Non-transmembrane Protein Tyrosine Phosphatase Research Articles

PTPN7 Tyrosine Phosphatase Negatively Regulates Thromboxane Generation and ERK Phosphorylation in Platelets

Background: Thromboxane is a short-lived lipid mediator which recruits more platelets to site of vascular injury. Dysregulation of thromboxane generation can lead to several thrombotic complications such as stroke and myocardial infarction. In platelets, thromboxane generation is regulated by extracellular signal-regulated protein kinase 1/2 (ERK1/2) and therefore it is important to understand how ERK is regulated in platelets. In lymphocytes, ERK phosphorylation is regulated by a non-transmembrane protein tyrosine phosphatase PTPN7. In this study we investigated the presence of PTPN7 in platelets and effect of PTPN7 deficiency on platelet activation.Results: Western blot analysis revealed that PTPN7 is present in both human and murine platelets (Fig. 1). PTPN7 KO murine platelets show increased platelet functional responses such as aggregation, dense granule secretion and thromboxane generation upon stimulation with both GPCR and GPVI agonists (Fig. 2). In the presence of COX inhibitor indomethacin, the PTPN7 KO murine platelets aggregate and secrete to the same extent as the WT. This suggests that elevated thromboxane A2 levels are responsible for potentiation of platelet functional responses. ERK phosphorylation is also potentiated in PTPN7 KO (Fig.3) and the activity of signaling molecules upstream of ERK such as MEK is unaffected. This shows that specificity of the PTPN7 towards ERK in platelets. Lastly, in pulmonary embolism model PTPN7 KO mice have shorter time to death due to pulmonary thrombosis.Conclusions: PTPN7 is a negative regulator ERK phosphorylation and thromboxane generation in platelets. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of protein tyrosine phosphatase 1B by flavonoids: A structure - activity relationship study

The classical non-transmembrane protein tyrosine phosphatase 1B (PTP1B) has emerged as a key negative regulator of insulin signaling pathways that leads to insulin resistance, turning this enzyme a promising therapeutic target in the management of type 2 diabetes mellitus (T2DM). In the present work, the in vitro inhibitory activity of a panel of structurally related flavonoids, for recombinant human PTP1B was studied and the type of inhibition of the most active compounds further evaluated. The majority of the studied flavonoids was tested in this work for the first time, including flavonoid C13, which was the most potent inhibitor. It was observed that the ability to inhibit PTP1B depends on the nature, position and number of substituents in the flavonoid structure, as the presence of both 7- and 8-OBn groups in the A ring, together with the presence of both 3′ and 4′-OMe groups in the B ring and the 3-OH group in the C ring; these substituents increase the flavonoids' ability to inhibit PTP1B. In conclusion, some of the tested flavonoids seem to be promising PTP1B inhibitors and potential effective agents in the management of T2DM, by increasing insulin sensitivity.

Progress of relationship between phosphatase of regeneration liver-3 and tumors

Phosphatase of regenerating liver-3 (PRL-3) is a kind of non-transmembrane protein-tyrosine phosphatase, characterized by its promotion of cell proliferation, migration and invasion. It is highly expressed in many tumors and has a close correlation with tumorigenesis, metastasis and prognosis. With the further research in recently years, more and more studies have suggested that PRL-3 has a wide applicative prospect in clinic, serving as a important clinical biological marker to predict tumor metastasis and prognosis. This paper will review the structure of PRL-3, mechanism and relationship between PRL-3 and tumors. Key words: Phosphatase of regeneration liver-3; Neoplasm metastasis; Prognosis

Transmembrane Phosphatases and Cancer Development, the Role of Protein Tyrosine Phosphatase-kappa (PTPκ) and Protein Tyrosine Phosphatase-mu (PTPμ)

Protein tyrosine phosphatases (PTPs) are important in the regulation of diverse cellular functions including proliferation, migration and invasion; aberration of these cellular events is crucial for the development and progression of cancer. PTPs family comprises of two groups, classic PTPs and dual specificity phosphatases. The classic PTPs include both non-transmembrane PTPs and transmembrane receptor-like PTPs (RPTPs). RPTPs are composed of extracellular regions, transmembrane domains and intracellular phosphatase domains. The extracellular regions of RPTPs are similar to cell adhesion molecules and can interact homophilically and heterophilically. There are eight subgroups in the RPTPs separated according to the differences in their extracellular regions. PTPRK and PTPRM belong to the R2B subfamily of RPTPs and both perform homophilic interactions and regulate cell-cell aggregation and adhesion. Furthermore, both PTPRK and PTPRM can interact with the catenin/cadherin complex to regulate cell proliferation and migration. The current review discusses the present knowledge on RPTPs and their potential implication in the development and progression of cancer.

Protein tyrosine phosphatase 1B inhibitory activities of ursane- and lupane-type triterpenes from Sorbus pohuashanensis

Protein tyrosine phosphatase 1B (PTP1B) is a non-transmembrane protein tyrosine phosphatase, and has received much attention as a molecular target for the treatment of insulin resistance diseases because of its critical roles in negatively regulating insulin- and leptin-signaling cascades. Six ursane-type triterpenes, 3β-acetoxy-urs-12-ene-28-oic acid (1), pomolic acid-3β-acetate (2), pomolic acid (3), ursolaldehyde (4), euscaphic acid (5) and 3β-acetoxy-urs-11-en-28,13-olide (6), and a lupane-type triterpene, betulinic acid (7), from the fruits of Sorbus pohuashanensis, exhibited significant PTP1B inhibitory activity, with IC50 values ranging from 3.5 to 54.8 μM. Kinetics analyses revealed that compounds 2, 3, and 7 are non-competitive PTP1B inhibitors, and compounds 1 and 6 are mixed-type PTP1B inhibitors.

Overexpression of PTP1B in human colorectal cancer and its association with tumor progression and prognosis

Protein tyrosine phosphatase 1B (PTP1B) is a non-transmembrane protein tyrosine phosphatase that has been implicated in cancer pathogenesis. However, the expression level and the role of PTP1B in the development and prognosis of colorectal cancer (CRC) remain unclear. In this study, the expression of PTP1B in CRC tissues and matched noncancerous tissues were detected by using immunohistochemistry, real-time PCR and Western blotting. The correlations between PTP1B expression level and clinicopathologic characteristics and patient survival were analyzed. We found that PTP1B expression was significantly higher in CRC tissues compared with matched non-tumour tissues. Statistical analysis showed that the PTP1B expression was correlated with tumor differentiation, tumor invasion, lymph node metastasis, and TNM stage. Patients with higher expressions of PTP1B had the lower survival (P = 0.012). Taken together, our results suggest that PTP1B expression might play a critical role in the progression of CRC and may serve as a valuable prognostic biomarker for CRC.

PTP1B expression is an independent positive prognostic factor in human breast cancer

Protein tyrosine phosphatase 1B (PTP1B) is a non-transmembrane protein tyrosine phosphatase that has come into focus as a critical regulator of multiple signaling pathways. The role of PTP1B in breast cancer remains unclear with evidence suggesting that PTP1B can exert both tumor-suppressing and tumor-promoting effects. To better define the role of PTP1B in human breast cancer, and its relationship with HER2, we performed immunohistochemical studies on a large cohort of functionally annotated primary breast cancer specimens. 683 of 1,402 (49 %) evaluable primary breast cancers are positive for PTP1B. There is no statistically significant association between PTP1B expression and age, tumor size, T stage, histologic grade, lymph node status, or histological subtype. Of note, there is no significant association between PTP1B expression and HER2 expression (PTP1B expression 53.1 % in HER2(+) cancers vs. 47.5 % in HER2(-) cancers, p = 0.0985). However, PTP1B expression is significantly associated with estrogen receptor expression (PTP1B expression 50.7 % in ER(+) cancers vs. 43.1 % in ER(-) cancers, p = 0.0137) and intrinsic molecular subtype (PTP1B expression 53.9 % in the luminal B HER2(+) subtype and 37.9 % in the basal-like subtype). Of note, multivariate analyses demonstrate that PTP1B is an independent predictor of improved survival in breast cancer (HR 0.779, p = 0.006). Taken together, we demonstrate in the largest study to date that (1) PTP1B is commonly expressed in breast cancer, (2) there is no association or functional impact of PTP1B expression in HER2(+) breast cancer, and (3) PTP1B expression in breast cancer is associated with significantly improved clinical outcome. Until additional studies are performed, caution should be exercised in using PTP1B inhibitors in human breast cancer.

The tyrosine phosphatase HD-PTP (PTPN23) is degraded by calpains in a calcium-dependent manner

HD-PTP (PTPN23) is a non-transmembrane protein tyrosine phosphatase which contributes to the signal transduction pathways involved in the regulation of cell migration and invasion. We here demonstrate in T24 bladder carcinoma cells that HD-PTP undergoes calcium-dependent degradation which can be prevented by specific calpain inhibitors. In addition, treatment of the cells with the calpain inhibitor calpeptin results in the redistribution of endogenous HD-PTP to the periphery of the cells.Since (i) calpains are overexpressed in some tumors and (ii) the downregulation of HD-PTP enhances cell migration and invasion, we propose that HD-PTP degradation by calpains might result in the acquisition of a more aggressive phenotype in neoplastic cells.

Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n=16) and hepatocellular carcinoma (n=169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6+ tumor-initiating cells (T-ICs) and high frequency of nuclear β-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/β-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.

HD-PTP Is a Catalytically Inactive Tyrosine Phosphatase Due to a Conserved Divergence in Its Phosphatase Domain

BackgroundThe HD-PTP protein has been described as a tumor suppressor candidate and based on its amino acid sequence, categorized as a classical non-transmembrane protein tyrosine phosphatase (PTP). To date, no HD-PTP phosphorylated substrate has been identified and controversial results concerning its catalytic activity have been recently reported.Methodology and ResultsHere we report a rigorous enzymatic analysis demonstrating that the HD-PTP protein does not harbor tyrosine phosphatase or lipid phosphatase activity using the highly sensitive DiFMUP substrate and a panel of different phosphatidylinositol phosphates. We found that HD-PTP tyrosine phosphatase inactivity is caused by an evolutionary conserved amino acid divergence of a key residue located in the HD-PTP phosphatase domain since its back mutation is sufficient to restore the HD-PTP tyrosine phosphatase activity. Moreover, in agreement with a tumor suppressor activity, HD-PTP expression leads to colony growth reduction in human cancer cell lines, independently of its catalytic PTP activity status.ConclusionIn summary, we demonstrate that HD-PTP is a catalytically inactive protein tyrosine phosphatase. As such, we identify one residue involved in its inactivation and show that its colony growth reduction activity is independent of its PTP activity status in human cancer cell lines.

The Association Between SHP-2 Mutations and Platelet Function in Noonan Syndrome.

Noonan syndrome (NS) is an autosomal dominant disorder characterized by variable expression of multiple defects including short stature, facial dysmorphias, congenital heart defects, developmental delay, and hematological abnormalities. A bleeding tendency has been variably described in patients with NS; Factor XI deficiency and platelet function abnormalities are reported most frequently. Recently, germline missense mutations in the PTPN11 gene have been identified in up to 50% of individuals with NS. PTPN11 encodes the ubiquitously expressed cell-signaling mediator SHP-2, a non-transmembrane protein tyrosine phosphatase involved in the normal activation of the Ras-MAPK signaling cascade. SHP-2 has a pivotal role in cell proliferation, differentiation, and survival, but also modulates platelet responses to some stimuli. Some PTPN11 mutations lead to altered SHP-2 phosphatase activity, and have been shown to impact cardiac development in a mouse model of NS.This study examined the potential association among PTPN11 mutations, platelet function abnormalities, and platelet SHP-2 activity in patients with NS. Eighteen patients with a clinical diagnosis of NS were studied: 14 (78%) were female and the mean age was 20.7 years (range 4–56 years). A validated bleeding questionnaire was administered to each participant, and platelet aggregometry was performed on platelet-rich plasma according to standard clinical practice. SHP-2 was isolated by immunoprecipitation from agoniststimulated platelets and phosphatase activity measured spectrophotometrically. PTPN11 mutations were identified by dHPLC and defined by DNA sequencing in each participant to characterize the presence and the precise nature of a mutation associated with their NS.Seven (39%) participants were classified as bleeders on the basis of the questionnaire; of these, four had no hemostasis abnormality identified. One participant had mild Factor XI deficiency. Seven participants had abnormalities of platelet aggregation, and four participants had decreased platelet dense granule numbers and ATP release. PTPN11 mutations were identified in 12 (67%) participants: seven were the commonly described mutation in Exon 8; four members of a three-generation family had a previously described Exon 3 mutation; and one individual had a mutation in Exon 12 which is usually associated with LEOPARD syndrome. Mutations in other genes associated with NS were not evaluated in this study. Six patients with PTPN11 mutations were clinically classified as bleeders, but no correlation was found between PTPN11 mutation and agonist-induced platelet aggregation or SHP-2 phosphatase activity. Three patients with platelet function abnormalities had no PTPN11 mutation identified.Not all patients with NS have a clinical bleeding profile or platelet function abnormalities. Neither clinical bleeding history nor platelet function abnormalities correlated with PTPN11 mutations, suggesting that additional factors may be required for a bleeding phenotype associated with PTPN11 mutations. Mutations in other NS-associated genes may contribute to a bleeding phenotype. Abnormalities in platelet aggregometry could not be explained on the basis of SHP-2 activity, although it is possible that changes in enzyme activity downstream in the Ras-MAPK pathway may impact platelet function in some patients with NS. Ultimately, the identification of a molecular basis for the frequently observed platelet aggregation defects could provide a means to predict the risk of bleeding in patients with NS.

The novel protein PTPIP51 is expressed in human keratinocyte carcinomas and their surrounding stroma

Background: The novel protein PTPIP51 (SwissProt accession code Q96SD6) is known to interact with two non-transmembrane protein-tyrosine phosphatases, PTP1B and TCPTP in vitro. Overexpression of the full-length protein induces apoptosis in HeLa and HEK293T cells (Lv et al. 2006). PTPIP51 shows a tissue-specific expression pattern and is associated with cellular differentiation and apoptosis in some mammalian tissues, especially in human follicular and interfollicular epidermis. PTPIP51 protein is expressed in all suprabasal layers of normal epidermis, whereas the basal layer contains PTPIP51 mRNA only but lacks the protein. Objectives: The expression of PTPIP51 was investigated in keratinocyte carcinomas, that is human basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) as well as Bowen's disease (BD) and keratoacanthomas (KAs) on a transcriptional (mRNA) and translational (immunohistochemical) level. Methods: Formalin-fixed, paraffin-embedded sections of BCCs, SCCs, KAs and BD, respectively, were analysed by RT-PCR, as well as immunohistochemistry and subsequent fluorescence microscopy. PTPIP51-positive cells of the tumour and the surrounding stroma were identified on the basis of specific morphological features by means of H & E staining. To obtain further information about a putative function of PTPIP51, a possible association of PTPIP51 with apoptotic cells, as well as an assumed negative correlation with proliferating cells was investigated by means of an in-situ TUNEL assay and Ki67/MIB-1 antigen staining, respectively. Co-immunostainings with PTPIP51 were performed for the following antigens: TCPTP, PTP1B and β-catenin. Results: PTPIP51-expression was detected in BCCs and SCCs of the skin, as well as in KAs and BD. Both types of keratinocyte carcinoma revealed a specific localization pattern of PTPIP51 in malignant keratinocytes. Whereas PTPIP51 -positive cells of BCC were found to form two cluster types with a different subcellular localization of the protein, i.e. cytoplasmic and nuclear or predominantly membranous, investigation of SCC revealed a meshwork-like appearance of PTPIP51-positive malignant keratinocytes, created by a mainly membranous localization. BD and KA resembled the findings of PTPIP51-expression in SCC. Furthermore, we observed a partial co-localization of PTP1B and PTPIP51 in BCC. SCC and BCC showed a co-expression and partial co-localization of PTPIP51 with β-catenin. Some PTPIP51-positive cells were found to undergo apoptosis. PTPIP51 was also expressed in cells comprising the surrounding stromal microenvironment. This was particularly noticed for endothelial cells lining peritumoural vessels as well as for infiltrating cells of both, the innate and the adaptive immune system. Conclusions: The results showed a distinct mainly membranous expression pattern of PTPIP51 in BCCs and SCCs. Since PTPIP51 was also detected in the peritumoural tissue, the protein may play a crucial role in keratinocyte tumour development.

Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity.

Mouse PTP-BL is a large, nontransmembrane protein tyrosine phosphatase of unclear physiological function that consists of a KIND domain, a FERM domain, five PDZ domains, and a COOH-terminal catalytic PTP domain. PTP-BL and its human ortholog PTP-BAS have been proposed to play a role in the regulation of microfilament dynamics, cytokinesis, apoptosis, and neurite outgrowth. To investigate the biological function of PTP-BL enzyme activity, we have generated mice that lack the PTP-BL PTP moiety. These PTP-BL(DeltaP/DeltaP) mice are viable and fertile and do not present overt morphological alterations. Although PTP-BL is expressed in most hematopoietic cell lineages, no alterations of thymocyte development in PTP-BL(DeltaP/DeltaP) mice could be detected. Sciatic nerve lesioning revealed that sensory nerve recovery is unaltered in these mice. In contrast, a very mild but significant impairment of motor nerve repair was observed. Our findings exclude an essential role for PTP-BL as a phosphotyrosine phosphatase and rather are in line with a role as scaffolding or anchoring molecule.

Role of the CD47-SHPS-1 system in regulation of cell migration.

SHPS-1 is a transmembrane protein whose extracellular region interacts with CD47 and whose cytoplasmic region undergoes tyrosine phosphorylation and there by binds the protein tyrosine phosphatase SHP-2. Formation of this complex is implicated in regulation of cell migration by an unknown mechanism. A CD47-Fc fusion protein or antibodies to SHPS-1 inhibited migration of human melanoma cells or of CHO cells overexpressing SHPS-1. Overexpression of wild-type SHPS-1 promoted CHO cell migration, whereas expression of the SHPS-1-4F mutant, which lacks the phosphorylation sites required for SHP-2 binding, had no effect. Antibodies to SHPS-1 failed to inhibit migration of CHO cells expressing SHPS-1-4F. SHPS-1 ligands induced the dephosphorylation of SHPS-1 and dissociation of SHP-2. Antibodies to SHPS-1 also enhanced Rho activity and induced both formation of stress fibers and adoption of a less polarized morphology in melanoma cells. Our results suggest that engagement of SHPS-1 by CD47 prevents the positive regulation of cell migration by this protein. The CD47- SHPS-1 system and SHP-2 might thus contribute to the inhibition of cell migration by cell-cell contact.

In vivo by reversible oxidation" chap="23">Analysis of the Regulation of Protein Tyrosine Phosphatases in Vivo by Reversible Oxidation

The chapter presents analysis of the regulation of protein tyrosine phosphatases (PTPs) in vivo by reversible oxidation. Consistent with an important function as regulators of signal transduction, the activity of the PTPs is tightly controlled in vivo. Various mechanisms of regulation are described, from ligand binding to the receptor-like PTPs and targeting of nontransmembrane PTPs to defined subcellular locations, to regulation of PTP function by covalent modification, such as phosphorylation and proteolysis. It has become apparent that the production of ROS in nonphagocytic cells is important for propagating an optimal response to stimulation by growth factors, cytokines, and hormones. The unique features of the active site of the PTPs and their mechanism of catalysis have drawn attention to their potential for regulation by reversible oxidation. In order to promote the anaerobic environment and ensure that the oxidation status of the PTPs in the lysate reflects accurately that of the intact cell, degassed buffers should be used for these studies. The chapter describes the measurement of PTP activity using the in-gel assay and the stimulus-induced oxidation of PTPs.

Interaction between chicken protein tyrosine phosphatase 1 (CPTP1)-like rat protein phosphatase 1 (PTP1) and p60(v-src) in v-src-transformed Rat-1 fibroblasts.

CPTP1 is a nontransmembrane chicken protein tyrosine phosphatase having 92% sequence homology to the corresponding 321 amino acids of human protein tyrosine phosphatase 1B (HPTP1B). Using anti-CPTP1 antibody, we identified CPTP1-like rat PTP1 of 51 kDa in Rat-1 and v-src-transformed Rat-1 fibroblasts. Here we show that CPTP1-like rat PTP1 binds to p60(v-src) in vivo and CPTP1 also can associate with p60(v-src) in cell lysate of v-src- transformed Rat-1 fibroblasts. Interaction between HPTP1B-type PTPs, CPTP1-like rat PTP1 and CPTP1, and p60(v-src) was reduced by vanadate treatment for 13 h due to down regulation of the protein level of p60(v-src) in vivo. Interestingly, CPTP1-like rat PTP1 was coimmunoprecipitated with a 70-kDa protein which has a possibility to be tyrosine- phosphorylated by p60(v-src) in v-src-transformed Rat-1 fibroblasts. These results suggest that HPTP1B-type PTPs may play an important role in p60(src) dependent signal pathway in eucaryotic cells.

The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif

Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.

Regulation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B activity by casein kinase II- and p56lck-mediated phosphorylation.

Protein tyrosine phosphorylation and dephosphorylation are important in the regulation of cell proliferation and signaling cascade. In order to examine whether phosphatase activity of CPTP1 and HPTP1B, typical nontransmembrane protein tyrosine phosphatase, could be controlled by phosphorylation, affinity-purified PTPs were phosphorylated by CKII and p56lck in vitro. Phosphoamino acid analysis revealed that CPTP1 was phosphorylated on both serine and threonine residues by CKII, and tyrosine residue by p56lck. Phosphatase activity of CPTP1 was gradually increased by three-fold concomitant with phosporylation by CKII. Phosphorylation of HPTP1B by CKII resulted in quick two-fold enhancement of its phosphatase activity within 5 min of incubation and remained in that state. In the presence of CKII inhibitor, heparin or poly(Glu.Tyr), both phosphorylation and enhancement of phosphatase activity of CPTP1 and HPTP1B were mostly blocked. p56lck catalyzed tyrosine phosphorylation of CPTP1 and HPTP1B was only observed by inhibiting the intrinsic tyrosine phosphatase activity. Taken together, these results indicate that CPTP1 or HPTP1B possesses a capability to regulate its phosphatase activity through phosphorylation processes and may participate in the cellular signal cascades.

Human 70-kDa SHP-1L Differs from 68-kDa SHP-1 in Its C-terminal Structure and Catalytic Activity

Human 70-kDa SHP-1L Differs from 68-kDa SHP-1 in Its C-terminal Structure and Catalytic Activity

Human 70-kDa SHP-1L Differs from 68-kDa SHP-1 in Its C-terminal Structure and Catalytic Activity

The tyrosine phosphatase SHP-1 functions as a negative regulator in hematopoietic cell development, proliferation, and receptor-mediated cellular activation. In Jurkat T cells, a major 68-kDa band and a minor 70-kDa band were immunoprecipitated by a monoclonal antibody against the SHP-1 protein-tyrosine phosphatase domain, while an antibody against the SHP-1 C-terminal 19 amino acids recognized only the 68-kDa SHP-1. The SDS-gel-purified 70-kDa protein was subjected to tryptic mapping and microsequencing, which was followed by molecular cloning. It revealed that the 70-kDa protein, termed SHP-1L, is a C-terminal alternatively spliced form of SHP-1. SHP-1L is 29 amino acids longer than SHP-1, and its 66 C-terminal amino acids are different from SHP-1. The C terminus of SHP-1L contains a proline-rich motif PVPGPPVLSP, a potential Src homology 3 domain-binding site. In contrast to SHP-1, tyrosine phosphorylation of SHP-1L is not detected upon stimulation in Jurkat T cells. This is apparently due to the lack of a single in vivo tyrosine phosphorylation site, which only exists in the C terminus of SHP-1 (Y564). COS cell-expressed glutathione S-transferase-SHP-1L can dephosphorylate tyrosine-phosphorylated ZAP70. At pH 7.4, SHP-1L was shown to be more active than SHP-1 in the dephosphorylation of ZAP70. At pH 5.4, SHP-1L and SHP-1 exhibited similar catalytic activity. It is likely that these two isoforms play different roles in the regulation of hematopoietic cell signal transduction.