Distribution of prostatic markers in glands of the female urethra and anterior vaginal wall-a rapid autopsy study.

BACKGROUND: There are varying reports of immunohistochemically detected prostatic marker protein distribution in glands associated with the female urethra that may be related to tissue integrity at the time of fixation. AIM: In this study we used tissue derived from rapid autopsies of female patients to determine the distribution of glandular structures expressing prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) along the female urethra and in surrounding tissues, including the anterior vaginal wall (AVW). METHODS: Tissue blocks from 7 donors that contained the entire urethra and adjacent AVW were analyzed. These tissue samples were fixed within 4-12 hours of death and divided into 5-mm transverse slices that were paraffin embedded. Sections cut from each slice were immunolabeled for PSA or PSAP and a neighboring section was stained with hematoxylin and eosin. The sections were reviewed by light microscopy and analyzed using QuPath software. OBSERVATIONS: In tissue from all donors, glandular structures expressing PSA and/or PSAP were located within the wall of the urethra and were present along its whole length. RESULTS: In the proximal half of the urethra from all donors, small glands expressing PSAP, but not PSA, were observed adjacent to the and emptying into the lumen. In the distal half of the urethra from 5 of the 7 donors, tubuloacinar structures lined by a glandular epithelium expressed both PSA and PSAP. In addition, columnar cells at the surface of structures with a multilayered transitional epithelium in the distal half of the urethra from all donors expressed PSAP. No glands expressing PSA or PSAP were found in tissues surrounding the urethra, including the AVW. CLINICAL IMPLICATIONS: Greater understanding of the distribution of urethral glands expressing prostatic proteins in female patients is important because these glands are reported to contribute to the female sexual response and to urethral pathology, including urethral cysts, diverticula, and adenocarcinoma. STRENGTHS AND LIMITATIONS: Strengths of the present study include the use of rapid autopsy to minimize protein degradation and autolysis, and the preparation of large tissue sections to demonstrate precise anatomical relations within all the tissues surrounding the urethral lumen. Limitations include the sample size and that all donors had advanced malignancy and had undergone previous therapy which may have had unknown tissue effects. CONCLUSION: Proximal and distal glands expressing prostate-specific proteins were observed in tissue from all donors, and these glands were located only within the wall of the urethra.

Mesenchymal and Phosphatase of Regenerating Liver-3 Status in Circulating Tumor Cells May Serve as a Crucial Prognostic Marker for Assessing Relapse or Metastasis in Postoperative Patients With Colorectal Cancer.

INTRODUCTION: Circulating tumor cells (CTCs) and phosphatase of regenerating liver-3 (PRL-3) have been considered to be significant prognostic indicators in metastatic colorectal cancer (CRC). This study discusses the prognostic significance of mesenchymal CTCs with PRL-3 (M+ PRL-3+ CTCs) in postoperative patients with CRC. METHODS: We detected CTC subtypes (including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs) and PRL-3 in CTCs from the peripheral blood samples of 156 patients. Receiver operating characteristic curve analysis, Kaplan-Meier analysis, and Cox proportional hazards regression analysis were performed to identify the prognostic value of mesenchymal CTCs with PRL-3+. Immunohistochemistry was used to detect the expression of PRL-3 in tumor tissues from some of the patients to explore the connection between CTCs and tissues. RESULTS: All CTCs were positive in all samples, both mesenchymal CTCs and PRL-3-positive cells. The count of mesenchymal and PRL-3+ CTCs was significantly associated with recurrence, and the optimal cutoff value was 2 (area under the curve = 0.690, P < 0.001). In addition, these patients had a significantly shorter median disease-free survival than those who did not fulfill the criteria (8.5 vs 24 months, P < 0.001) according to multivariable and multinomial logistic regression. Immunohistochemistry was applied to explore the associations between PRL-3 expression and significant prognostic risk factors, including recurrence (R = 0.566; P < 0.001), and M+ PRL-3+ status in CTCs (R = 0.452; P = 0.001). DISCUSSION: The status of M+ PRL-3+ in CTCs may serve as a crucial prognostic marker for assessing clinical outcomes in CRC.

Label-Free Assay of Protein Tyrosine Phosphatase Activity in Single Cells.

Populations of cells exhibit variations in biochemical activity, resulting from many factors including random stochastic variability in protein production, metabolic and cell-cycle states, regulatory mechanisms, and external signaling. The development of methods for the analysis of single cells has allowed for the measurement and understanding of this inherent heterogeneity, yet methods for measuring protein activities on the single-cell scale lag behind their genetic analysis counterparts and typically report on expression rather than activity. This paper presents an approach to measure protein tyrosine phosphatase (PTP) activity in individual cells using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry. Using flow cytometry, individual cells are first sorted into a well plate containing lysis buffer and a phosphopeptide substrate. After lysis and incubation-during which the PTP enzymes act on the peptide substrate-the reaction substrate and product are immobilized onto arrays of self-assembled monolayers, which are then analyzed using mass spectrometry. PTP activities from thousands of individual cells were measured and their distributions analyzed. This work demonstrates a general method for measuring enzyme activities in lysates derived from individual cells and will contribute to the understanding of cellular heterogeneity in a variety of contexts.

Assessment of Protein Carbonylation and Protein Tyrosine Phosphatase (PTP) Oxidation in Vascular Smooth Muscle Cells (VSMCs) Using Immunoblotting Approaches.

Post-translational modification of proteins, such as phosphorylation and oxidation, plays a major role in cellular signaling by influencing protein structure and function. In vascular cells, in addition to influencing phosphorylation, angiotensin II (Ang II) induces oxidation of proteins, important in redox signaling in the cardiovascular and renal systems. The present chapter describes immunoblotting approaches to assess irreversible protein carbonylation and protein tyrosine phosphatase (PTPs) oxidation status in the proteome of vascular smooth muscle cells (VSMC).Protein carbonylation is generally measured using the OxyBlot™ approach, whereby derivatization of protein carbonyl groups (C = O) on oxidized amino acids by dinitrophenylhydrazine (DNPH) results in the formation of a stable dinitrophenyl (DNP) hydrazone product. The samples are analyzed by SDS-PAGE and a primary antibody raised against the DNP moiety is used to determine levels of irreversible protein carbonylation in the sample by immunoblotting.Oxidation of PTPs can be evaluated using a monoclonal antibody against the "hyperoxidized" (SO3H) catalytic site of these enzymes. The described methodology offers the ability to discriminate between irreversible (SO3H) and reversible (SOH) PTP oxidation states. Initially, the free unmodified PTP-thiols (S-) are alkylated and the sample is split into two. One part is used to assess the PTP-SO3H form. In the other part reversibly modified PTP-thiols are first reduced and then hyperoxidized by pervanadate (PV). Both untreated and PV-treated samples are analyzed by SDS-PAGE and "hyperoxidized" PTPs are detected by immunoblotting. The proportion of reversibly oxidized PTP-SOH fraction is determined by the difference between the signals in untreated and the PV-treated samples.The above immunoassays provide general approaches to detect and quantify global levels of irreversible protein oxidation and of irreversibly/reversibly oxidized PTPs in any (patho)physiological context. Characterization of the global redox status is essential to better understand the redox-sensitive mechanisms underlying chronic diseases associated with oxidative stress. This is particularly important in systems influenced by the renin angiotensin system, because Ang II is a potent inducer of oxidative stress and redox signaling.

Do phosphatase of regenerating liver-3, matrix metalloproteinases-2, matrix metalloproteinases-9, and epidermal growth factor receptor-1 predict response to therapy and survival in glioblastoma multiforme?

CONTEXT: Poor survival of the glioblastoma multiforme (GBM) has been attributed in part to the invasive nature of the lesion making complete surgical removal near impossible. Phosphatase of regenerating liver-3 (PRL-3), matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9), and epidermal growth factor receptor (EGFR-1) play a role in invasive nature of tumor cells. AIMS: This study was conducted to evaluate PRL-3, MMP-2, MMP-9, and EGFR-1 (markers) expression in cases to GBM and to correlate their expression with therapy response and survival. SETTINGS AND DESIGN: GBM cases (n = 62) underwent surgery followed by radiation (n = 34) and chemoradiation (n = 28). Using WHO Response Evaluation Criteria in Solid Tumors criteria response to therapy was assessed at 3 months and cases followed up for survival. SUBJECTS AND METHODS: Expression of markers was assessed by immunohistochemistry as a percentage of positive tumor cells in hot spots. STATISTICAL ANALYSIS USED: Kaplan-Meier, ANOVA, Chi-square test, univariate, and multivariate Cox-regression analysis was done. RESULTS: Response to therapy was evident in 54.8% cases of responders with the mean survival of 494.03 ± 201.13 days and 45.2% cases of non responders (278.32 ± 121.66 days) with P = 0.001. Mean survival for the patient's opted chemoradiation was 457.43 ± 222.48 days which was approximately 3 months greater than those who opted radiation alone (P = 0.029). We found PRL-3 overexpression was an independent, significant, poor prognostic factor for survival by multivariate analysis (P = 0.044). Cases negative for MMP's and EGFR showed increased survival, but the difference was insignificant. CONCLUSION: PRL-3 expression appears to be related to an adverse disease outcome.

Localizing PRL-2 expression and determining the effects of dietary Mg(2+) on expression levels.

The phosphatase of regenerating liver (PRL) is a group of protein tyrosine phosphatases that play a key role in cancer progression and metastasis. We previously showed that PRL-2 modulates intracellular Mg(2+) levels and sustains cancer phenotypes by binding to the Mg(2+) transporter CNNM3. However, the physiological functions of PRL-2 in animals remain largely unknown. To better understand which cell types are associated with PRL-2 function, we characterized its expression in mouse tissues using a PRL-2 ß-galactosidase reporter mouse model. Our results demonstrated that PRL-2 was ubiquitously expressed, with the highest expression levels observed in the hippocampal pyramidal neurons, ependymal cells, cone and rod photoreceptor cells, endocardium, vascular and bronchial smooth muscle, and collecting ducts in the kidney. On the other hand, PRL-2 expression was undetectable or very low in the parenchymal cells of the liver and pancreas. Our results also indicated that PRL-2 is involved in cell-type-specific Mg(2+) homeostasis and that PRL-2 expression is potentially inversely regulated by dietary Mg(2+) levels.

Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants.

Redox-based probes as tools to monitor oxidized protein tyrosine phosphatases in living cells.

Reversible oxidation of protein tyrosine phosphatases (PTPs) has emerged as an important regulatory mechanism whereby reactive oxygen species (ROS) inactivates the PTP and promotes phosphorylation and induction of the signaling cascade. The lack of sensitive and robust methods to directly detect oxidized PTPs has made it difficult to understand the effects that PTP oxidative inactivation play in redox signaling. We report the use of redox-based probes to directly detect oxidized PTPs in a cellular context, which highlights the importance of direct approaches to assist in the study of physiological and pathophysiological PTP activity in redox regulation. We also demonstrate, as a proof-of-concept, that these redox-based probes serve as prototypes for the design and development of a new class of inhibitors for phosphatases. We envision a nucleophile reacting with the oxidized inactive catalytic cysteine to generate an irreversible thioether adduct which prevents the phosphatase from being reactivated and ultimately fortifies the signaling cascade. Our results reveal the potential of translation of our redox-based probes, which are used to understand redox cell circuitry and disease biology, to small-molecule nucleophile-based inhibitors, which may treat diseases associated with redox stress. This may have implications in the treatment of type 2 diabetes and cancer.

Prostatic acid phosphatase is the main acid phosphatase with 5'-ectonucleotidase activity in the male mouse saliva and regulates salivation.

We have previously shown that in addition to the well-known secreted isoform of prostatic acid phosphatase (sPAP), a transmembrane isoform exists (TMPAP) that interacts with snapin (a SNARE-associated protein) and regulates the endo-/exocytic pathways. We have also shown that PAP has 5'-ectonucleotidase and thiamine monophosphatase activity and elicits antinociceptive effects in mouse models of chronic inflammatory and neuropathic pain. Therefore, to determine the physiological role of PAP in a typical exocrine organ, we studied the submandibular salivary gland (SMG) of PAP(-/-) and wild-type C57BL/6J mice by microarray analyses, microRNA sequencing, activity tests, immunohistochemistry, and biochemical and physiological analyses of saliva. We show that PAP is the main acid phosphatase in the wild-type male mouse saliva, accounting for 50% of the total acid phosphatase activity, and that it is expressed only in the granular convoluted tubules of the SMGs, where it is the only 5'-ectonucleotidase. The lack of PAP in male PAP(-/-) mice was associated with a significant increase in the salivation volume under secretagogue stimulation, overexpression of genes related to cell proliferation (Mki67, Aurkb, Birc5) and immune response (Irf7, Cxcl9, Ccl3, Fpr2), and upregulation of miR-146a in SMGs. An increased and sustained acinar cell proliferation was detected without signs of glandular hyperplasia. Our results indicate that in PAP(-/-) mice, SMG homeostasis is maintained by an innate immune response. Additionally, we suggest that in male mice, PAP via its 5'-ectonucleotidase activity and production of adenosine can elicit analgesic effects when animals lick their wounds.

Ex vivo chemical cytometric analysis of protein tyrosine phosphatase activity in single human airway epithelial cells.

We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min(-1) mg(-1)) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (76%, 69%, and 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min(-1) mg(-1) and was also effectively inhibited by preincubation of the cells with the inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min(-1) mg(-1) resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34 to 36 pmol min(-1) mg(-1) (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues.

Measurement of protein tyrosine phosphatase activity in single cells by capillary electrophoresis.

A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn(2+) and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min(-1) mg(-1)) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min(-1) mg(-1)). Zn(2+), 1,2-naphthoquinone, and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 µM, and 100 nM, respectively. Dephosphorylation of the reporter, following loading into living single cells, occurred at rates of at least 2 pmol min(-1) mg(-1). When single cells were exposed to 1,2-naphthoquinone (50 µM), Zn(2+) (100 µM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens.

PTPIP51: a new interaction partner of the insulin receptor and PKA in adipose tissue.

AIMS: Our previous experiments revealed an association of PTPIP51 (protein tyrosine phosphatase interacting protein 51) with the insulin signalling pathway through PTP1B and 14-3-3beta. We aimed to clarify the role of PTPIP51 in adipocyte metabolism. METHODS: Four groups of ten C57Bl/6 mice each were used. Two groups were fed a standard diet; two groups were fed a high-fat diet. Two groups (one high-fat diet and one standard diet) were submitted to endurance training, while the remaining two groups served as untrained control groups. After ten weeks, we measured glucose tolerance of the mice. Adipose tissue samples were analyzed by immunofluorescence and Duolink proximity ligation assay to quantify interactions of PTPIP51 with either insulin receptor (IR) or PKA. RESULTS: PTPIP51 and the IR and PTPIP51 and PKA, respectively, were colocalized in all groups. Standard diet animals that were submitted to endurance training showed low PTPIP51-IR and PTPIP51-PKA interactions. The interaction levels of both the IR and PKA differed between the feeding and training groups. CONCLUSION: PTPIP51 might serve as a linking protein in adipocyte metabolism by connecting the IR-triggered lipogenesis with the PKA-dependent lipolysis. PTPIP51 interacts with both proteins, therefore being a potential gateway for the cooperation of both pathways.

Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science.

In forensic casework analysis, identification of the biological matrix and the species of a forensic trace, preferably without loss of DNA, is of major importance. The biological matrices that can be encountered in a forensic context are blood (human or non-human), saliva, semen, vaginal fluid, and to a lesser extent nasal secretions, feces, and urine. All these matrices were applied on swabs and digested with trypsin in order to obtain peptides. These peptides were injected on a mass spectrometer (ESI Q-TOF) resulting in the detection of several biomarkers that were used to build a decision tree for matrix identification. Saliva and blood were characterized by the presence of alpha-amylase 1 and hemoglobin, respectively. In vaginal fluid, cornulin, cornifin, and/or involucrin were found as biomarkers while semenogelin, prostate-specific antigen, and/or acid phosphatase were characteristic proteins for semen. Uromodulin or AMBP protein imply the presence of urine, while plunc protein is present in nasal secretions. Feces could be determined by the presence of immunoglobulins without hemoglobin. The biomarkers for the most frequently encountered biological matrices (saliva, blood, vaginal fluid, and semen) were validated in blind experiments and on real forensic samples. Additionally, by means of this proteomic approach, species identification was possible. This approach has the advantage that the analysis is performed on the first "washing" step of the chelex DNA extraction, a solution which is normally discarded, and that one single test is sufficient to determine the identity and the species of the biological matrix, while the conventional methods require cascade testing. This technique can be considered as a useful additional tool for biological matrix identification in forensic science and holds the promise of further automation.

[Prostatic tissue in a mature cystic teratoma of the ovary: report of one case]. / Tejido prostático en teratoma quístico maduro del ovario: caso clínico.

Male accessory sexual glands arising in ovarian cystic teratoma are exceedingly rare. We report a 56-year-old female subjected to an ovariohysterectomy due to a left ovarian mass. The pathological study of the surgical piece revealed a tumor composed of different mature tissue elements and well defined nodules of benign prostatic tissue.

The expression of the phosphatase regenerating liver 3 gene is associated with outcome in patients with colorectal cancer.

BACKGROUND/AIMS: The phosphatase of regenerating liver-3 (PRL-3) is over expressed in several human cancers and associated with tumor progression, invasion and metastasis. However, the correlation between PRL-3 expression and clinical outcome in colorectal cancer (CRC) has not been investigated. This study examined the relationship between the relative expression of the PRL-3 gene to the clinicopathological factors and outcomes in patients with CRC. METHODOLOGY: Surgical specimens of cancer tissue and adjacent normal mucosa were obtained from 202 patients with untreated CRC. The relative expression level of PRL-3 mRNA in cancer and in the normal adjacent mucosa was measured using the quantitative real-time reverse-transcriptase PCR. RESULTS: PRL-3 expression was higher in cancer tissue than in the adjacent normal mucosa. The tumor location and liver metastasis were found to be related to the PRL-3 expression level. The overall survival differed significantly between patients with high PRL-3 expression and those with low expression. CONCLUSIONS: High expression of the PRL-3 gene might be a useful predictor of poor postoperative outcome in patients with CRC.

Human aqueous humor phosphatase activity in cataract and glaucoma.

PURPOSE: To investigate the presence and activity of protein phosphatase-2A (PPase2A), protein phosphatase-2C (PPase2C), and protein tyrosine phosphatases (PTPs) in the human aqueous humor (AH) of patients with primary open-angle glaucoma (POAG) and cataract and to study the correlation between these phosphatases and the redox state of the AH. METHODS: Eighty-six cataract patients and 29 POAG patients who were scheduled for cataract surgery with or without glaucoma surgery were enrolled in the study. PPase2A, PPase2C, and PTPs levels in AH were measured by enzyme-linked immunosorbent assays, Western blot analyses, and spectral METHODS: The redox state was measured by spectral and fluorescent methods. RESULTS: Phosphatase activity-positive results were significantly higher in AH samples from the POAG group (PP2A χ(2)(1) = 11.754, P < 0.01; PP2C χ(2)(1) = 8.754, P < 0.01; PTP χ(2)(1) = 11.073, P < 0.01). Western blot analysis revealed higher PP2C levels in the AH of glaucoma patients compared with PP2C levels in the AH of cataract patients (P = 0.012). Both oxidized/reduced glutathione ratios and superoxide dismutase levels in the AH were significantly higher in the glaucoma group than in the cataract group. Finally significant correlations were found between PP2A and PP2C, PP2A and PTP, and total antioxidant activity and PTP levels. CONCLUSIONS: There is a statistically significant difference between phosphatase levels in the AH of POAG patients and cataract patients. The phosphatase content of the AH represents tissue pathology, but their presence in the AH may be attributed to cell debris or to active signaling to other molecular events.

Tejido prostático en teratoma quístico maduro del ovario: Caso clínico / Prostatic tissue in a mature cystic teratoma of the ovary: Report of one case

Male accessory sexual glands arising in ovarian cystic teratoma are exceedingly rare. We report a 56-year-old female subjected to an ovariohysterectomy due to a left ovarian mass. The pathological study of the surgical piece revealed a tumor composed of different mature tissue elements and well defined nodules of benign prostatic tissue.

PTPIP51, a positive modulator of the MAPK/Erk pathway, is upregulated in glioblastoma and interacts with 14-3-3ß and PTP1B in situ.

Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumour. Protein tyrosine phosphatase interacting protein 51 (PTPIP51) is an interaction partner of 14-3-3ß, which correlates with the grade of malignancy in gliomas. In this study PTPIP51 and its interacting partners 14-3-3ß, PTP1B, c-Src, Raf-1 as well as EGFR were investigated in human glioblastoma. Twenty glioblastoma samples were analyzed on transcriptional and translational level by immunohistochemistry, in situ hybridization and RT-PCR. To compare PTPIP51 expression in gliomas of different malignancies, quantitative RT-PCR for grade II astrocytoma and GBM samples was employed. Additionally, we analyzed the correlation between PTPIP51 and 14-3-3ß transcription, and checked for in situ interaction between PTPIP51 and 14-3-3ß and PTP1B, respectively. PTPIP51 and 14-3-3ß mRNA showed a tumour grade dependent upregulation in gliomas. Glioblastoma cells displayed a strong immunoreaction of PTPIP51, which co-localized with 14-3-3ß and PTP1B. The duolink proximity ligation assay corroborated a direct in situ interaction of PTPIP51 with both proteins, known to interact with PTPIP51 in vitro. The in vitro interacting partners Raf-1 and c-Src showed a partial co-localization. Besides, immune cells located in capillaries or infiltrating the tumour tissue and endothelial cells of pseudoglomerular vessels revealed a high PTPIP51 expression. The upregulation of PTPIP51 and its connection with the EGFR/MAPK pathway by 14-3-3ß via Raf-1 and by PTP1B via c-Src, argue for a functional role of PTPIP51 in the pathogenesis of human glioblastoma.

Global proteomic assessment of the classical protein-tyrosine phosphatome and "Redoxome".

Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H(2)O(2). Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.

Recent advances in protein tyrosine phosphatase detection using chemical probes.

Protein tyrosine phosphatases (PTPs) are key regulatory enzymes in signal transduction pathways and their aberrancy has been implicated in various diseases such as cancers, metabolic syndrome, and autoimmune disorders. In spite of its great importance, determination of the functional significance of PTPs remains a major challenge, and efficient methodologies are needed to specifically delineate PTP functions. Besides the strategy to use potent and selective PTP inhibitors to study the physiological roles of the enzymes, measurement of PTP activities using specific PTP substrates or activity-based probes (ABPs) has been reported. This review focused on the recent development of small molecular probes to detect PTP activities, consisting of five sub-categories: 1. Conventional fluorescent substrates; 2. Ratiometric fluorescent PTP substrates; 3. Fluorescence substrates with selectivity to a single PTP or a class of PTPs; 4. ABPs specific for PTPs; and 5. A real-time imaging of PTP-substrate complex using a small molecule PTP probe which, offers a measurement of a real-time activity of a certain PTP in cells.