The expression of the phosphotyrosine phosphatase DEP-1/PTPeta dictates the responsivity of glioma cells to somatostatin inhibition of cell proliferation

Here we characterize the intracellular effectors of the antiproliferative activity of somatostatin in glioma cell lines and post-surgical specimens. The responsiveness to somatostatin correlated with the expression of the phosphotyrosine phosphatase DEP-1/PTPeta, identified in C6 and U87MG cells, in which somatostatin inhibited cell growth. The expression of a dominant negative mutant of DEP-1/PTPeta in C6 cells abolished somatostatin effects, confirming the involvement of this phosphotyrosine phosphatase in such effects. Somatostatin treatment increased the activity of DEP-1/PTPeta and inhibited ERK1/2 activation. Conversely, basic fibroblast growth factor-dependent MEK phosphorylation was not affected, suggesting a direct effect on ERK1/2. In vitro experiments showed that PTPeta was able to interact and dephosphorylate ERK1/2 activated by basic fibroblast growth factor. Furthermore, by transfecting PTPeta in the somatostatin-unresponsive, DEP-1/PTPeta-deficient U373MG cells, the somatostatin-dependent control of cell proliferation was recovered. Finally we evaluated the requirement for DEP-1/PTPeta in somatostatin inhibition of cell proliferation in post-surgical specimens derived from different grade human gliomas. Although all of the glioma analyzed expressed somatostatin receptor mRNA, DEP-1/PTPeta expression was limited to 8 of 22 of the tumors. Culturing seven gliomas, a correlation between the expression of DEP-1/PTPeta and the somatostatin antiproliferative effects was identified. In conclusion we propose that the expression and activation of DEP-1/PTPeta is required for somatostatin inhibition of glioma proliferation.     

Somatostatin receptor 1 (SSTR1)-mediated inhibition of cell proliferation correlates with the activation of the MAP kinase cascade: role of the phosphotyrosine phosphatase SHP-2.

The mitogen activated protein (MAP) kinase cascade represents one of the major regulator of cell growth by hormones and growth factors. However, although the activation of this intracellular pathway has been often regarded as mediator of cell proliferation, in many cell types the increase in MAP kinase (also called extra-cellular signal regulated kinase: ERK) activity may result in cell growth arrest, depending on the length or the intensity of the stimulation. In this review we examine recent data concerning the effects of somatostatin on the MAP kinase cascade through one of its major receptor subtype, the somatostatin receptor 1 (SSTR1), stably expressed in CHO-K1 cells. Somatostatin inhibits the proliferative effects of basic FGF (bFGF) in CHO-SSTR1 cell line. However, in these cells, somatostatin robustly activates the MAP kinase and augments bFGF-induced stimulation of ERK. We show that the activation of ERK via SSTR1 is mediated by the betagamma subunit of a pertussis toxin-sensitive G-protein and requires both the small G protein Ras and the serine/threonine kinase Raf-1. Moreover the phosphatidyl inositol-3kinase and the cytosolic tyrosine kinase c-src participate in the signal transduction regulated by SSTRI to activate ERK, as well as it is involved the protein tyrosine phosphatase (PTP) SHP-2. Previous studies have suggested that somatostatin-stimulated PTP activity mediates the growth inhibitory actions of somatostatin, in CHO-SSTR1 cells. Thus, the activation of SHP-2 by SSTR1 may mediate the antiproliferative activity of somatostatin. SHP-2 may. in turn, regulate the activity of kinases upstream of ERK that require tyrosine dephosphorylation to be activated, such as c-src. Finally, the synergism between somatostatin and bFGF in the activation of ERK results in an increased expression of the cyclin-dependent kinase inhibitor p21cip/WAF1 as molecular effector of the antiproliferative activity of somatostatin.     

An intracellular multi-effector complex mediates somatostatin receptor 1 activation of phospho-tyrosine phosphatase eta

The receptor-like phosphotyrosine phosphatase eta (PTPeta) is an important intracellular effector of the cytostatic action of SST. Here we characterize, in Chinese hamster ovary-k1 cells, the intracellular pathway that from somatostatin receptor 1 (SSTR1), leads to the activation of PTPeta and that involves, in a multimeric complex and sequential activation, the tyrosine kinases Janus kinase (JAK) 2 and Src, and the cytosolic phosphotyrosine phosphatase SHP-2. We show that inhibitors of JAK2 and Src and dominant-negative mutants of SHP-2 and Src abolished the SSTR1-mediated PTPeta activation, suggesting that all these effectors participate in the activation of PTPeta. In basal conditions, JAK2 forms a multimeric complex with SHP-2, Src and PTPeta. In response to SST, JAK2 is activated in a G protein-dependent manner, dissociates from and phosphorylates SHP-2, increasing its activity. Subsequently, SHP-2 dissociates from Src, dephosphorylates the Src inhibitory tyrosine-529, and causes an autocatalytical increase of the phosphorylation of Src tyrosine 418, located inside its kinase activation loop. Active Src, in turn, controls the activity of PTPeta, via a direct interaction and phosphorylation of the phosphatase. These data for the first time depict an intracellular pathway involving a precise sequence of interactions and cross-activation among tyrosine phosphatases and kinases acting upstream of PTPeta. In particular the sequential activation of JAK2, SHP-2, and Src conveys the molecular signaling from SSTR1 to the activation of this phosphatase that is responsible for the final biological effects of SST.     

Pertussis toxin blocks somatostatin's inhibition of stimulated cyclic AMP accumulation in anterior pituitary tumor cells

Somatostatin inhibits both forskolin and (-) isoproterenol-stimulated cyclic AMP accumulation in AtT-20 cells. Pretreatment of these cells with pertussis toxin prevents somatostatin's inhibitory effects on cyclic AMP production. This pretreatment also enhances the cyclic AMP response to forskolin and (-) isoproterenol without affecting basal cyclic AMP levels. The blockade of somatostatin's inhibitory effect was dependent both on the time of preincubation and concentration of pertussis toxin used. The rise in forskolin-stimulated cyclic AMP formation following pertussis toxin treatment preceded the blockade of somatostatin's inhibitory actions. The results suggest that somatostatin acts through an inhibitory guanine nucleotide regulatory protein to affect adenylate cyclase activity.     

Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase

STATc becomes tyrosine phosphorylated and accumulates in the nucleus when Dictyostelium cells are exposed to the prestalk cell inducer Differentiation inducing factor 1 (DIF-1), or are subjected to hyper-osmotic stress. We show that the protein tyrosine phosphatase PTP3 interacts directly with STATc and that STATc is refractory to activation in PTP3 overexpressing cells. Conversely, overexpression of a dominant inhibitor of PTP3 leads to constitutive tyrosine phosphorylation and ectopic nuclear localisation of STATc. Treatment of cells with DIF-1 or exposure to hyper-osmotic stress induces a decrease in biochemically assayable PTP3 activity and both agents also induce serine-threonine phosphorylation of PTP3. These observations suggest a novel mode of STAT activation, whereby serine-threonine phosphorylation of a cognate protein tyrosine phosphatase results in the inhibition of its activity, shifting the phosphorylation-dephosphorylation equilibrium in favour of phosphorylation.     

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.     

Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/Protein kinase B activation

Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. In adipose tissue and muscle of obese humans and rodents, PTP1B expression is increased, which led to the hypothesis that PTP1B plays a role in the pathogenesis of insulin resistance. Consistent with this, mice in which the PTP1B gene was disrupted exhibit increased insulin sensitivity. To test whether increased expression of PTP1B in an insulin-sensitive cell type could contribute to insulin resistance, we overexpressed wild-type PTP1B in 3T3L1 adipocytes using adenovirus-mediated gene delivery. PTP1B expression was increased approximately 3-5-fold above endogenous levels at 16 h, approximately 14-fold at 40 h, and approximately 20-fold at 72 h post-transduction. Total protein-tyrosine phosphatase activity was increased by 50% at 16 h, 3-4-fold at 40 h, and 5-6-fold at 72 h post-transduction. Compared with control cells, cells expressing high levels of PTP1B showed a 50-60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Akt phosphorylation and activity were unchanged. Phosphorylation of p42 and p44 MAP kinase (MAPK) was reduced approximately 32%. Overexpression of PTP1B had no effect on basal, submaximally or maximally (100 nm) insulin-stimulated glucose transport or on the EC(50) for transport. Our results suggest that: 1) insulin stimulation of glucose transport in adipocytes requires 相似文献   

Vitamin E deficiency impairs the somatostatinergic receptor–effector system and leads to phosphotyrosine phosphatase overactivation and cell death in the rat hippocampus

Vitamin E plays an essential role in maintaining the structure and function of the nervous system, and its deficiency, commonly associated with fat malabsorption diseases, may reduce neuronal survival. We previously demonstrated that the somatostatinergic system, implicated in neuronal survival control, can be modulated by α-tocopherol in the rat dentate gyrus, increasing cyclic adenosine monophosphate response element binding protein phosphorylation. To gain a better understanding of the molecular actions of tocopherols and examine the link among vitamin E, somatostatin and neuronal survival, we have investigated the effects of a deficiency and subsequent administration of tocopherol on the somatostatin signaling pathway and neuronal survival in the rat hippocampus. No changes in somatostatin expression were detected in vitamin-E-deficient rats. These rats, however, showed a significant increase in the somatostatin receptor density and dissociation constant, which correlated with a significant increase in the protein levels of somatostatin receptors. Nevertheless, vitamin E deficiency impaired the ability of the somatostatin receptors to couple to the effectors adenylyl cyclase and phosphotyrosine phosphatase by diminishing Gi protein functionality. Furthermore, vitamin E deficiency significantly increased phosphotyrosine phosphatase activity and PTPη expression, as well as PKCδ activation, and decreased extracellular-signal-regulated kinase phosphorylation. All these changes were accompanied by an increase in neuronal cell death. Subsequent α-tocopherol administration partially or completely reversed all these values to control levels. Altogether, our results prove the importance of vitamin E homeostasis in the somatostatin receptor–effector system and suggest a possible mechanism by which this vitamin may regulate the neuronal cell survival in the adult hippocampus.     

The activated insulin-like growth factor I receptor induces depolarization in breast epithelial cells characterized by actin filament disassembly and tyrosine dephosphorylation of FAK, Cas, and paxillin.

Insulin-like growth factor I (IGF-I) promotes the motility of different cell types. We investigated the role of IGF-I receptor (IGF-IR) signaling in locomotion of MCF-7 breast cancer epithelial cells overexpressing the wild-type IGF-IR (MCF-7/IGF-IR). Stimulation of MCF-7/IGF-IR cells with 50 ng/ml IGF-I induced disruption of the polarized cell monolayer followed by morphological transition toward a mesenchymal phenotype. Immunofluorescence staining of the cells with rhodamine-phalloidin revealed rapid disassembly of actin fibers and development of a cortical actin meshwork. Activation of phosphatidylinositol (PI)3-kinase downstream of the IGF-IR was necessary for this process, as blocking PI 3-kinase activity with the specific inhibitor LY 294002 at 10 microM prevented disruption of the filamentous actin. In parallel, IGF-IR activation induced rapid and transient tyrosine dephosphorylation of focal adhesion proteins p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (Cas), and paxillin. This process required phosphotyrosine phosphatase (PTP) activity, since pretreatment of the cells with 5 microM phenylarsine oxide (PAO), an inhibitor of PTPs, rescued FAK and its associated proteins Cas and paxillin from IGF-I-induced dephosphorylation. In addition, PAO-pretreated cells were refractory to IGF-I-induced morphological transition. Thus, our findings reveal a new function of the IGF-IR, the ability to depolarize epithelial cells. In MCF-7 cells, mechanisms of IGF-IR-mediated cell depolarization involve PI 3-kinase signaling and putative PTP activities.     

The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2, and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase.     

Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3α in A431 cells

Modulation of protein kinase FA /glycogen synthase kinase-3α (kinase FA /GSK-3α) by reversible tyrosine phosphorylation/dephosphorylation was investigated. In addition to genistein, other protein tyrosine kinase (PTK) inhibitors, such as tyrphostin A47 and B42, also could induce tyrosine dephosphorylation and inactivation of kinase FA /GSK-3α in A431 cells, and this process was found to be reversible. Pretreatment of the cells with 100 μM orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor, could diminish significantly the effects of PTK inhibitors on both enzyme activity and phosphotyrosine content of the kinase, suggesting that the PTK inhibitors induced tyrosine dephosphorylation/inactivation of this kinase is mediated by orthovanadate-sensitive PTP(s) in A431 cells. Moreover, the phosphotyrosine moiety of kinase FA /GSK-3α was found to be highly turned over in resting cells. Interestingly, we found that the less active, tyrosine-dephosphorylated form of kinase FA /GSK-3α immunoprecipitated from genistein-treated cells was able to reactivate partially with concomitant rephosphorylation of tyrosine residue in vitro. Taken together, these findings demonstrate that tyrosine phosphorylation and concomitant activation of kinase FA /GSK-3α can be carried out both in vitro and in vivo and an in vivo phosphatase activity may function in antagonism to PTK activation of kinase FA /GSK-3α. J. Cell. Physiol. 171:95–103, 1997. © 1997 Wiley-Liss, Inc.     

Small molecule peptidomimetics containing a novel phosphotyrosine bioisostere inhibit protein tyrosine phosphatase 1B and augment insulin action

Protein tyrosine phosphatase 1B (PTP1B) attenuates insulin signaling by catalyzing dephosphorylation of insulin receptors (IR) and is an attractive target of potential new drugs for treating the insulin resistance that is central to type II diabetes. Several analogues of cholecystokinin(26)(-)(33) (CCK-8) were found to be surprisingly potent inhibitors of PTP1B, and a common N-terminal tripeptide, N-acetyl-Asp-Tyr(SO(3)H)-Nle-, was shown to be necessary and sufficient for inhibition. This tripeptide was modified to reduce size and peptide character, and to replace the metabolically unstable sulfotyrosyl group. This led to the discovery of a novel phosphotyrosine bioisostere, 2-carboxymethoxybenzoic acid, and to analogues that were >100-fold more potent than the CCK-8 analogues and >10-fold selective for PTP1B over two other PTP enzymes (LAR and SHP-2), a dual specificity phosphatase (cdc25b), and a serine/threonine phosphatase (calcineurin). These inhibitors disrupted the binding of PTP1B to activated IR in vitro and prevented the loss of tyrosine kinase (IRTK) activity that accompanied PTP1B-catalyzed dephosphorylation of IR. Introduction of these poorly cell permeant inhibitors into insulin-treated cells by microinjection (oocytes) or by esterification to more lipophilic proinhibitors (3T3-L1 adipocytes and L6 myocytes) resulted in increased potency, but not efficacy, of insulin. In some instances, PTP1B inhibitors were insulin-mimetic, suggesting that in unstimulated cells PTP1B may suppress basal IRTK activity. X-ray crystallography of PTP1B-inhibitor complexes revealed that binding of an inhibitor incorporating phenyl-O-malonic acid as a phosphotyrosine bioisostere occurred with the mobile WPD loop in the open conformation, while a closely related inhibitor with a 2-carboxymethoxybenzoic acid bioisostere bound with the WPD loop closed, perhaps accounting for its superior potency. These CCK-derived peptidomimetic inhibitors of PTP1B represent a novel template for further development of potent, selective inhibitors, and their cell activity further justifies the selection of PTP1B as a therapeutic target.     

The roles of K5 and K14 head, tail, and R/K L L E G E domains in keratin filament assembly in vitro

A rat cDNA encoding a 51-kD protein tyrosine phosphatase (PTP1) was cloned into a mammalian expression vector and transfected into normal and v-src-transformed mouse NIH 3T3 fibroblasts. In the stable subclones isolated, PTP1 expression at the mRNA level was elevated twofold to 25-fold. The highest constitutive level of phosphotyrosine-specific dephosphorylating activity observed without cytotoxic effects or significant clonal instability was approximately 10-fold over the endogenous activity. The expressed PTP1 was found to be associated with the particulate fraction of the fibroblasts. Subcellular fractionation and immunofluorescent microscopic examination of PTP1-overexpressing cells has shown the phosphatase to be localized to the reticular network of the ER. PTP1 was readily solubilized by detergents, but not by high salt. Limited proteolysis of membrane-associated PTP1 resulted in the release of lower molecular mass (48 and 37 kD) forms of the enzyme to the cytosol. Thermal phase partitioning of isolated membranes with Triton X-114 indicated that the full-length PTP1 was strongly integrated into the membrane in contrast to the proteolytically derived fragments of PTP1. Overexpression of PTP1 caused little apparent change in the rate of cell proliferation, but did induce changes in fibroblast morphology. A substantial increase in the proportion of bi- and multinucleate cells in PTP1-expressing cell populations was observed, and, in the case of the v-src-transformed cells, cell flattening and loss of refractibility occurred. Although no apparent difference in the tyrosine phosphorylation of pp60v-src was noted in v-src-transformed control and PTP1-overexpressing fibroblasts, the phosphotyrosine content of a 70-kD polypeptide was decreased in PTP1-overexpressing cells.     

The eta isoform of protein kinase C inhibits UV-induced activation of caspase-3 in normal human keratinocytes

Protein kinase C (PKC) fulfills a central role in the decision of cell fate in keratinocytes. Both PKC delta and PKC eta induce growth inhibition and differentiation of normal human keratinocytes (NHK). Here we show that PKC delta and PKC eta play opposite roles in UVB-induced apoptosis in NHK. PKC delta enhanced UVB-induced caspase-3 activity, while overexpression of PKC eta reduced it. In keeping with these observations, the dominant negative mutant of PKC delta significantly inhibited the activation of caspase-3, whereas dominant negative PKC eta increased it in a dose (MOI)-dependent manner. Unlike PKC delta, cleavage and translocation to mitochondria of PKC eta were not observed, resulting in no detection of cytochorome c release. Furthermore, UV-induced activation of p38 MAP kinase, which suppressed the caspase-3 activity in NHK, was blocked by dominant negative PKC eta. These findings suggest that PKC eta negatively regulates UV-induced apoptosis through its localization, resistance to cleavage, and the p38 MAPK pathway.     

Somatostatin interferes with thyrotropin-induced G1-S transition mediated by cAMP-dependent protein kinase and phosphatidylinositol 3-kinase. Involvement of RhoA and cyclin E x cyclin-dependent kinase 2 complexes

cAMP-mediated cell proliferation is a complex process that involves multiple pathways. Using a cAMP-dependent cell system, FRTL-5 thyroid cells, we have previously demonstrated the existence of a precise autocrine loop in the control of cell proliferation that involves the positive effector thyrotropin (TSH) and the general inhibitor somatostatin. In search of the regulatory mechanisms responsible for the TSH and somatostatin control of cell proliferation, we analyzed the cell cycle regulatory proteins and the cellular pathways involved in the action of both signals. The results show that specific inhibition of cAMP-dependent protein kinase (PKA) and phosphatidylinositol (PI) 3-kinase blocks independently TSH-induced FRTL-5 cell proliferation and that somatostatin interferes with both signals. Each pathway activates different proteins required for G(1)/S progression. Thus, PKA is responsible for the TSH-induction of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels, RhoA activation, and down-regulation of p27(kip1). These correlated events are necessary for FRTL-5 cell proliferation after TSH stimulation. Moreover, TSH through PKA pathway increases cyclin-dependent kinase 2 levels, whereas PI 3-kinase signaling increases cyclin E levels. Together, both pathways finally converge, increasing the formation and activation of cyclin E x cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G(1) to S phase in growth-stimulated cells. Somatostatin exerts its antiproliferative effect inhibiting more upstream the TSH stimulation of PKA and PI 3-kinase, interfering with the TSH-mediated increases of intracellular cAMP levels by inactivation of adenylyl cyclase activity. Together, these results suggest the existence of a PKA-dependent pathway and a new PKA-independent PI 3-kinase pathway in the TSH/cAMP-mediated proliferation of FRTL-5 thyroid cells.     

Characterization of a prostate-specific tyrosine phosphatase by mutagenesis and expression in human prostate cancer cells

The cellular form of human prostatic acid phosphatase (PAcP) is a neutral protein-tyrosine phosphatase (PTP) and may play a key role in regulating the growth and androgen responsiveness of prostate cancer cells. The functional role of the enzyme is at least due in part to its dephosphorylation of c-ErbB-2, an in vivo substrate of the enzyme. In this study, we investigated the molecular mechanism of phosphotyrosine dephosphorylation by cellular PAcP. We mutated several amino acid residues including one cysteine residue that was proposed to be involved in the PTP activity of the enzyme by serving as the phosphate acceptor. The cDNA constructs of mutant enzymes were transiently transfected into C-81 LNCaP and PC-3 human prostate cancer cells that lack the endogenous PAcP expression. The phosphotyrosine level of ErbB-2 in these transfected cells was subsequently analyzed. Our results demonstrated that the phosphotyrosine level of ErbB-2 in cells expressing H12A or D258A mutant PAcP is similar to that in control cells without PAcP expression, suggesting that these mutants are incapable of dephosphorylating ErbB-2. In contrast, cells expressing C183A, C281A, or wild-type PAcP had a decreased phosphotyrosine level of ErbB-2, compared with the control cells. Similar results were obtained from in vitro dephosphorylation of immunoprecipitated ErbB-2 by these mutant enzymes. Furthermore, transient expression of C183A, C281A, or the wild-type enzyme, but not H12A or D258A, decreased the growth rate of C-81 LNCaP cells. The data collectively indicate that His-12 and Asp-258, but not Cys-183 or Cys-281, are required for the PTP activity of PAcP.