PTP Activity Research Articles

Impact of different severity hyperglycemia on erythrocyte rheological properties1.

The triad "insulin resistance, prediabetes, diabetes" is three independent neologies with characteristic features and development. In addition, each are characterized by progression and the possibility of transition from one form to other. Due to the fact that diabetes is one of the common diseases associated with high rates of disability, it is necessary to improve diagnostic methods and educational regimens for successful prevention and treatment of the disease. We investigated Band 3 protein (B3p) level, osmotic resistance of erythrocytes, the total antioxidant activity (TAA) of blood serum, level of HbA1 in group patients with insulin resistance (IR), prediabetes, and Type 2 Diabetes Mellitus (T2DM) and comparative with health control group. We used original, accurate research methods that measure the essence of the studied quantities. Disruptions of glucose and insulin homeostasis ay lead to the initiation of oxidative stress (in our study demonstrated by a decrease of TAA of blood serum) increased redox-sensitive PTP activity and aberrant band 3 phosphorylation, potentially leading to reduced erythrocyte deformability. At the same time glycation of Hb during T2DM may affect its cross-link with membrane proteins, in particular with B3p, and although appears to limit its cross-linking and decrease its clusterization ability, induces alterations in the cytoskeletal matrix, and thereby decrease erythrocytes' osmotic resistance making them more susceptible to hemolysis. The osmotic resistance of the erythrocytes can be used as a sensitive marker for the detection of the early stages of hyperglycemia (prediabetes). This set of clinical trials will make it possible to identify diseases that make up the triad at an early stage. Early detection of disorders and continued research in this direction will help in the development of a diagnostic scheme for the prevention of such patients. Based on our data, research into anti-oxidation drugs is very important. With the help of the array of studies described in the article and antioxidant treatment, the likelihood of successful treatment will increase.

Probing the Immunoreceptor Tyrosine-Based Inhibition Motif Interaction Protein Partners with Proteomics.

Phosphorylation of tyrosine is the basic mode of protein function and signal transduction in organisms. This process is regulated by protein tyrosine kinases (PTKs) and protein tyrosinases (PTPs). Immunoreceptor tyrosine-based inhibition motif (ITIM) has been considered as regulating the PTP activity through the interaction with the partner proteins in the cell signal pathway. The ITIM sequences need to be phosphorylated first to active the downstream signaling proteins. To explore potential regulatory mechanisms, the ITIM sequences of two transmembrane immunoglobulin proteins, myelin P0 protein-related protein (PZR) and programmed death 1 (PD-1), were analyzed to investigate their interaction with proteins involved in regulatory pathways. We discovered that phosphorylated ITIM sequences can selectively interact with the tyrosine phosphatase SHP2. Specifically, PZR-N-ITIM (pY) may be critical in the interaction between the ITIM and SH2 domains of SHP2, while PD1-C-ITSM (pY) may play a key role in the interaction between the ITIM and SH2 domains of SHP2. Quite a few proteins were identified containing the SH2 domain, exhibiting phosphorylation-mediated interaction with PZR-ITIM. In this study, 14 proteins with SH2 structural domains were identified by GO analysis on 339 proteins associated to the affinity pull-down of PZR-N-ITIM (pY). Through the SH2 domains, these proteins may interact with PZR-ITIM in a phosphorylation-dependent manner.

Pan-cancer analyses of classical protein tyrosine phosphatases and phosphatase-targeted therapy in cancer.

Protein tyrosine phosphatases function in dephosphorylating target proteins to regulate signaling pathways that control a broad spectrum of fundamental physiological and pathological processes. Detailed knowledge concerning the roles of classical PTPs in human cancer merits in-depth investigation. We comprehensively analyzed the regulatory mechanisms and clinical relevance of classical PTPs in more than 9000 tumor patients across 33 types of cancer. The independent datasets and functional experiments were employed to validate our findings. We exhibited the extensive dysregulation of classical PTPs and constructed the gene regulatory network in human cancer. Moreover, we characterized the correlation of classical PTPs with both drug-resistant and drug-sensitive responses to anti-cancer drugs. To evaluate the PTP activity in cancer prognosis, we generated a PTPscore based on the expression and hazard ratio of classical PTPs. Our study highlights the notable role of classical PTPs in cancer biology and provides novel intelligence to improve potential therapeutic strategies based on pTyr regulation.

PTPs: Degrading the Undruggable.

By using a proteolysis-targeting chimera (PROTAC) strategy that couples an allosteric, reversible PTP inhibitor with an E3 ligase targeting ligand through a well-designed linker, the first molecule to successfully inhibit PTP activity through degradation has been developed.

Gain-of-Function SHP2 E76Q Mutant Rescuing Autoinhibition Mechanism Associated with Juvenile Myelomonocytic Leukemia.

Juvenile myelomonocytic leukemia (JMML) is an invasive myeloproliferative neoplasm and is a childhood disease with very high clinical lethality. The SHP2 is encoded by the PTPN11 gene, which is a nonreceptor (pY)-phosphatase and mutation causes JMML. The structural hierarchy of SHP2 includes protein tyrosine phosphatase domain (PTP) and Src-homology 2 domain (N-SH2 and C-SH2). Somatic mutation (E76Q) in the interface of SH2-PTP domain is the most commonly identified mutation found in up to 35% of patients with JMML. The mechanism of this mutant associated with JMML is poorly understood. Here, molecular dynamics simulation was performed on wild-type and mutant (E76Q) of SHP2 to explore the precise impact of gain-of-function on PTP's activity. Consequently, such impact rescues the SHP2 protein from autoinhibition state through losing the interface interactions of Q256/F7 and S502/Q76 or weakening interactions of Q256/R4, Q510/G60, and Q506/A72 between N-SH2 and PTP domains. The consequences of these interactions further relieve the D'E loop away from the PTP catalytic site. The following study would provide a mechanistic insight for better understanding of how individual SHP2 mutations alter the PTP's activity at the atomic level.

The Two SH2 Domains of SHP-2 Bridge Two PD-1 Molecules Resulting in SHP-2 Activation and PD-1-Mediated Inhibition

Programmed cell death-1 (PD-1) is a checkpoint inhibitory receptor member of the B7-CD28 family, which promotes peripheral tolerance and restrains anti-viral and anti-tumor immunity. Although PD-1 blockade leads to durable clinical responses in a fraction of patients, the majority of patients display only transient responses, emphasizing the need for better understanding of the mechanism of PD-1-mediated T cell inhibition. In contrast to other CD28 family family members, which form disulfide-linked homodimers, PD-1 lacks the juxtamembrane cysteine residue responsible for homodimer formation and has been shown to exist as a monomer. The cytoplasmic tail of PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM), centered on Y223 residue, and one immunoreceptor tyrosine-based switch motif (ITSM), centered on Y248 residue. Mutational studies have shown that PD-1-mediated inhibition relies on the interaction of the ITSM with SHP-2 but the mechanism by which PD-1 induces SHP-2 activation is unknown. In this study we sought to determine how PD-1: SHP-2 interaction leads to inhibition of T-cell responses. SHP-2 contains a phosphatase (PTP) domain and two SH2 domains, N-SH2 and C-SH2. To determine whether PD-1 selectively interacts with a specific SH2 domain of SHP-2, we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Surprisingly, pull-down assays with GST-fusion proteins using lysates from primary human T cells or Jurkat T cell line revealed that PD-1 interacted with SHP-2 via both SH2 domains of SHP-2. Biacore assays confirmed that phosphorylated ITSM Y248 interacted with SHP-2 N-SH2 and C-SH2. To determine whether one of the SHP-2 SH2 domains might preferentially interact with PD-1 in live cells, we mutagenized the functional sites of the N-SH2 or C-SH2 domain at arginine R32 and R138, respectively, and transfected COS cells with cDNA of SHP-2 wild type or each SHP-2 SH2 mutant, together with PD-1 and the TCR proximal kinase Fyn, required for PD-1 phosphorylation and interaction with SHP-2. Immunoprecipitation and immunoblot assays showed that mutagenesis of either SH2 domain abrogated interaction of SHP-2 with PD-1 ITSM Y248, providing evidence that both SH2 domains of SHP-2 are involved in the interaction with PD-1. Because each PD-1 molecule has only one ITSM, these results indicate that SHP-2 interacts via its two SH2 domains with two PD-1 molecules. Assessment of PD-1: PD-1 dimer formation in live cells by split luciferase complementation, using NanoBiT proximity assays, showed that upon PD-1 phosphorylation, PD-1: PD1 interaction occurs only in the presence of SHP-2 with intact N-SH2 and C-SH2 domains. The SH2 domains of SHP-2 have a crucial and distinct role in regulating SHP-2 PTPase activity. In the absence of a tyrosine-phosphorylated binding ligand, N-SH2 is bound to the PTP domain leading to an auto-inhibitory closed conformation that blocks the PTP active site. Phosphorylation of Y542 in the SHP-2 C-terminus tail leads to intramolecular interaction of Y542 with the N-SH2 domain to relieve binding to PTP domain. Similarly, intermolecular interaction of the N-SH2 domain with specific phosphorylated ligands disrupts its PTP recognition surface reversing the auto-inhibitory conformation and activates the phosphatase. We determined that monomeric PD-1 ITIM-pY223 or ITSM-pY248 peptides did not activate the PTP. A dimeric phosphopeptide generated by covalent joining of two PD-1 ITSM-pY248 phosphopeptides with a linker that matches the distance between the binding sites of the two SHP-2 SH2 domains induced rapid activation of SHP-2. In contrast, similarly designed dimeric peptides joining either two PD-1 ITIM-pY223 or ITIM-pY223-and-ITSM-pY248 phosphopeptides did not activate PTP activity of SHP-2. The ability of PD1 ITSM-pY248 to induce SHP-2 activation was correlated with inhibition of antigen-mediated IL-2 production, which was abrogated when Y248 was mutagenized to phenylalanine. Our results reveal the geometry of PD-1: SHP-2 interaction that leads to SHP-2 activation and have implications for the development of PD-1-binding compounds to selectively suppress T cell responses by dimerizing PD-1 or enhance T cell responses by disrupting PD-1 dimerization and SHP-2 activation. DisclosuresFreeman:EMD-Serono: Patents & Royalties; Xios: Membership on an entity's Board of Directors or advisory committees; Origimed: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Patents & Royalties; Bristol-Myers-Squibb: Patents & Royalties; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees; Roche: Patents & Royalties; Dako: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Merck: Patents & Royalties; Novartis: Patents & Royalties.

Activation of Engineered Protein Tyrosine Phosphatases with the Biarsenical Compound AsCy3-EDT2.

Methods for activating signaling enzymes hold significant potential for the study of cellular signal transduction. Here we present a strategy for engineering chemically activatable protein tyrosine phosphatases (actPTPs). To generate actPTP1B, we introduced three cysteine point mutations in the enzyme's WPD loop. Biarsenical compounds were screened for the capability to bind actPTP1B's WPD loop and increase its phosphatase activity. We identified AsCy3-EDT2 as a robust activator that selectively targets actPTP1B in proteomic mixtures and intact cells. Introduction of the corresponding mutations in T-cell PTP also generates an enzyme (actTCPTP) that is strongly activated by AsCy3-EDT2 . Given the conservation of WPD-loop structure among the classical PTPs, our results potentially provide the groundwork of a widely generalizable approach for generating actPTPs as tools for elucidating PTP signaling roles as well as connections between dysregulated PTP activity and human disease.

SHP2 is required for BCR-ABL1-induced hematologic neoplasia.

BCR-ABL1-targeting tyrosine kinase inhibitors (TKIs) have revolutionized treatment of Philadelphia chromosome-positive (Ph+) hematologic neoplasms. Nevertheless, acquired TKI resistance remains a major problem in chronic myeloid leukemia (CML), and TKIs are less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). GAB2, a scaffolding adaptor that binds and activates SHP2, is essential for leukemogenesis by BCR-ABL1, and a GAB2 mutant lacking SHP2 binding cannot mediate leukemogenesis. Using a genetic loss-of-function approach and bone marrow transplantation (BMT) models for CML and BCR-ABL1+ B-ALL, we show that SHP2 is required for BCR-ABL1-evoked myeloid and lymphoid neoplasia. Ptpn11 deletion impairs initiation and maintenance of CML-like myeloproliferative neoplasm, and compromises induction of BCR-ABL1+ B-ALL. SHP2, and specifically, its SH2 domains, PTP activity and C-terminal tyrosines, is essential for BCR-ABL1+, but not WT, pre-B cell proliferation. The MEK/ERK pathway is regulated by SHP2 in WT and BCR-ABL1+ pre-B cells, but is only required for the proliferation of BCR-ABL1+ cells. SHP2 is required for SRC family kinase (SFK) activation only in BCR-ABL1+ pre-B cells. RNAseq reveals distinct SHP2-dependent transcriptional programs in BCR-ABL1+ and WT pre-B cells. Our results suggest that SHP2, via SFKs and ERK, represses MXD3/4 to facilitate a MYC-dependent proliferation program in BCR-ABL1-transformed pre-B cells.

Titanium‐released from dental implant enhances pre‐osteoblast adhesion by ROS modulating crucial intracellular pathways

It is important to understand the cellular and molecular events that occur at the cell-material interface of implants used for bone repair. The mechanisms involved in the initial stages of osteoblast interactions with the surface of the implant material must be decisive for cell fating surrounding them. In order to address this issue, we decided to investigate if conditioned medium for dental implants was able to modulate murine pre-osteoblast metabolism. First, we determined the concentration of titanium (Ti)-containing conditioned medium and found that it was 2-fold increased (p < 0.0001). We have reported that this conditioned medium significantly up-modulated pre-osteoblast adhesion up to 24 h (p < 0.0001). In parallel, our results showed that both phosphorylations of FAK (focal adhesion kinase) at Y397 (p < 0.0011) and Cofilin at Ser03 (p < 0.0053) were also up-modulated, as well as for Rac1 expression (p < 0.0175); both of them are involved with cell adaptation by rearranging cytoskeleton actin filaments. Thereafter, Ti-containing medium stimulated ROS (reactive oxygen species) production by pre-osteoblast cells, and it is very possible that ROS compromised PTP-1B (protein tyrosine phosphatase 1B) activation since PTP1B was down-phosphorylated (p < 0.0148). The low PTP activity guarantees the phosphorylation of FAK at Y-residue, causing better pre-osteoblast adhesion in response to Ti-containing medium. Altogether, these data indicate that ROS indirectly modulate FAK phosphorylation in response to Ti-released from dental implants. Taken the results in account, these data showed for the first time that the implanted dental device is able to dynamically affect surrounding tissues, mainly by promoting a better performance of the pre-osteoblast cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2968-2976, 2017.

The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN−) oxidation by H2O2 to form hypothiocyanous acid (HOSCN), an oxidant that targets Cys residues. Dysregulated phosphorylation and elevated MPO levels have been associated with chronic inflammatory diseases where HOSCN can be generated. Previous studies have shown that HOSCN inhibits isolated PTP1B and induces cellular dysfunction in cultured macrophage-like cells. The present study extends this previous work and shows that physiologically-relevant concentrations of HOSCN alter the activity and structure of other members of the wider PTP family (including leukocyte antigen-related PTP, PTP–LAR; T-cell PTP, TC–PTP; CD45 and Src homology phosphatase-1, Shp-1) by targeting Cys residues. Isolated PTP activity, and activity in lysates of human monocyte-derived macrophages (HMDM) was inhibited by 0–100µM HOSCN with this being accompanied by reversible oxidation of Cys residues, formation of sulfenic acids or sulfenyl-thiocyanates (detected by Western blotting, and LC–MS as dimedone adducts), and structural changes. LC–MS/MS peptide mass-mapping has provided data on the modified Cys residues in PTP–LAR. This study indicates that inflammation-induced oxidants, and particularly myeloperoxidase-derived species, can modulate the activity of multiple members of the PTP superfamily via oxidation of Cys residues to sulfenic acids. This alteration of the balance of PTP/kinase activity may perturb protein phosphorylation and disrupt cell signaling with subsequent induction of apoptosis at sites of inflammation.

Direct Chemical Activation of a Rationally Engineered Signaling Enzyme.

Few chemical strategies for activating enzymes have been developed. Here we show that a biarsenical compound (FlAsH) can directly activate a rationally engineered protein tyrosine phosphatase (Shp2 PTP) by disrupting autoinhibitory interactions between Shp2's N-terminal SH2 domain and its PTP domain. We found that introducing a tricysteine motif at a loop of Shp2's N-SH2 domain confers affinity for FlAsH; binding of FlAsH to the cysteine-enriched loop relieves Shp2's inhibitory interdomain interaction and substantially increases the enzyme's PTP activity. Activation of engineered Shp2 is substrate independent and is observed in the contexts of both purified enzyme and complex proteomes. A chemical means for activating Shp2 could be useful for investigating its roles in signaling and oncogenesis, and the loop-targeting strategy described herein could provide a blueprint for the development of target-specific activators of other autoinhibited enzymes.

Strategies to make protein serine/threonine (PP1, calcineurin) and tyrosine phosphatases (PTP1B) druggable: Achieving specificity by targeting substrate and regulatory protein interaction sites

The established dogma is that protein serine/threonine (PSPs) and tyrosine (PTPs) phosphatases are unattainable drug targets. This is because natural product inhibitors of PSP active sites are lethal, while the active sites of PTPs are exceptionally conserved and charged, making it nearly impossible to develop PTP inhibitors that are selective. However, due to a series of recent structural and functional studies, this view of phosphatases is about to undergo a radical change. Rather than target active sites, these studies have demonstrated that targeting PSP/PTP protein (substrate/regulatory) interaction sites, which are distal from the active sites, are highly viable and suitable drugs targets. This is especially true for calcineurin (CN), in which the blockbuster immunosuppressant drugs FK506 and cyclosporin A were recently demonstrated to bind and block one of the key CN substrate interaction sites, the LxVP site. Additional studies show that this approach—targeting substrate and/or regulatory protein interaction sites—also holds incredible promise for protein phosphatase 1 (PP1)-related diseases. Finally, domains outside PTP catalytic domains have also recently been demonstrated to directly alter PTP activity. Collectively, these novel insights offer new, transformative perspectives for the therapeutic targeting of PSPs by interfering with the binding of PIPs or substrates and PTPs by targeting allosteric sites outside their catalytic domains.

Identification of a novel stress regulated FERM domain containing cytosolic protein having PTP activity in Setaria cervi, a bovine filarial parasite

A 67 kDa cytosolic FERM domain containing protein having significant protein tyrosine phosphatases activity (PTPL) has been purified to homogeneity from Setaria cervi, a bovine filarial parasite. The MALDI-MS/MS analysis of the purified protein revealed 16 peptide peaks showing nearest match to Brugia malayi Moesin/ezrin/radixin homolog 1 protein and one peptide showing significant similarity with a region lying in the catalytic domain of human PTPD1. PTPL showed significant cross reactivity with the human PTP1B antibody and colocalize with actin in the coelomyrian cells of hypodermis in the parasite. PTPL was stress regulated as it showed marked decrease in the expression when exposed to Aspirin, an antifilarial drug and Phenylarsine Oxide, PTP inhibitor.

Proteinase-activated receptor 1- and 4-promoted migration of Hep3B hepatocellular carcinoma cells depends on ROS formation and RTK transactivation.

There is growing evidence for a role of proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, in cancer. We have previously shown that PAR1 and PAR4 are able to promote the migration of hepatocellular carcinoma (HCC) cells suggesting a function in HCC progression. In this study, we assessed the underlying signalling mechanisms. Using Hep3B liver carcinoma cells, RTK activation was assessed by Western blot employing phospho-RTK specific antibodies, ROS level were estimated by H2DCF-DA using confocal laser scanning microscopy, and measurement of PTP activity was performed in cell lysates using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a substrate. Thrombin, the PAR1 selective agonist peptide TFLLRN-NH2 (PAR1-AP), and the PAR4 selective agonist peptide, AYPGKF-NH2 (PAR4-AP), induced a significant increase in Hep3B cell migration that could be blocked by inhibitors targeting formation of reactive oxygen species (ROS), or activation of hepatocyte-growth factor receptor (Met), or platelet-derived growth factor receptor (PDGFR), respectively. The involvement of these intracellular effectors in PAR1/4-initiated migratory signalling was further supported by the findings that individual stimulation of Hep3B cells with the PAR1-AP and the PAR4-AP induced an increase in ROS production and the transactivation of Met and PDGFR. In addition, PAR1- and PAR4-mediated inhibition of total PTP activity and specifically PTP1B. ROS inhibition by N-acetyl-L-cysteine prevented the inhibition of PTP1B phosphatase activity induced by PAR1-AP and the PAR4-AP, but had no effect on PAR1/4-mediated activation of Met and PDGFR in Hep3B cells. Collectively, our data indicate that PAR1 and PAR4 activate common promigratory signalling pathways in Hep3B liver carcinoma cells including activation of the receptor tyrosine kinases Met and PDGFR, the formation of ROS and the inactivation of PTP1B. However, PAR1/4-triggered Met and PDGFR transactivation seem to be mediated independently from the ROS-PTP1B signalling module.

Protein tyrosine phosphatase-PEST (PTP-PEST) regulates mast cell-activating signals in PTP activity-dependent and -independent manners

Aggregation of the high-affinity IgE receptor (FcεRI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Tyrosine phosphorylation of proteins in response to FcεRI aggregation has been implicated in mast cell activation. Here, we determined the role of PTP-PEST (encoded by PTPN12) in the regulation of mast cell activation using the RBL-2H3 rat basophilic leukemia cell line as a model. PTP-PEST expression was significantly induced upon FcεRI-crosslinking, and aggregation of FcεRI induced the phosphorylation of PTP-PEST at Ser39, thus resulting in the suppression of PTP activity. By overexpressing a phosphatase-dead mutant (PTP-PEST CS) and a constitutively active mutant (PTP-PEST SA) in RBL-2H3 cells, we showed that PTP-PEST decreased degranulation and enhanced IL-4 and IL-13 transcription in FcεRI-crosslinked RBL-2H3 cells, but PTP activity of PTP-PEST was not necessary for this regulation. However, FcεRI-induced TNF-α transcription was increased by the overexpression of PTP-PEST SA and suppressed by the overexpression of PTP-PEST CS. Taken together, these results suggest that PTP-PEST is involved in the regulation of FcεRI-mediated mast cell activation through at least two different processes represented by PTP activity-dependent and -independent pathways.

Aspirin inhibits lipopolysaccharide-induced COX-2 expression and PGE2production in porcine alveolar macrophages by modulating protein kinase C and protein tyrosine phosphatase activity

Aspirin has been demonstrated to be effective in inhibiting COX-2 and PGE2 in Alveolar macrophages (AMs). However, the mechanisms have not been fully understood. In the present study, we found that pretreatment with aspirin inhibited LPS-induced COX-2 and PGE2 upregulation, IκBα degradation, NFκB activation and the increase of PKC activity, but elevated LPS-induced the decrease of PTP activity. The PKC inhibitor calphostin C dramatically reduced the COX-2 mRNA and PGE2 levels, but the PTP inhibitor peroxovanadium (POV) significantly increased the COX-2 mRNA and PGE2 levels. Furthermore, the PTP inhibitor mitigated the inhibitory effect of aspirin on COX-2 and PGE2 upregulation and NF-κB activation, whereas the PKC inhibitor enhanced the inhibitory effects of aspirin on the production of COX-2 and PGE2. Our data indicate a novel mechanism by which aspirin acts as a potent anti-inflammatory agent in alveolus macrophages and ALI. [BMB Reports 2014; 47(1): 45-50]

Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells: FLT3 ITD sustains high levels of DUSP6 expression

Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation.

Activation of Src and transformation by an RPTPα splice mutant found in human tumours

Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ∼30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.

Effect of diethylcarbamazine, butylated hydroxy anisole and methyl substituted chalcone on filarial parasite Setaria cervi: Proteomic and biochemical approaches

For survival, parasite exerts several lines of defense of which drug neutralization is one of the major phenomena. Lack of phase I cytochrome P450 in some of the nematode render them depend on the phase II detoxification system involving GST as a major detoxifying enzymes. In present study, the antifilarial DEC, phenolic compound BHA and methyl chalcone have been evaluated for proteomic and biochemical studies in Setaria cervi. BHA and methyl chalcone showed cytotoxic effect leading to irreversible inhibition in motility and viability of parasites. These drugs showed marked alteration in proteomic profile of S. cervi at 100 μM concentration with 10.82, 8.52 and 6.75% downregulated (<0.5) and 7.64, 31.78 and 24.32% upregulated (>1.5) in DEC, BHA and methyl chalcone treatment respectively. Significant depletion in GSH level with increase in NO production was observed. Amongst these compounds, methyl chalcone demonstrated significant inhibitory effect (p<0.05) on GST, PGHS and PTP activity leading to loss of metabolic homeostasis and parasite death. The cytotoxic response and altered expression profile of major enzymes under drug exposure suggested the oxidative stress induced apoptosis as a major cause of parasite killing which was further supported by DNA fragmentation in BHA and methyl chalcone.

Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth

Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr(1221/2) correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr(1221/2). Concurrently, Tyr(317) phosphorylation of p52(Shc), proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr(1221/2). Its downstream p52(Shc), ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr(1221/2) phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr(1221/2) and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas.