Tyrosine-specific Phosphatase Research Articles

Downregulation of INPP4B is Associated with Poor Prognosis in Epithelial Ovarian Carcinoma.

INPP4B is a tyrosine-specific phosphatase in the human body, which plays an important role in the developing process of carcinogenesis. However, The correlation between INPP4B and epithelial ovarian cancer is rarely explored. In this study, the expression of INPP4B in human epithelial ovarian carcinoma and normal ovaries was detected, to explore the correlation between INPP4B expression and clinicopathological risk factors of epithelial ovarian carcinoma and to clarify its significance in the developing process of and prognosis of epithelial ovarian carcinoma. The expression of INPP4B in various tumors was detected by bioinformatics method, and the expression in epithelial ovarian cancer and normal control group was detected by Elisa. The immunohistochemical method was used in this experiment to analyze the expression of INPP4B in specimens of 100 cases of epithelial ovarian carcinoma and 20 cases of normal ovaries. Analysis of clinicopathological risk factors and related survival analysis was carried out on the expression of INPP4B in 100 cases of epithelial ovarian carcinoma. The results showed that the positive expressed INPP4B protein in epithelial ovarian carcinoma was significantly less, compared with that in normal ovaries (P < 0.05). The expression of INPP4B was significantly associated with many clinicopathologic factors, such as tumor differentiation (P < 0.001), FIGO stage (P < 0.001), lymph node metastasis (P < 0.001) and distant metastasis at recurrence (P=0. 009), but not with age, pathologic type of tumor, serum CA125 at recurrence and chemotherapy sensitivity. In epithelial ovarian carcinoma, there is a downregulation of INPP4B expression, which may be related to poor tumor differentiation, late FIGO stage, lymph node metastasis, distant metastasis at recurrence and insensitivity to chemotherapy. Under-expression of INPP4B, lymph node metastasis, FIGO stage, and distant metastasis at recurrence are factors of poor prognostic. The under-expression level of INPP4B may be involved in the progression of epithelial ovarian carcinoma.

Oxidation of the Mycobacterium tuberculosis key virulence factor protein tyrosine phosphatase A (MptpA) reduces its phosphatase activity.

The Mycobacterium tuberculosis tyrosine-specific phosphatase MptpA and its cognate kinase PtkA are prospective targets for anti-tuberculosis drugs as they interact with the host defense response within the macrophages. Although both are structurally well-characterized, the functional mechanism regulating their activity remains poorly understood. Here, we investigate the effect of post-translational oxidation in regulating the function of MptpA. Treatment of MptpA with H2 O2 /NaHCO3 , mimicking cellular oxidative stress conditions, leads to oxidation of the catalytic cysteine (C11) and to a conformational rearrangement of the phosphorylation loop (D-loop) by repositioning the conserved tyrosine 128 (Y128) and generating a temporarily inactive preclosed state of the phosphatase. Thus, the catalytic cysteine in the P-loop acts as a redox switch and regulates the phosphatase activity of MptpA.

128-OR: DOC2b Boosts Insulin Secretion via a Yes Kinase-Dependent Mechanism

Exocytosis regulatory protein DOC2b is a crucial regulator of SNARE assembly in β-cells and enrichment of DOC2b enhances glucose-stimulated insulin secretion (GSIS). However, the underlying mechanism of how DOC2b boosts GSIS remains to be explored. We hypothesize that tyrosine-phosphorylation of DOC2b’s functionally indispensable C2AB domain is required for DOC2b’s beneficial function on GSIS. Biochemical analysis revealed that DOC2b is tyrosine-phosphorylated specifically by glucose within 1-2 min of stimulation in MIN6 β-cells and human islets. Pervanadate (pV) treatment, which inhibits tyrosine-specific phosphatases, validated the phosphorylation event to be tyrosine-specific. Further, the Src family kinases (SFKs) were implicated in this phosphorylation event, based upon the ability of SFK inhibitor SU-6656 to markedly attenuate the level of tyrosine-phosphorylated DOC2b under pV treated conditions. Phosphorylated/activated SFKY416 co-immunoprecipitated with DOC2b from pV treated β-cells; SU-6656 abrogated the interaction. YES kinase, an SFK, was also co-immunoprecipitated with DOC2b following glucose stimulation or pV treatment, suggesting YES kinase to be the specific SFK that phosphorylates DOC2b in β-cells. All-atom molecular dynamics simulations identified a specific tyrosine residue present in DOC2b’s C2B domain, Y301, as solvent-exposed and available for modification. Our results using phosphodeficient and phosphomimetic point mutants validated the involvement of the Y301 phosphorylation in for the boosting effect of DOC2b on GSIS. Taken together, these results resolve the first glucose-induced post-translational modification of DOC2b in β-cells, pinpointing the kinase and the site of action. These findings support a new model for DOC2b in GSIS: 1) glucose stimulation of the β-cell rapidly phosphorylates DOC2b on residue Y301 via YES kinase at the plasma membrane, yielding 2) an activated form of DOC2b that regulates insulin release. Disclosure D. Chatterjee bhowmick: None. A. Aslamy: None. S. Bhattacharya: None. E. Oh: None. M. Ahn: None. D. C. Thurmond: Consultant; Self; Immutics. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK067912, DK112917, DK102233 to D.C.T.); JDRF (3-PDF-2020-934-A-N to D.C.B.)

The Role of Protein Tyrosine Phosphatases in Inflammasome Activation.

Inflammasomes are multi-protein complexes that mediate the activation and secretion of the inflammatory cytokines IL-1β and IL-18. More than half a decade ago, it has been shown that the inflammasome adaptor molecule, ASC requires tyrosine phosphorylation to allow effective inflammasome assembly and sustained IL-1β/IL-18 release. This finding provided evidence that the tyrosine phosphorylation status of inflammasome components affects inflammasome assembly and that inflammasomes are subjected to regulation via kinases and phosphatases. In the subsequent years, it was reported that activation of the inflammasome receptor molecule, NLRP3, is modulated via tyrosine phosphorylation as well, and that NLRP3 de-phosphorylation at specific tyrosine residues was required for inflammasome assembly and sustained IL-1β/IL-18 release. These findings demonstrated the importance of tyrosine phosphorylation as a key modulator of inflammasome activity. Following these initial reports, additional work elucidated that the activity of several inflammasome components is dictated via their phosphorylation status. Particularly, the action of specific tyrosine kinases and phosphatases are of critical importance for the regulation of inflammasome assembly and activity. By summarizing the currently available literature on the interaction of tyrosine phosphatases with inflammasome components we here provide an overview how tyrosine phosphatases affect the activation status of inflammasomes.

OsPFA-DSP2 Encodes a Functional Tyrosine-Specific Phosphatase, and Negatively Regulates the Responses to Xanthomonas Oryzae pv. Oryzae in Overexpressing Rice Plants

Atypical dual-specific phosphatases (PFA-DSP) is a kind of special phosphatase in plants, and play key roles in physiological processes. OsPFA-DSP2 is a member of PFA-DSP family, and negatively regulates Magnaporthe grisea responses in when overexpressed in rice. Whereas, their activity and function response to bacterial pathogen stress are still unknown. The results showed that OsPFA-DSP2 was a tyrosine-specific phosphatase, and its phosphatase activity was regulated by redox condition. The overexpressing OX13/21 lines were sensitive to Xanthomonas oryzae pv. Oryzae (Xoo) and enhanced the expression levels of OsCEBiP in leaves, but showed little difference on the H2O2 production compared with control. Interestingly, OsPFA-DSP2 physically interacted with OsMAPK9 in vitro. These findings indicated that OsPFA-DSP2 was a tyrosine-specific phosphatase that negatively regulates Xoo response in overexpressing rice lines, which may execute through the mitogen-activated protein kinase (MAPK) signaling pathway.

Verruculosins A-B, New Oligophenalenone Dimers from the Soft Coral-Derived Fungus Talaromyces verruculosus.

In an effort to discover new bioactive anti-tumor lead compounds, a specific tyrosine phosphatase CDC25B and an Erb family receptor EGFR were selected as drug screening targets. This work led to the investigation of the soft coral-derived fungus Talaromyces verruculosus and identification of two new oligophenalenone dimers, verruculosins A–B (1–2), along with three known analogues, bacillisporin F (3), duclauxin (4), and xenoclauxin (5). Compound 1 was the first structure of the oligophenalenone dimer possessing a unique octacyclic skeleton. The detailed structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic data, X-ray crystallography, optical rotation, Electronic Circular Dichroism (ECD) analysis, and nuclear magnetic resonance (NMR) calculations. Among which, compounds 1, 3, and 5 exhibited modest inhibitory activity against CDC25B with IC50 values of 0.38 ± 0.03, 0.40 ± 0.02, and 0.26 ± 0.06 µM, respectively.

Src signaling in a low-complexity unicellular kinome

Creolimax fragrantissima is a member of the ichthyosporean clade, the earliest branching holozoan lineage. The kinome of Creolimax is markedly reduced as compared to those of metazoans. In particular, Creolimax possesses a single non-receptor tyrosine kinase: CfrSrc, the homolog of c-Src kinase. CfrSrc is an active tyrosine kinase, and it is expressed throughout the lifecycle of Creolimax. In animal cells, the regulatory mechanism for Src involves tyrosine phosphorylation at a C-terminal site by Csk kinase. The lack of Csk in Creolimax suggests that a different mode of negative regulation must exist for CfrSrc. We demonstrate that CfrPTP-3, one of the 7 tyrosine-specific phosphatases (PTPs) in Creolimax, suppresses CfrSrc activity in vitro and in vivo. Transcript levels of CfrPTP-3 and two other PTPs are significantly higher than that of CfrSrc in the motile amoeboid and sessile multinucleate stages of the Creolimax life cycle. Thus, in the context of a highly reduced kinome, a pre-existing PTP may have been co-opted for the role of Src regulation. Creolimax represents a unique model system to study the adaptation of tyrosine kinase signaling and regulatory mechanisms.

In vitro characterization and identification of potential substrates of a low molecular weight protein tyrosine phosphatase in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen responsible for significant mortality and morbidity worldwide. Within the annotated genome of the pneumococcus lies a previously uncharacterized protein tyrosine phosphatase which shows homology to low molecular weight protein tyrosine phosphatases (LMWPTPs). LMWPTPs modulate many processes critical for the pathogenicity of a number of bacteria including capsular polysaccharide biosynthesis, stress response and persistence in host macrophages. Here, we demonstrate that Spd1837 is indeed a LMWPTP, by purifying the protein, and characterizing its phosphatase activity. Spd1837 showed specific tyrosine phosphatase activity, and it did not form higher order oligomers in contrast to many other LMWPTPs. Substrate-trapping assays using the wild-type and the phosphatase-deficient Spd1837 identified potential substrates/interacting proteins including major metabolic enzymes such as ATP-dependent-6-phosphofructokinase and Hpr kinase/phosphorylase. Given the tight association between the bacterial basic physiology and virulence, this study hopes to prompt further investigation of how the pneumococcus controls its metabolic flux via the LMWPTP Spd1837.

Abstract P6-12-14: Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells

Abstract Background:Aberrant signaling through HER2 and other members of the HER family has been identified as mediator of endocrine resistance in estrogen receptor alpha (ERα) positive breast cancer. On the other hand, ERα co-expression has been shown to attenuate the efficiency of anti-HER2 targeted therapies. These findings indicate that HER2 and ERα synergize to allow breast cancer cells to escape from both anti-ERα and anti-HER2-targeted therapies. Rationally designed clinical trials that combine endocrine therapy with anti-HER2 agents to interfere with HER2/ERα cross-talk have been conducted. However, the outcome of these trials suggests that novel therapeutic approaches are needed to further improve inhibition of HER2 and other HER family members in conjunction with a more efficient ERα blockade. We examined the ability of proteasome inhibitors (PIs) to disrupt HER2/ERα cross-talk in HER2+/ER+ breast cancer (BC) cells. Furthermore we investigated the potential of PIs to suppress the activity of a constitutively active HER2 variant resistant to trastuzumab and lapatinib. Methode: HER2+/ER+ BC cells and fulvestrant resistant ER+ BC cells that overexpress a constitutively active HER2 variant resistant to trastuzumab and lapatinib have been treated with the PIs carfilzomib and bortezomib. The potential of these PIs to suppress ERα expression, to block HER2 activation and to inhibit the HER2 downstream pathways PI3K/Akt and Ras/MAPK was monitored by western blotting. Induction of cell death upon PI treatment was measured by quantification of SubG1 cells using propidium iodide staining or the use of colony formation assays. Results: Carfilzomib and bortezomib markedly inhibit bi-directional HER2/ERα signaling pathways in HER2/ER+ BC cells. Both PIs suppress ERα expression, inhibit HER2 activity and subsequently suppress the HER2 downstream pathways PI3K/Akt and Ras/MAPK that are major executors for endocrine resistance. Furthermore we observed that both PIs stabilize the HER2 specific tyrosine phosphatase BDP1 (PTPN18), thereby suppressing the activity of even a constitutive active HER2 variant that cause resistance to trastuzumab and lapatinib. Based on these findings we hypothesize that PIs inhibits ERα and HER2 activity through different mechanisms as currently used therapeutic regiments. Conclusion: These findings demonstrate that PIs disrupt the cross-talk between HER2 and ERα signaling pathways and therefore might have the potential to expand treatment opportunities for HER2+/ER+ and possibly also for other groups of BC patients. Citation Format: Thaler S, Schmidt M, Thiede G, Schad A, Sleeman JP. Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-14.

Mycobacterium tuberculosis Low Molecular Weight Phosphatases (MPtpA and MPtpB): From Biological Insight to Inhibitors.

Mycobacterium tuberculosis (Mtb), the main aetiological agent of tuberculosis (TB) in humans, is estimated to cause nearly two million deaths every year. Despite their huge therapeutic value, existing antitubercular drugs have several shortcomings, such as for instance the insurgence of drug resistance, which is mostly triggered by lack of compliance during the lengthy treatment. Novel and more effective drugs against Mtb acting on new molecular targets are therefore in demand in order to reduce treatment time and address the severe issue related to the progressive loss of antibiotic efficacy. Mtb encodes for two low molecular weight tyrosine specific phosphatases (MPtpA and MPtpB) that are crucially involved in Mtb pathogenesis. While MPtpA interferes with phagosome acidification blocking its maturation, MPtpB disrupts host signal transduction cascades, causing immune response subversion in the host. The important role played by both MPtpA and MPtpB in host-pathogen interaction makes them appealing targets for TB drug discovery. Here, we provide an exhaustive review of the current knowledge on MPtpA and MPtpB characterization and role in TB pathogenesis. In particular, special emphasis is placed on all class of inhibitors that have been developed and studied to date; their binding mode, design strategies, biological activities, main pharmacophore features as well as the efforts to overcome the poor druggability of their target are summarized in detail.

MiR-155 releases the brakes on antitumor T cells

Homeostatic γC cytokines are essential to support the expansion and function of tumor-specific T cells, but their effects are constrained by suppressor of cytokine signaling (SOCS) proteins as well as phosphoinositide and tyrosine-specific phosphatases. The microRNA miR-155 counteracts these inhibitory hurdles to potentiate intracellular cytokine signaling and T cell antitumor immunity.

Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1.

Biofilms are important for cell communication and growth in most bacteria, and are responsible for a number of human clinical infections and diseases. TpbA (PA3885) is a dual specific tyrosine phosphatase (DUSP) that negatively regulates biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa PAO1 by converting extracellular quorum sensing signals into internal gene cascade reactions that result in reduced biofilm formation. We have determined the three-dimensional crystal structure of wild-type TpbA from P. aeruginosa PAO1 in the phosphate-bound state and a TpbA (C132S) mutant with phosphotyrosine. Comparison between the phosphate-bound structure and the previously reported ligand-free TpbA structure reveals the extent of conformational changes that occur upon substrate binding. The largest changes occur in the functional loops that define the substrate binding site, including the PTP, general acid and α4-α5 loops. We further show that TpbA efficiently catalyzes the hydrolysis of two phosphotyrosine peptides derived from the periplasmic domain of TpbB (YfiN, PA1120), with a strong preference for dephosphorylating Tyr48 over Tyr62. This work adds to the small repertoire of DUSP structures in both the ligand-free and ligand-bound states, and provides a starting point for further study of the role of TpbA in biofilm formation.

Regulation of hERG and hEAG Channels by Src and by SHP-1 Tyrosine Phosphatase via an ITIM Region in the Cyclic Nucleotide Binding Domain

Members of the EAG K+ channel superfamily (EAG/Kv10.x, ERG/Kv11.x, ELK/Kv12.x subfamilies) are expressed in many cells and tissues. In particular, two prototypes, EAG1/Kv10.1/KCNH1 and ERG1/Kv11.1/KCNH2 contribute to both normal and pathological functions. Proliferation of numerous cancer cells depends on hEAG1, and in some cases, hERG. hERG is best known for contributing to the cardiac action potential, and for numerous channel mutations that underlie ‘long-QT syndrome’. Many cells, particularly cancer cells, express Src-family tyrosine kinases and SHP tyrosine phosphatases; and an imbalance in tyrosine phosphorylation can lead to malignancies, autoimmune diseases, and inflammatory disorders. Ion channel contributions to cell functions are governed, to a large degree, by post-translational modulation, especially phosphorylation. However, almost nothing is known about roles of specific tyrosine kinases and phosphatases in regulating K+ channels in the EAG superfamily. First, we show that tyrosine kinase inhibitor, PP1, and the selective Src inhibitory peptide, Src40-58, reduce the hERG current amplitude, without altering its voltage dependence or kinetics. PP1 similarly reduces the hEAG1 current. Surprisingly, an ‘immuno-receptor tyrosine inhibitory motif’ (ITIM) is present within the cyclic nucleotide binding domain of all EAG-superfamily members, and is conserved in the human, rat and mouse sequences. When tyrosine phosphorylated, this ITIM directly bound to and activated SHP-1 tyrosine phosphatase (PTP-1C/PTPN6/HCP); the first report that a portion of an ion channel is a binding site and activator of a tyrosine phosphatase. Both hERG and hEAG1 currents were decreased by applying active recombinant SHP-1, and increased by the inhibitory substrate-trapping SHP-1 mutant. Thus, hERG and hEAG1 currents are regulated by activated SHP-1, in a manner opposite to their regulation by Src. Given the widespread distribution of these channels, Src and SHP-1, this work has broad implications in cell signaling that controls survival, proliferation, differentiation, and other ERG1 and EAG1 functions in many cell types.

Effect of sorafenib on cathepsin B-dependent BID-mediated apoptosis in cancer cells.

e15515 Background: Several tyrosine kinase inhibitors (TKIs), have been developed and approved for clinical use in multi-targeted cancer therapy. Among these, sorafenib is an orally available multikinase inhibitor approved for the treatment of the advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Aim of our study was to evaluate the mechanisms responsible for the cytotoxic effects induced by in vitro use of µM doses of sorafenib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with the anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Apoptosis, (ΔΨm) dissipation and ROS generation were determined by Annexin-V/PI, JC-1 and DCFDA staining, respectively and cytofluorimetric analysis. The cathepsin B activation was evaluated by western blot using an anti-cathepsin B antibody; the cathepsin B proteolytic activity was determined using the fluorogenic Z-Arg-Arg-AMC peptide and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin was detected by a SpectraMax Gemini XPS microplate reader. Results: We found that sorafenib markedly reduced at µM dose the viability of BC cells. Treatment for 24h with 20µM of sorafenib, triggered “Incomplete autophagy”, that induced apoptosis of BC cells. Sorafenib by inducing an increased cathepsin B activity and pro-apoptotic protein BID activation, triggered a ROS-mediated-mitochondrial-dependent apoptosis of BC cells. Moreover, the increase of cathepsin B activity induced by sorafenib was inhibited by a specific tyrosine phosphatase inhibitor (e.g., orthovanadate) strongly suggesting for a contribute of tyrosine-phosphatases in sorafenib-induced apoptosis. Conclusions: Sorafenib by triggering incomplete autophagy, stimulates a cathepsin B-induced-BID-mediated-ROS- and mitochondrial-dependent apoptosis of BC cells, which is likely regulated by tyrosine-phosphatases.

Tyrosine phosphatase PTP1B functions to limit cytokine responses and leukocyte trafficking during allergic inflammation (P6057)

Abstract Allergic inflammation is orchestrated by an intricate cellular communication network operated by cytokines. Members of protein tyrosine phosphatase family are potentially critical in balancing cytokine-triggered cellular activation. However, virtually nothing is known about function of specific tyrosine phosphatases in immune-mediated diseases. We interrogated role of protein tyrosine phosphatase PTP1B during allergic inflammation using ovalbumin asthma models in PTP1B KO and WT mice. Flow cytometry and intravital microscopy were used to characterize trafficking of leukocytes to the lung. In this study, we observed exaggerated allergen-driven eosinophilopoiesis and early-induced alterations in leukocyte recruitment to the lungs of KO mice. Intravital microscopy revealed profound differences in kinetics of leukocyte binding to endothelium between challenged WT (in hours) and KO mice (in minutes). In concert with stimulated eosinophilopoiesis and enhanced leukocyte trafficking, there was an increase in eotaxins and a number of Th2 and tissue-derived cytokines in KO mice compared to WT mice. Eosinophils and mast cells cultured from bone marrow of PTP1B KO mice exhibited cytokine-induced hyperproliferation, hyperactivation, as well as an increased chemotaxis and upregulation of markers for migration. In summary, phosphatase PTP1B is a critical endogenous negative regulator of inflammation and a possible target for cytokine-oriented therapies in asthma.

NEURONAL FUNCTIONS OF PROTEIN PHOSPHATASE 2A

Reversible protein phosphorylation is a common and important form of protein posttranslational modification which determines the proteins’ activities, substrate specificity and inhibitions or degradations, depending on the specific amino acid residue, or combination of residues, being targeted. While protein phosphorylations by kinases have been well studied, the importance of protein dephosphorylation by phosphatases has emerged over the past two decades in numerous cell functions, including cell cycle, development, regulation of signal transduction pathways, mitochondrial fussion and fission and disease states of cells. So far, there are five classes of phosphatases: tyrosine-specific phosphatases, serine/threonine specific phosphatases, dual specificity phosphatases, histidine phosphatase and lipid phosphatase. The subject of this review, Protein Phosphatase 2 (PP2, aka PP2A), is a member of the serine/threonine specific phosphatases which dephosphorylate a wide range of proteins including many of the phosphatases and kinases. In eukaryotic proteins, 86 and 12% are phosphorylated on Ser and Thr, respectively and the reversible phosphorylation of these proteins is key to their dynamic functions. The research done on PP2A has exploded in recent years and there are many excellent detail reviews each with a specific PP2A topic. This article presents a brief overview of PP2A functions in four aspects: Their substrate specificity, roles in Tau hyperphosphorylation, roles in mitochondria fusion and fission and nine examples of their roles in neuronal functions.

Imatinib mesylate and nilotinib affect MHC-class I presentation by modulating the proteasomal processing of antigenic peptides.

Imatinib (IM) has been described to modulate the function of dendritic cells and T lymphocytes and to affect the expression of antigen in CML cells. In our study, we investigated the effect of the tyrosine kinase inhibitors IM and nilotinib (NI) on antigen presentation and processing by analyzing the proteasomal activity in CML cell lines and patient samples. We used a biotinylated active site-directed probe, which covalently binds to the proteasomally active beta-subunits in an activity-dependent fashion. Additionally, we analyzed the cleavage and processing of HLA-A3/11- and HLA-B8-binding peptides derived from BCR-ABL by IM- or NI-treated isolated 20S immunoproteasomes using mass spectrometry. We found that IM treatment leads to a reduction in MHC-class I expression which is in line with the inhibition of proteasomal activity. This process is independent of BCR-ABL or apoptosis induction. In vitro digestion experiments using purified proteasomes showed that generation of epitope-precursor peptides was significantly altered in the presence of NI and IM. Treatment of the immunoproteasome with these compounds resulted in an almost complete reduction in the generation of long precursor peptides for the HLA-A3/A11 and -B8 epitopes while processing of the short peptide sequences increased. Treatment of isolated 20S proteasomes with serine-/threonine- and tyrosine-specific phosphatases induced a significant downregulation of the proteasomal activity further indicating that phosphorylation of the proteasome regulates its function and antigen processing. Our results demonstrate that IM and NI can affect the immunogenicity of malignant cells by modulating proteasomal degradation and the repertoire of processed T cell epitopes.

Protein tyrosine phosphatase 1B restrains mammary alveologenesis and secretory differentiation

Tyrosine phosphorylation plays a fundamental role in mammary gland development. However, the role of specific tyrosine phosphatases in controlling mammary cell fate remains ill defined. We have identified protein tyrosine phosphatase 1B (PTP1B) as an essential regulator of alveologenesis and lactogenesis. PTP1B depletion increased the number of luminal mammary progenitors in nulliparous mice, leading to enhanced alveoli formation upon pregnancy. Mechanistically, Ptp1b deletion enhanced the expression of progesterone receptor and phosphorylation of Stat5, two key regulators of alveologenesis. Furthermore, glands from Ptp1b knockout mice exhibited increased expression of milk proteins during pregnancy due to enhanced Stat5 activation. These findings reveal that PTP1B constrains the number of mammary progenitors and thus prevents inappropriate onset of alveologenesis in early pregnancy. Moreover, PTP1B restrains the expression of milk proteins during pregnancy and thus prevents premature lactogenesis. Our work has implications for breast tumorigenesis because Ptp1b deletion has been shown to prevent or delay the onset of mammary tumors.

Estrogens and PTP1B Function in a Novel Pathway to Regulate Aromatase Enzymatic Activity in Breast Cancer Cells

Local estrogen production by aromatase is an important mechanism of autocrine stimulation in hormone-dependent breast cancer. We have previously shown that 17-β estradiol (E(2)) rapidly enhances aromatase enzymatic activity through an increase of tyrosine protein phosphorylation controlled by the activity of the c-Src kinase in breast cancer cells. Here, we investigated the protein tyrosine phosphatase PTP1B (protein tyrosine phosphatase 1B) as a potential regulator of aromatase activity. We demonstrated a specific association between PTP1B and aromatase at protein-protein level and a reduction of aromatase activity in basal and E(2)-treated MCF-7 and ZR75 breast cancer cells when PTP1B was overexpressed. Indeed, a specific tyrosine phosphatase inhibitor increased basal and E(2)-induced enzymatic activity as well as tyrosine phosphorylation status of the purified aromatase protein. Moreover, E(2) through phosphatidylinositol 3 kinase/Akt activation caused a significant decrease of PTP1B catalytic activity along with an increase in its serine phosphorylation. Concomitantly, the phosphatidylinositol 3 kinase inhibitor LY294002 or a dominant negative of Akt was able to reduce the E(2) stimulatory effects on activity and tyrosine phosphorylation levels of aromatase. Taken together, our results suggest that E(2) can impair PTP1B ability to dephosphorylate aromatase, and thus it increases its enzymatic activity, creating a positive feedback mechanism for estradiol signaling in breast cancer.

Biochemical and Functional Studies of Lymphoid-Specific Tyrosine Phosphatase (Lyp) Variants S201F and R266W

The Lymphoid specific tyrosine phosphatase (Lyp) has elicited tremendous research interest due to the high risk of its missense mutation R620W in a wide spectrum of autoimmune diseases. While initially characterized as a gain-of-function mutant, R620W was thought to lead to autoimmune diseases through loss-of-function in T cell signaling by a recent study. Here we investigate the biochemical characters and T cell signaling functions of two uncharacterized Lyp variants S201F and R266W, together with a previously characterized Lyp variant R263Q, which had reduced risk in several autoimmune diseases, including systemic lupus erythematosus (SLE), ulcerative colitis (UC) and rheumatoid arthritis (RA). Our kinetic and functional studies of R263Q polymorphism basically reproduced previous findings that it was a loss-of-function mutant. The other variant S201F reduced Lyp phosphatase activity moderately and decreased Lyp function in T cell slightly, while R266W severely impaired phosphatase activity and was a loss-of-function variant in T cell signaling. A combined kinetic and structure analysis suggests that the R266W variant may decrease its phosphatase activity through perturbing either the Q-loop or the WPD loop of Lyp. As both R266W and R263Q significantly change their phosphatase activity and T cell functions, future work could be considered to evaluate these mutants in a broader spectrum of autoimmune diseases.