as a fusion protein containing five extra amino acids. The purified fusion enzyme catalyzed the removal of phosphate from p-nitrophenylphosphate (PNPP), phosphotyrosine (PY), and a commercial phosphopeptide containing a single phosphotyrosine residue but did not cleave phosphoserine or phosphothreonine. The pH optima for PNPP and PY hydrolysis by PtpA were 6.0 and 6.5, respectively. The Km values for hydrolysis of PNPP and PY by PtpA were 0.75 mM (pH 6.0, 37 degrees C) and 2.7 mM (pH 6.5, 37 degrees C), respectively. Hydrolysis of PNPP by S. coelicolor PtpA were 0.75 mM (pH 6.0, 37 degrees C) and 2.7 mM (pH 6.5, 37 degrees C), respectively. Hydrolysis of PNPP by S. coelicolor PtpA was competitively inhibited by dephostatin with a Ki of 1.64 microM; the known PTPase inhibitors phenylarsine oxide, sodium vanadate, and iodoacetate also inhibited enzyme activity. Apparent homologs of ptpA were detected in other streptomycetes by Southern hybridization; the biological functions of PtpA and its putative homologs in streptomycetes are not yet known.
Project description:Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 184.108.40.206). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/reducing conditions) of nickel-affinity purified protein revealed the presence of a near-homogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 x 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., 'nonspecific' substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.
Project description:4-Nitrophenyl phosphate (p-nitrophenyl phosphate, pNPP) is widely used as a small molecule phosphotyrosine-like substrate in activity assays for protein tyrosine phosphatases. It is a colorless substrate that upon hydrolysis is converted to a yellow 4-nitrophenolate ion that can be monitored by absorbance at 405 nm. Therefore, the pNPP assay has been widely adopted as a quick and simple method to assess phosphatase activity and is also commonly used in assays to screen for inhibitors. Here, the first crystal structure is presented of a dual-specificity phosphatase, human dual-specificity phosphatase 22 (DUSP22), in complex with pNPP. The structure illuminates the molecular basis for substrate binding and may also facilitate the structure-assisted development of DUSP22 inhibitors.
Project description:During the purification of annexin VI from pig lung, we previously reported the isolation of another 67 kDa protein (protein 67E) differing from the former by immunological reactivity, amino acid composition, inability to interact with anionic phospholipids in the presence of Ca2+ and inability to inhibit phospholipase A2 [Fauvel, Vicendo, Roques, Ragab-Thomas, Granier, Vilgrain, Chambaz, Rochat, Chap & Douste-Blazy (1987) FEBS Lett. 221, 397-402]. Attempts to phosphorylate protein 67E by the protein tyrosine kinase of epidermal-growth-factor receptor revealed a dramatic inhibition of receptor autophosphorylation, which was also observed with insulin receptor. This inhibitory effect was found to be supported by a phosphatase active towards p-nitrophenyl phosphate, phosphotyrosine, [32P]phosphotyrosyl histones and [32P]phosphotyrosyl poly(Glu,Tyr), but inactive towards phosphoserine, phosphothreonine and [32P]phosphoseryl histones. Although not purified to complete homogeneity, the enzyme was purified 273-fold over EGTA extracts from pig lung and corresponded to a monomeric protein displaying an apparent molecular mass of 67 kDa. With [32P]phosphotyrosyl poly(Glu,Tyr) as substrate, the purified enzyme displayed Km and Vmax. values of 10 microM and 1.93 mumol/min per mg respectively, which compare reasonably well with other recently described phosphotyrosyl protein phosphatases. From these data and from its sensitivity to various inhibitors, it is concluded that protein fraction 67E contains a novel phosphotyrosyl protein phosphatase, the association of which with annexin extract might offer a clue to the understanding of its possible targeting to membrane substrates.
Project description:Tyrosyl phosphorylation plays an important regulatory role in osteoclast formation and activity. Phosphotyrosyl phosphatases (PTPs), in addition to tyrosyl kinases, are key determinants of intracellular tyrosyl phosphorylation levels. To identify the PTP that might play an important regulatory role in osteoclasts, we sought to clone an osteoclast-specific PTP. A putative full-length clone encoding a unique PTP (referred to as PTP-oc) was isolated from a 10-day-old rabbit osteoclastic cDNA library and sequenced. A single open reading frame predicts a protein with 405 amino acid residues containing a putative extracellular domain, a single transmembrane region, and an intracellular portion. PTP-oc is structurally unique in that, unlike most known transmembrane PTPs, it has a short extracellular region (eight residues), lacks a signal peptide proximal to the N-terminus, and contains only a single 'PTP catalytic domain'. The PTP catalytic domain shows 45-50% sequence identity with the catalytic domain of human HPTP beta and with the first catalytic domain of LCA. The PTP-oc gene exists as a single copy in the rabbit genome. The corresponding mRNA (3.8 kb) is expressed in osteoclasts but not in other bone-derived cells (e.g. osteoblasts and stromal cells). The 3.8 kb PTP-oc mRNA transcript was also expressed in the rabbit brain, kidney and spleen. However, the brain and kidney, but not osteoclasts or spleen, also expressed a larger transcript (6.5 kb). The PTP catalytic domain of PTP-oc was expressed as a GST-cPTP-oc fusion protein. In vitro phosphatase assays indicated that the purified fusion protein exhibited phosphatase activities at neutral pH values toward p-nitrophenyl phosphate, phosphotyrosyl Raytide, and phosphotyrosyl histone, whereas it had no appreciable activity toward phosphoseryl casein. In summary, we have: (a) cloned and sequenced the putative full-length cDNA of a unique PTP (PTP-oc) from rabbit osteoclasts; (b) shown that the mature 3.8 kb PTP-oc mRNA was expressed primarily in osteoclasts and the spleen; and (c) shown that the PTP-oc fusion protein exhibited a phosphotyrosine-specific phosphatase activity. In conclusion, PTP-oc represents a structurally unique subfamily of transmembrane PTPs.
Project description:The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.
Project description:A novel extracellular alkaline phosphatase/phosphodiesterase from the structural protein family PhoD that encoded by the genome sequence of the marine bacterium Cobetia amphilecti KMM 296 (CamPhoD) has been expressed in Escherichia coli cells. The calculated molecular weight, the number of amino acids, and the isoelectric point (pI) of the mature protein's subunit are equal to 54832.98 Da, 492, and 5.08, respectively. The salt-tolerant, bimetal-dependent enzyme CamPhoD has a molecular weight of approximately 110 kDa in its native state. CamPhoD is activated by Co2+, Mg2+, Ca2+, or Fe3+ at a concentration of 2 mM and exhibits maximum activity in the presence of both Co2+ and Fe3+ ions in the incubation medium at pH 9.2. The exogenous ions, such as Zn2+, Cu2+, and Mn2+, as well as chelating agents EDTA and EGTA, do not have an appreciable effect on the CamPhoD activity. The temperature optimum for the CamPhoD activity is 45 °C. The enzyme catalyzes the cleavage of phosphate mono- and diester bonds in nucleotides, releasing inorganic phosphorus from p-nitrophenyl phosphate (pNPP) and guanosine 5'-triphosphate (GTP), as determined by the Chen method, with rate approximately 150- and 250-fold higher than those of bis-pNPP and 5'-pNP-TMP, respectively. The Michaelis-Menten constant (Km), Vmax, and efficiency (kcat/Km) of CamPhoD were 4.2 mM, 0.203 mM/min, and 7988.6 S-1/mM; and 6.71 mM, 0.023 mM/min, and 1133.0 S-1/mM for pNPP and bis-pNPP as the chromogenic substrates, respectively. Among the 3D structures currently available, in this study we found only the low identical structure of the Bacillus subtilis enzyme as a homologous template for modeling CamPhoD, with a new architecture of the phosphatase active site containing Fe3+ and two Ca2+ ions. It is evident that the marine bacterial phosphatase/phosphidiesterase CamPhoD is a new structural member of the PhoD family.
Project description:By use of the autophosphorylated epidermal-growth-factor receptor and the synthetic peptide RRLIE-DAEY(P)AARG, representing an autophosphorylation site of the transforming protein of Rous-sarcoma virus, it is demonstrated that the phosphotyrosyl phosphatase activity of the polycation-stimulated phosphatases is substantially increased by an enzyme-directed effect of ATP or PPi. Concomitant with this increase in phosphotyrosyl phosphatase activity, the phosphorylase phosphatase activity is decreased, thus dramatically changing the substrate specificity of these enzymes. The dephosphorylation of four different phosphotyrosyl sites of the epidermal-growth-factor receptor is neither consecutive nor at random, but a preferred dephosphorylation of the P1 site over the P3 greater than P2 greater than P4 sites is observed. This phosphatase activity represents a substantial fraction of the total phosphotyrosyl phosphatase activity in the post-mitochondrial supernatant of Xenopus laevis oocytes.
Project description:A [phosphotyrosine]protein phosphatase (PTPPase) was purified almost to homogeneity from rat brain, with [32P]p130gag-fps, an oncogene product of Fujinami sarcoma virus, as substrate. The characteristics of the purified preparation of PTPPase were as follows: the enzyme was a monomer with a molecular mass of 23 kDa; its optimum pH was 5.0-5.5; its activity was not dependent on bivalent cations; its activity was strongly inhibited by sodium vanadate, but was not inhibited by ZnCl2, L(+)-tartrate or NaF; it catalysed the dephosphorylation of [32P]p130gag-fps, [[32P]Tyr]casein, p-nitrophenyl phosphate and L-phosphotyrosine, but did not hydrolyse [[32P]Ser]tubulin, L-phosphoserine, DL-phosphothreonine, 5'-AMP, 2'-AMP or beta-glycerophosphate significantly. During the purification, most of the PTPPase activity was recovered in distinct fractions from those of conventional low-molecular-mass acid phosphatase (APase), which was reported to be a major PTPPase [Chernoff & Li (1985) Arch. Biochem. Biophys. 240, 135-145], from DE-52 DEAE-cellulose column chromatography, and those two enzymes could be completely separated by Sephadex G-75 column chromatography. APase also showed PTPPase activity with [32P]p130gag-fps, but the specific activity was lower than that of PTPPase with molecular mass of 23 kDa, and it was not sensitive to sodium vanadate. These findings suggested that PTPPase (23 kDa) was the major and specific PTPPase in the cell.
Project description:Abstract The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.