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1.
Scand J Rheumatol ; 48(3): 173-177, 2019 May.
Article in English | MEDLINE | ID: mdl-30474480

ABSTRACT

OBJECTIVE: FURIN is a proprotein convertase enzyme that inhibits the proinflammatory function of T cells and myeloid cells. Elevated FURIN expression levels have been reported in immune-mediated diseases, such as primary Sjögren's syndrome. Here, we investigated the levels of FURIN in peripheral blood (PB) and synovial fluid (SF) leucocytes from patients with rheumatoid arthritis (RA). METHOD: FURIN mRNA expression in PB and SF cells was determined by quantitative reverse transcription-polymerase chain reaction and FURIN plasma levels were measured using enzyme-linked immunosorbent assay. Associations between FURIN levels and demographic and clinical characteristics of the patients were determined. RESULTS: FURIN levels were significantly elevated in PB and SF mononuclear cells, T cells, and monocytes from RA patients compared to healthy controls. High FURIN levels were significantly associated with the prevailing prednisolone treatment, higher prednisolone doses, and increased C-reactive protein levels and Health Assessment Questionnaire values. CONCLUSION: FURIN is significantly upregulated in RA PB and SF leucocytes, suggesting that it may have a role in the pathogenesis of RA. In addition, our results suggest that elevated FURIN expression is associated with the indicators of more severe RA.


Subject(s)
Arthritis, Rheumatoid , Furin , Leukocytes, Mononuclear , Monocytes , Prednisolone/therapeutic use , Synovial Membrane , Anti-Inflammatory Agents/therapeutic use , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/diagnosis , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/epidemiology , Correlation of Data , Female , Finland/epidemiology , Furin/blood , Furin/metabolism , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Monocytes/drug effects , Monocytes/metabolism , RNA, Messenger , Synovial Fluid/immunology , Synovial Membrane/immunology , Synovial Membrane/pathology , Up-Regulation/drug effects , Up-Regulation/immunology
2.
Clin Exp Immunol ; 181(1): 29-38, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25736842

ABSTRACT

The expression and phosphorylation of signal transducer and activator of transcription-1 (STAT-1) have been shown to be markedly increased in the salivary gland epithelial cells of patients with primary Sjögren's syndrome (pSS). The present aim was to investigate the activation status of different STAT proteins in peripheral blood (PB) lymphocytes and monocytes, and their correlations with clinical parameters in patients with pSS. To this end, PB samples were drawn from 16 patients with active pSS and 16 healthy blood donors, and the phosphorylation of STAT-1, -3, -4, -5 and -6 proteins was studied in T cells, B cells and monocytes using multi-colour flow cytometry. In addition, mRNA expression of STAT molecules in PB mononuclear cells (PBMC) was studied with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Basal phosphorylation of STAT-5 was found to be significantly higher in PB T cells, B cells and monocytes in patients with pSS than in healthy controls. The expression of STAT-5 mRNA was not increased in PBMC. pSTAT-5 levels in B cells and monocytes showed a significant correlation with serum immunoglobulin (Ig)G levels and anti-SSB antibody titres. Constitutive STAT-5 activation in monocytes and CD4(+) T cells was associated with purpura. There were no major differences in the activation of other STATs between pSS patients and healthy controls. In conclusion, STAT-5 is activated constitutively in PB leucocytes in patients with pSS, and basal STAT-5 phosphorylation seems to associate with hypergammaglobulinaemia, anti-SSB antibody production and purpura.


Subject(s)
Autoantigens/immunology , CD4-Positive T-Lymphocytes/immunology , Immunoglobulin G/blood , Ribonucleoproteins/immunology , STAT5 Transcription Factor/metabolism , Sjogren's Syndrome/immunology , Adult , Aged , Aged, 80 and over , B-Lymphocytes/immunology , Enzyme Activation , Female , Humans , Leukocytes, Mononuclear/immunology , Male , Middle Aged , Phosphorylation , RNA, Messenger/biosynthesis , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/metabolism , STAT5 Transcription Factor/genetics , STAT6 Transcription Factor/genetics , STAT6 Transcription Factor/metabolism , SS-B Antigen
3.
Oncogene ; 33(29): 3794-802, 2014 Jul 17.
Article in English | MEDLINE | ID: mdl-23995791

ABSTRACT

Splicing abnormalities have profound impact in human cancer. Several splicing factors, including SAM68, have pro-oncogenic functions, and their increased expression often correlates with human cancer development and progression. Herein, we have identified using mass spectrometry proteins that interact with endogenous SAM68 in prostate cancer (PCa) cells. Among other interesting proteins, we have characterized the interaction of SAM68 with SND1, a transcriptional co-activator that binds spliceosome components, thus coupling transcription and splicing. We found that both SAM68 and SND1 are upregulated in PCa cells with respect to benign prostate cells. Upregulation of SND1 exerts a synergic effect with SAM68 on exon v5 inclusion in the CD44 mRNA. The effect of SND1 on CD44 splicing required SAM68, as it was compromised after knockdown of this protein or mutation of the SAM68-binding sites in the CD44 pre-mRNA. More generally, we found that SND1 promotes the inclusion of CD44 variable exons by recruiting SAM68 and spliceosomal components on CD44 pre-mRNA. Inclusion of the variable exons in CD44 correlates with increased proliferation, motility and invasiveness of cancer cells. Strikingly, we found that knockdown of SND1, or SAM68, reduced proliferation and migration of PCa cells. Thus, our findings strongly suggest that SND1 is a novel regulator of alternative splicing that promotes PCa cell growth and survival.


Subject(s)
Alternative Splicing , Gene Expression Regulation, Neoplastic , Nuclear Proteins/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Transcriptional Activation , Adaptor Proteins, Signal Transducing/metabolism , Cell Line, Tumor , Cell Movement/genetics , DNA-Binding Proteins/metabolism , Endonucleases , Exons , Gene Knockdown Techniques , Humans , Hyaluronan Receptors/genetics , Male , Protein Binding , RNA Polymerase II/metabolism , RNA-Binding Proteins/metabolism
4.
Scand J Rheumatol ; 39(2): 118-26, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20001767

ABSTRACT

OBJECTIVES: Interleukin (IL)-10 functions as an anti-inflammatory cytokine in rheumatoid arthritis (RA). New IL-10 family cytokines IL-19, IL-20, IL-22, IL-24, and IL-26 have recently been discovered. Information concerning the expression and function of these cytokines in autoimmune diseases is currently limited. The aim of this study was to investigate their expression in RA. METHODS: mRNA levels of the cytokines were studied using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MCs), purified T cells, and monocytes/macrophages from RA patients and healthy volunteers, and synovial tissues from patients with RA or osteoarthritis (OA), were examined. The expression of IL-19 protein in T cells and monocytes/macrophages was studied by flow cytometry. RESULTS: IL-10 and IL-19 mRNA levels were significantly elevated in SFMCs from patients with RA compared with PBMCs from RA patients or healthy volunteers. IL-20 and IL-22 mRNA levels were also upregulated in RA SFMCs but their level of expression was lower than that of IL-10 or IL-19. Importantly, synovial tissue IL-19 levels in RA were increased when compared with OA. IL-19 expression was upregulated in both T cells and macrophages derived from patients with RA. IL-1beta increased IL-19 levels in PBMCs, suggesting that elevated levels of IL-1 in RA joints may contribute to upregulated IL-19 expression. CONCLUSIONS: The majority of the IL-10 family cytokines are expressed in RA. IL-19 demonstrated the highest expression in rheumatoid joints, and could thus be involved in the regulation of synovial inflammation in RA.


Subject(s)
Arthritis, Rheumatoid/metabolism , Bursa, Synovial/metabolism , Interleukins/metabolism , Synovial Fluid/metabolism , Arthritis, Rheumatoid/genetics , Bursa, Synovial/immunology , Cells, Cultured , Flow Cytometry , Humans , Interleukins/genetics , Interleukins/immunology , Monocytes/metabolism , Osteoarthritis/genetics , Osteoarthritis/metabolism , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Statistics, Nonparametric , Synovial Fluid/immunology , T-Lymphocytes/metabolism
5.
Rheumatology (Oxford) ; 46(10): 1538-46, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17726036

ABSTRACT

OBJECTIVES: Cytokines play a key pathogenic role in rheumatoid arthritis (RA). Several cytokines signal through the JAK-STAT pathway, which is negatively regulated by the suppressors of cytokine signalling (SOCS) proteins. Since SOCS protein levels can profoundly modulate cellular responses to cytokines, we have investigated their expression in chronic RA. METHODS: The levels of SOCS1-3 and CIS1 mRNA in peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MCs), purified T cells and monocytes from RA patients and healthy volunteers were studied using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). SOCS mRNA and protein expression in synovial tissues were examined by RT-PCR and immunohistochemistry. RESULTS: The levels of SOCS1 and SOCS3 were significantly increased in PBMCs from RA patients when compared with healthy volunteers. These differences were mainly due to up-regulation of SOCS1 in PB T cells and of SOCS3 in PB monocytes. In addition, SOCS2 was up-regulated in PB T cells. Interestingly, SF T cells expressed lower and SF macrophages higher levels of SOCS molecules than their PB counterparts. Similarly, while a significant portion of macrophages in synovial tissues expressed SOCS1 and SOCS3 proteins, the majority of T cells remained SOCS negative. Finally, SOCS1 was up-regulated in the synovial membranes from patients with RA when compared with osteoarthritis. CONCLUSIONS: SOCS expression levels are profoundly altered in RA, and the profile of SOCS expression is dependent on both the cell type as well as the cellular compartment.


Subject(s)
Arthritis, Rheumatoid/metabolism , Suppressor of Cytokine Signaling Proteins/metabolism , Adult , Aged , Aged, 80 and over , Cells, Cultured , Cytokines/pharmacology , Female , Gene Expression Regulation , Humans , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Monocytes/metabolism , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/genetics , Synovial Fluid/metabolism , Synovial Membrane/metabolism , T-Lymphocyte Subsets/metabolism , Up-Regulation
6.
Oncogene ; 26(48): 6905-14, 2007 Oct 18.
Article in English | MEDLINE | ID: mdl-17486069

ABSTRACT

Cleavable isoforms of the ErbB4 receptor tyrosine kinase release a soluble intracellular domain (ICD) that may translocate to the nucleus and regulate signaling. However, ErbB4 gene is alternatively spliced generating CYT-1 and CYT-2 isoforms with different cytoplasmic tails. Here, we addressed whether the two alternative ErbB4 ICDs of either CYT-1 (ICD1) or CYT-2 (ICD2) type differ in signaling to the nucleus. Confocal microscopy and extraction of nuclear cell fractions indicated that significantly more ICD2 translocated to the nuclei when compared to ICD1. Unlike the membrane-anchored 80 kDa fragments derived from full-length ErbB4 isoforms, the two ICDs did not differ from each other in metabolic stability or ubiquitylation. However, ICD2 was phosphorylated at tyrosine residues to a higher extent and demonstrated greater in vitro kinase activity than ICD1. Mutating the ATP-binding site within ICD2 kinase domain (ICD2 K751R) blocked its tyrosine phosphorylation and significantly reduced its nuclear translocation. When expressed in the context of full-length ErbB4, ICD2 was also more efficient than ICD1 in promoting transcriptional activation of the STAT5 target gene beta-casein. These findings indicate that the two alternative ICDs of ErbB4 differ in their nuclear accumulation, and that the mechanism involves differential kinase activity but not ubiquitin-regulated ICD stability.


Subject(s)
Alternative Splicing , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , ErbB Receptors/metabolism , Adenosine Triphosphate/metabolism , Animals , Binding Sites , COS Cells , Caseins/genetics , Caseins/metabolism , Chlorocebus aethiops , ErbB Receptors/genetics , Humans , Kidney/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Isoforms , Receptor, ErbB-4 , STAT5 Transcription Factor , Signal Transduction , Tyrosine/metabolism , Ubiquitin/metabolism
7.
Scand J Gastroenterol ; 37(6): 667-73, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12126244

ABSTRACT

BACKGROUND: Disturbances in the function of sphincter of Oddi (SO) may prevent normal bile flow and thus enhance the probability of common bile duct stone (CBDS) formation. Previously, we have shown increased prevalence of hypothyroidism in CBDS patients. METHODS: In animal (pig) experiments, thyroxine (T4) and triiodothyronine have a specific inhibitory effect on SO contractility, which raises the possibility that the lack of this prorelaxing effect in hypothyroidism could, at least in part, explain the increased prevalence of CBDS. The aims of the present study were to investigate, whether human SO reacts similarly to T4, and to study the mechanisms of the T4 prorelaxing effect. RESULTS: We found that T4 had similar inhibitory effects on both human and pig SO contractions. The T4 effect was dose-dependent, and maximum was observed in 30 min. The maximal prorelaxing effect was achieved with 0.1 nM T4 concentration, the effect of the physiological T4 concentration (0.01 nM) being about half of the maximal effect. Addition of alpha-adrenoceptor antagonist phentolamine, beta-adrenoceptor antagonist propranolol, nitric oxide (NO)-synthesis inhibitor L-NAME, nerve conductance blocker tetrodotoxin, or cyclooxygenase inhibitor diclofenac did not affect the T4-induced inhibition of contraction. Addition of transcription inhibitor actinomycin D or translation inhibitor cyclophosphamide partially reversed the T4-induced inhibition of contraction. Addition of K+ channel blocker glibenclamide totally reversed the T4-induced inhibition of contraction. In Western blotting, the thyroid hormone receptor (TR) antibody recognized 53 kDa and 58 kDa proteins, corresponding to beta1 and beta2 isoforms of TR, in the human SO tissue. CONCLUSIONS: We conclude that T4 has a direct prorelaxing effect on human SO that expresses TR beta1 and beta2. This effect is mediated through a transcriptional mechanism that requires new mRNA and protein synthesis and subsequently leads to the activation of K+ channels.


Subject(s)
Muscle Relaxation/drug effects , Sphincter of Oddi/drug effects , Thyroxine/pharmacology , Triiodothyronine/pharmacology , Acetylcholine/pharmacology , Analysis of Variance , Animals , Blotting, Western , Culture Techniques , Dose-Response Relationship, Drug , Drug Interactions , Humans , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle Relaxation/physiology , Potassium Chloride/pharmacology , Probability , Species Specificity , Sphincter of Oddi/physiology , Swine
8.
Mol Endocrinol ; 14(12): 1986-2000, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11117529

ABSTRACT

Steroid receptors mediate their actions by using various coregulatory proteins. We have recently characterized ARIP3/PIASx alpha as an androgen receptor (AR)-interacting protein (ARIP) that belongs to the PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] protein family implicated in the inhibition of cytokine signaling. We have analyzed herein the roles that four different PIAS proteins (ARIP3/PIASx alpha, Miz1/PIASx beta, GBP/PIAS1, and PIAS3) play in the regulation of steroid receptor- or STAT-mediated transcriptional activation. All PIAS proteins are able to coactivate steroid receptor-dependent transcription but to a differential degree, depending on the receptor, the promoter, and the cell type. Miz1 and PIAS1 are more potent than ARIP3 in activating AR function on minimal promoters. With the natural probasin promoter, PIAS proteins influence AR function more divergently, in that ARIP3 represses, but Miz1 and PIAS1 activate it. Miz1 and PIAS1 possess inherent transcription activating function, whereas ARIP3 and PIAS3 are devoid of this feature. ARIP3 enhances glucocorticoid receptor-dependent transcription more efficiently than Miz1 or PIAS1, and all PIAS proteins also activate estrogen receptor- and progesterone receptor-dependent transcription but to a dissimilar degree. The same amounts of PIAS proteins that modulate steroid receptor-dependent transcription influence only marginally transactivation mediated by various STAT proteins. It remains to be established whether the PIAS proteins play a more significant physiological role in steroid receptor than in cytokine signaling.


Subject(s)
Carrier Proteins/physiology , DNA-Binding Proteins/physiology , Nuclear Proteins/physiology , Proteins/physiology , Receptors, Steroid/metabolism , Transcription Factors/physiology , Transcriptional Activation , Amino Acid Sequence , Carrier Proteins/genetics , Cell Line , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Molecular Sequence Data , Nuclear Proteins/genetics , Promoter Regions, Genetic , Protein Inhibitors of Activated STAT , Proteins/genetics , Receptors, Androgen/metabolism , Receptors, Glucocorticoid/physiology , STAT1 Transcription Factor , Sequence Homology, Amino Acid , Trans-Activators/genetics , Trans-Activators/metabolism , Trans-Activators/physiology , Transcription Factors/genetics , Ubiquitin-Protein Ligases
9.
Oncogene ; 19(36): 4151-8, 2000 Aug 24.
Article in English | MEDLINE | ID: mdl-10962576

ABSTRACT

Cytoplasmic protein tyrosine kinases play crucial roles in signaling via a variety of cell surface receptors. The Bmx tyrosine kinase, a member of the Tec family, is expressed in hematopoietic cells of the granulocytic and monocytic lineages. Here we show that Bmx is catalytically activated by interleukin-3 (IL-3) and granulocyte-colony stimulating factor (G-CSF) receptors. Activation of Bmx required phosphatidylinositol 3-kinase (PI-3K) as demonstrated by the ability of PI-3K inhibitors to block the activation signal. A green fluorescent protein (GFP) tagged Bmx was translocated to cellular membranes upon co-expression of a constitutively active form of PI-3K, further indicating a role for PI-3K in signaling upstream of Bmx. The expression of wild type Bmx in 32D myeloid progenitor cells resulted in apoptosis in the presence of G-CSF, while cells expressing a kinase dead mutant of Bmx differentiated into mature granulocytes. However, Bmx did not modulate IL-3-dependent proliferation of the cells. These results demonstrate distinct effects of Bmx in cytokine induced proliferation and differentiation of myeloid cells, and suggest that the stage specific expression of Bmx is critical for the differentiation of myeloid cells. Oncogene (2000) 19, 4151 - 4158


Subject(s)
Granulocyte Colony-Stimulating Factor/metabolism , Interleukin-3/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Apoptosis , Blotting, Northern , Blotting, Western , Cell Differentiation , Cell Line , Cell Membrane/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Regulation , Green Fluorescent Proteins , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Luminescent Proteins/genetics , Mice , Phosphorylation , Protein-Tyrosine Kinases/genetics , RNA, Messenger/analysis , Receptors, Granulocyte Colony-Stimulating Factor/metabolism , Receptors, Interleukin-3/metabolism , Recombinant Fusion Proteins/metabolism , Transfection
10.
J Immunol ; 164(11): 5689-97, 2000 Jun 01.
Article in English | MEDLINE | ID: mdl-10820245

ABSTRACT

IFN-gamma and glucocorticoids regulate inflammatory and immune responses through Stat1 and glucocorticoid receptor (GR) transcription factors, respectively. The biological responses to these polypeptides are determined by integration of various signaling pathways in a cell-type and promoter-dependent manner. In this study we have characterized the molecular basis for the functional cooperation between IFN-gamma and dexamethasone (Dex) in the induction of the high-affinity Fc gamma receptor I (Fc gamma RI) in monocytes. Dex did not affect IFN-gamma-induced Stat1 DNA binding activity or induce novel DNA-binding complexes to the Fc gamma RI promoter. By using cell systems lacking functional GR or Stat1, we showed that GR stimulated Stat1-dependent transcription in a ligand-dependent manner, while Stat1 did not influence GR-dependent transcription. The cooperation required phosphorylation of Tyr701, DNA binding, and the trans-activation domain of Stat1, but did not involve Ser727 phosphorylation of Stat1 or physical interaction between GR and Stat1. The costimulatory effect of Dex was not dependent on a consensus glucocorticoid response element in the Stat1-responsive promoters, but required the DNA-binding and trans-activation functions of GR, and Dex-induced protein synthesis. GR activated the natural Fc gamma RI promoter construct, and this response required both Stat1 and the Ets family transcription factor PU.1. Previously, physical association between GR and Stat5 has been shown to enhance Stat5-dependent and suppress GR-dependent transcription. The results shown here demonstrate a distinct, indirect mechanism of cross-modulation between cytokine and steroid receptor signaling that integrates Stat1 and GR pathways with cell type-specific PU.1 transcription factor in the regulation of Fc gamma RI gene transcription.


Subject(s)
DNA-Binding Proteins/physiology , Milk Proteins , Monocytes/metabolism , Proto-Oncogene Proteins/physiology , Receptors, Glucocorticoid/physiology , Receptors, IgG/metabolism , Trans-Activators/physiology , Transcriptional Activation/immunology , DNA/metabolism , DNA-Binding Proteins/metabolism , Dexamethasone/pharmacology , Drug Synergism , Humans , Interferon-gamma/physiology , Monocytes/drug effects , Promoter Regions, Genetic/immunology , Protein Binding/drug effects , Protein Binding/immunology , Protein Structure, Tertiary , Receptors, Glucocorticoid/metabolism , Receptors, IgG/biosynthesis , Receptors, IgG/genetics , STAT1 Transcription Factor , STAT5 Transcription Factor , Signal Transduction/immunology , Trans-Activators/metabolism , Tumor Cells, Cultured
11.
Mol Cell Biol ; 20(10): 3387-95, 2000 May.
Article in English | MEDLINE | ID: mdl-10779328

ABSTRACT

Activation of Jak tyrosine kinases through hematopoietic cytokine receptors occurs as a consequence of ligand-induced aggregation of receptor-associated Jaks and their subsequent autophosphorylation. Jak kinases consist of a C-terminal tyrosine kinase domain, a pseudokinase domain of unknown function, and Jak homology (JH) domains 3 to 7, implicated in receptor-Jak interaction. We analyzed the functional roles of the different protein domains in activation of Jak2. Deletion analysis of Jak2 showed that the pseudokinase domain but not JH domains 3 to 7 negatively regulated the catalytic activity of Jak2 as well as Jak2-mediated activation of Stat5. Phosphorylation of Stat5 by wild-type Jak2 was dependent on the SH2 domain of Stat5; however, this requirement was lost upon deletion of the pseudokinase domain of Jak2. Investigation of the mechanisms of the pseudokinase domain-mediated inhibition of Jak2 suggested that this regulation did not involve protein tyrosine phosphatases. Instead, analysis of interactions between the tyrosine kinase domain and Jak2 suggested that the pseudokinase domain interacted with the kinase domain. Furthermore, coexpression of the pseudokinase domain inhibited the activity of the single tyrosine kinase domain. Finally, deletion of the pseudokinase domain of Jak2 deregulated signal transduction through the gamma interferon receptor by significantly increasing ligand-independent activation of Stat transcription factors. These results indicate that the pseudokinase domain negatively regulates the activity of Jak2, probably through an interaction with the kinase domain, and this regulation is required to keep Jak2 inactive in the absence of ligand stimulation. Furthermore, the pseudokinase domain may have a role in regulation of Jak2-substrate interactions.


Subject(s)
Milk Proteins , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , DNA-Binding Proteins , Enzyme Activation , Janus Kinase 2 , Phosphorylation , Protein-Tyrosine Kinases/genetics , Receptors, Interferon/metabolism , STAT5 Transcription Factor , Sequence Deletion , Signal Transduction , Trans-Activators , src Homology Domains , Interferon gamma Receptor
12.
Blood ; 95(5): 1626-32, 2000 Mar 01.
Article in English | MEDLINE | ID: mdl-10688817

ABSTRACT

Differentiation of macrophages from myeloid progenitor cells depends on a discrete balance between cell growth, survival, and differentiation signals. Interleukin-3 (IL-3) supports the growth and survival of myeloid progenitor cells through the activation of Jak2 tyrosine kinase, and macrophage differentiation has been shown to be regulated by protein kinase C (PKC). During terminal differentiation of macrophages, the cells lose their mitogenic response to IL-3 and undergo growth arrest, but the underlying signaling mechanisms have remained elusive. Here we show that in IL-3-dependent 32D myeloid progenitor cells, the differentiation-inducing PKC isoforms PKC-alpha and PKC-delta specifically caused rapid inhibition of IL-3-induced tyrosine phosphorylation. The target for this inhibition was Jak2, and the activation of PKC by 12-O-tetradecanoyl-phorbol-13-acetate treatment also abrogated IL-3-induced tyrosine phosphorylation of Jak2 in Ba/F3 cells. The mechanism of this regulation was investigated in 32D and COS7 cells, and the inhibition of Jak2 required catalytic activity of PKC-delta and involved the phosphorylation of Jak2 on serine and threonine residues by the associated PKC-delta. Furthermore, PKC-delta inhibited the in vitro catalytic activity of Jak2, indicating that Jak2 was a direct target for PKC-delta. In 32D cells, the inhibition of Jak2 either by PKC-delta, tyrosine kinase inhibitor AG490, or IL-3 deprivation caused a similar growth arrest. Reversal of PKC-delta-mediated inhibition by the overexpression of Jak2 promoted apoptosis in differentiating 32D cells. These results demonstrate a PKC-mediated negative regulatory mechanism of cytokine signaling and Jak2, and they suggest that it serves to integrate growth-promoting and differentiation signals during macrophage differentiation. (Blood. 2000;95:1626-1632)


Subject(s)
Interleukin-3/pharmacology , Isoenzymes/physiology , Macrophages/cytology , Protein Kinase C/physiology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Animals , Apoptosis/drug effects , Catalysis/drug effects , Cell Differentiation/drug effects , Cell Division/physiology , DNA Replication , Enzyme Activation , Enzyme Induction , Janus Kinase 2 , Macrophages/enzymology , Mice , Phosphorylation , Phosphoserine/metabolism , Phosphothreonine/metabolism , Phosphotyrosine/metabolism , Protein Kinase C-alpha , Protein Kinase C-delta , Protein Kinase C-epsilon , Protein Processing, Post-Translational , Recombinant Fusion Proteins/physiology , Tetradecanoylphorbol Acetate/pharmacology , Transfection
13.
Blood ; 95(2): 494-502, 2000 Jan 15.
Article in English | MEDLINE | ID: mdl-10627454

ABSTRACT

Stat6 transcription factor is a critical mediator of IL-4-specific gene responses. Tyrosine phosphorylation is required for nuclear localization and DNA binding of Stat6. The authors investigated whether Stat6-dependent transcriptional responses are regulated through IL-4-induced serine/threonine phosphorylation. In Ramos B cells, the serine/threonine kinase inhibitor H7 inhibited IL-4-induced expression of CD23. Treatment with H7 did not affect IL-4R-mediated immediate signaling events such as tyrosine phosphorylation of Jak1, Jak3, insulin receptor substrate (IRS)-1 and IRS-2, or tyrosine phosphorylation and DNA binding of Stat6. To analyze whether the H7-sensitive pathway was regulating Stat6-activated transcription, we used reporter constructs containing different IL-4 responsive elements. H7 abrogated Stat6-, as well as Stat5-, mediated reporter gene activation and partially reduced C/EBP-dependent reporter activity. By contrast, IL-4-induced transcription was not affected by wortmannin, an inhibitor of the phosphatidyl-inositol 3'-kinase pathway. Phospho-amino acid analysis and tryptic phosphopeptide maps revealed that IL-4 induced phosphorylation of Stat6 on serine and tyrosine residues in Ramos cells and in 32D cells lacking endogenous IRS proteins. However, H7 treatment did not inhibit the phosphorylation of Stat6. Instead, H7 inhibited the IL-4-induced phosphorylation of RNA polymerase II. These results indicate that Stat6-induced transcription is dependent on phosphorylation events mediated by H7-sensitive kinase(s) but that it also involves serine phosphorylation of Stat6 by an H7-insensitive kinase independent of the IRS pathway. (Blood. 2000;95:494-502)


Subject(s)
Gene Expression Regulation/immunology , Interleukin-4/pharmacology , Proto-Oncogene Proteins , Receptors, IgE/genetics , Trans-Activators/metabolism , Transcription, Genetic , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , B-Lymphocytes/immunology , Base Sequence , CCAAT-Enhancer-Binding Proteins , COS Cells , Cell Line , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Humans , Insulin Receptor Substrate Proteins , Intracellular Signaling Peptides and Proteins , Janus Kinase 1 , Janus Kinase 2 , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligodeoxyribonucleotides , Phosphoproteins/metabolism , Phosphorylation , Phosphotyrosine , Protein-Tyrosine Kinases/metabolism , Receptors, IgE/biosynthesis , Recombinant Proteins/biosynthesis , STAT6 Transcription Factor , Signal Transduction , Trans-Activators/genetics , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Transcriptional Activation , Transfection , Tumor Cells, Cultured , Tyrosine/metabolism
14.
Biochem J ; 345 Pt 3: 759-66, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10642538

ABSTRACT

Several growth factors activate signal transducers and activators of transcription (Stats) but the mechanism of Stat activation in receptor tyrosine kinase signalling has remained elusive. In the present study we have analysed the roles of different platelet-derived growth factor (PDGF)-induced tyrosine kinases in the activation of Stat5. Co-expression experiments in insect and mammalian cells demonstrated that both PDGF beta-receptor (PDGF beta-R) and Jak1, but not c-Src, induced the activation of Stat5. Furthermore, immune-complex-purified PDGF beta-R was able to phosphorylate Stat5 directly. The role of the cytoplasmic tyrosine kinases in the PDGF-induced activation of Stat5 was further investigated by overexpressing kinase-negative (KN) and wild-type Jak and c-Src kinases. Jak1-KN or Jak2-KN had no effect but both Src-KN and wild-type c-Src similarly decreased the PDGF-beta-R-induced activation of Stat5. The activation of both Src and Stat5 is dependent on the same tyrosine residues Tyr(579) and Tyr(581) in PDGF beta-R; thus the observed inhibition by Src might result from competition for binding of Stat5 to the receptor. Finally, fibroblasts derived from Src(-/-) and Fyn(-/-) mice showed normal pattern of PDGF-induced tyrosine phosphorylation of Stat5. Taken together, these results indicate that Stat5 is a direct substrate for PDGF beta-R and that the activation does not require Jak1, Jak2, c-Src or Fyn tyrosine kinases.


Subject(s)
DNA-Binding Proteins/metabolism , Milk Proteins , Protein-Tyrosine Kinases/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Trans-Activators/metabolism , src-Family Kinases/metabolism , Animals , Cells, Cultured , DNA-Binding Proteins/drug effects , Enzyme Activation , Insecta/cytology , Janus Kinase 1 , Janus Kinase 2 , Mammals , Mice , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fyn , STAT5 Transcription Factor , Signal Transduction , Trans-Activators/drug effects , Tyrosine/metabolism , src Homology Domains , src-Family Kinases/genetics
15.
Mol Cell Endocrinol ; 138(1-2): 1-10, 1998 Mar 16.
Article in English | MEDLINE | ID: mdl-9685210

ABSTRACT

A working model for haematopoietic cytokine signal transduction has been hypothesised as follows. Binding of cytokines to specific receptor molecules leads to phosphorylation and activation of receptor associated members of the Janus kinase family. This is followed by tyrosine phosphorylation of the associated receptor and members of the STAT (signal transducer and activator of transcription) family of DNA-binding transcription factors. Phosphorylation is accompanied by STAT dimerisation, nuclear transport and activation of gene transcription. Activation of gene transcription is mediated by the binding of STAT dimers to palindromic STAT response elements. A number of areas of confusion remain; not least the mechanism by which multiple cytokines signal via a limited number of STATs. A role has been suggested for phosphorylated receptor tyrosine residues as STAT docking sites on activated receptor-JAK complexes. According to this model the amino acid sequence context of key tyrosine residues confers receptor specificity upon STAT activation. There is some controversy as to whether this model applies to STAT 5. The heterologous expression of STAT 5 in Sf 9 insect cells using the baculovirus expression system is described here. Protein of the correct molecular weight was expressed and found to be phosphorylated on tyrosine residues and to bind to a STAT response DNA element. This binding was dependent upon the phosphorylation status of the STAT protein. DNA binding could be abolished in vitro by treatment with a phosphotyrosine phosphatase and restored in vitro by treatment with activated recombinant JAK 2. The protein was purified to near homogeneity using a simple ion exchange/gel filtration chromatography procedure. The interaction between purified recombinant STAT 5 and JAK 2, either expressed by baculovirus or endogenously expressed in Buffalo rat liver cells, was studied. In both cases STAT 5 in its non-phosphorylated form was found to form a stable complex with activated JAK 2. Non-activated JAK 2 and phosphorylated STAT 5 were unable to participate in complex formation. The results presented provide a mechanistic basis for the activation of STAT 5 by a wide range of cytokines capable of activating JAK 2.


Subject(s)
DNA-Binding Proteins/metabolism , Milk Proteins , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Trans-Activators/metabolism , Animals , Binding Sites , Cell Line , DNA/metabolism , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/isolation & purification , Janus Kinase 2 , Liver/enzymology , Models, Biological , Phosphorylation , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/isolation & purification , Rats , Rats, Inbred BUF , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , STAT5 Transcription Factor , Signal Transduction , Spodoptera , Substrate Specificity , Trans-Activators/biosynthesis , Trans-Activators/isolation & purification , Transfection , Tyrosine
16.
Oncogene ; 16(4): 505-15, 1998 Jan 29.
Article in English | MEDLINE | ID: mdl-9484840

ABSTRACT

Signal transducers and activators of transcription (Stats) are known to transduce signals from the cell surface to the nucleus in cytokine receptor signaling. We examined the capacity of platelet-derived growth factor (PDGF) receptor to interact with and activate Stat molecules. Activation of the PDGF beta-receptor led to tyrosine phosphorylation of Stat1, Stat3 and Stat5, which was accompanied by specific DNA-binding activities. These events were only weakly stimulated by the activated PDGF alpha-receptor. In cells expressing PDGF beta-receptors mutated at Tyr579, Tyr581 or Tyr775, tyrosine phosphorylation as well as DNA-binding activity of Stat5 was reduced. Immobilized peptides containing phosphorylated Tyr579, Tyr581 or Tyr775 bound Stat5, suggesting direct binding of Stat5 to these tyrosine residues of the PDGF beta-receptor. Members of the Janus kinase family were also shown to interact with the PDGF beta-receptor, and to a lesser extent with the alpha-receptor, but their importance for PDGF-induced Stat activation remains to be determined.


Subject(s)
DNA-Binding Proteins/metabolism , Milk Proteins , Platelet-Derived Growth Factor/physiology , Protein-Tyrosine Kinases/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction , Trans-Activators/metabolism , 3T3 Cells , Animals , COS Cells , DNA/metabolism , Janus Kinase 3 , Mice , Phosphorylation , Receptor, Platelet-Derived Growth Factor alpha , Receptor, Platelet-Derived Growth Factor beta , STAT1 Transcription Factor , STAT2 Transcription Factor , STAT3 Transcription Factor , STAT4 Transcription Factor , STAT5 Transcription Factor
17.
J Biol Chem ; 273(5): 3021-6, 1998 Jan 30.
Article in English | MEDLINE | ID: mdl-9446616

ABSTRACT

Signal transduction through the interferongamma (IFNgamma) receptor involves the formation of a ligand-dependent multimolecular association of receptor chains (alpha and beta), Janus tyrosine kinases (Jak1 and Jak2), and the transcription factor (signal transducers and activators of transcription 1alpha (STAT1alpha)) in addition to activation of mitogen-activated protein kinases (MAPK). Interactions between components of the Jak/STAT cascade and the p21(ras)/Raf-1/MAPK cascade are unexplored. Treatment of HeLa cells with IFNgamma resulted in the rapid and transient activation of Raf-1 and MAPK. Parallel activation of cells resulted in essentially no enhancement of p21(ras) activation despite marked enhancement after treatment with epidermal growth factor. In HeLa (E1C3) and fibrosarcoma (U4A) cell lines, both of which are deficient in Jak1 kinase, Raf-1 activation by IFNgamma was absent. Reconstitution of Raf-1 activity was observed only with kinase active Jak1 in both cell lines. In COS cells, transient expression of wild type or kinase-inactive Jak1 coimmunoprecipitated with Raf-1, but activation of Raf-1 activity was only observed in cells expressing kinase-active Jak1. These observations suggest that a kinase-active Jak1 is required for IFNgamma activation of Raf-1 that is p21(ras)-independent.


Subject(s)
Gene Expression Regulation , Interferon-gamma/physiology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , HeLa Cells , Humans , Janus Kinase 1 , Proto-Oncogene Proteins p21(ras)/metabolism , Signal Transduction
18.
J Biol Chem ; 272(48): 30237-43, 1997 Nov 28.
Article in English | MEDLINE | ID: mdl-9374508

ABSTRACT

STAT (signal transducers and activators of transcription) proteins are dual function proteins, which participate in cytokine-mediated signal transduction events at the cell surface and transcriptional regulation in the nucleus. We have exploited insights into the activation mechanism of STAT factors to derive constitutively active variants. Chimeric genes encoding fusion proteins of STAT5 and the kinase domain of JAK2 have been derived. The functional properties of the fusion proteins have been investigated in transiently transfected COS cells or in HeLa cells stably transfected with STAT5-JAK2 gene constructs regulated by a tetracycline-sensitive promoter. The STAT5-JAK2 proteins exhibit tyrosine kinase activity and are phosphorylated on tyrosine. The molecules are activated through an intramolecular or a cross-phosphorylation reaction and exhibit constitutive, STAT5-specific DNA binding activity. The transactivation potentials of three constitutively activated STAT5-JAK2 variants comprising different transactivation domains (TADs) derived from STAT5, STAT6, and VP16 were compared. The chimeric molecule containing the STAT5 TAD had no or only a very low, the molecule with the STAT6 TAD a medium, and the molecule with the VP16 TAD a very high transactivation potential. Transcription from STAT5-responsive gene promoter regions of the beta-casein, oncostatin M, and the cytokine-inducible Src homology 2 domain-containing protein genes was observed. These chimeric STAT molecules allow the study of the function of STAT5 independent of cytokine receptors and the activation of other signal transduction pathways.


Subject(s)
DNA-Binding Proteins/chemistry , Milk Proteins , Protein-Tyrosine Kinases/chemistry , Proto-Oncogene Proteins , Receptors, Cytokine/physiology , Trans-Activators/chemistry , Transcription Factors/chemistry , Animals , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation , HeLa Cells , Humans , Janus Kinase 2 , Recombinant Fusion Proteins , STAT5 Transcription Factor , Signal Transduction , Structure-Activity Relationship , Trans-Activators/metabolism , Transcription, Genetic
19.
Blood ; 90(11): 4341-53, 1997 Dec 01.
Article in English | MEDLINE | ID: mdl-9373245

ABSTRACT

Members of the hematopoietically expressed Tec tyrosine kinase family have an important role in hematopoietic signal transduction, as exemplified by the crucial role of Btk for B-cell differentiation and activation. Although a variety of cell surface receptors have been found to activate Tec tyrosine kinases, the specific signaling pathways and substrate molecules used by Tec kinases are still largely unknown. In this study a Tec family kinase, Bmx, was found to induce activation of the Stat signaling pathway. Bmx induced the tyrosine phosphorylation and DNA binding activity of all the Stat factors tested, including Stat1, Stat3, and Stat5, both in mammalian and insect cells. Bmx also induced transcriptional activation of Stat1- and Stat5-dependent reporter genes. Other cytoplasmic tyrosine kinases, Syk, Fyn, and c-Src, showed no or only weak ability to activate Stat proteins. Expression of Bmx in mammalian cells was found to induce activation of endogenous Stat proteins without activation of endogenous Jak kinases. We further analyzed the Bmx-mediated activation of Stat1, which was found to be regulated by protein kinase C delta (PKCdelta) isoform, but not beta 1, epsilon, or zeta isoforms, leading to inhibition of Stat1 tyrosine phosphorylation. In conclusion, these studies show that Bmx, a Tec family kinase, can function as an activator of the Stat signaling pathway and identify a role for PKCdelta in the regulation of Bmx signaling.


Subject(s)
DNA-Binding Proteins/metabolism , Isoenzymes/metabolism , Protein Kinase C/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Signal Transduction , Trans-Activators/metabolism , Animals , COS Cells , DNA/metabolism , Enzyme Activation , Janus Kinase 1 , Janus Kinase 2 , Protein Kinase C-delta , STAT1 Transcription Factor , STAT3 Transcription Factor
20.
Mol Cell Biol ; 17(7): 3833-40, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9199317

ABSTRACT

Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Interferon-beta/pharmacology , Mitogen-Activated Protein Kinase Kinases , Peptides/pharmacology , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Animals , COS Cells , DNA-Binding Proteins/metabolism , Enzyme Activation/drug effects , Epidermal Growth Factor/pharmacology , HeLa Cells , Humans , Janus Kinase 1 , MAP Kinase Kinase 1 , Oncostatin M , Proteins/metabolism , Proto-Oncogene Proteins c-raf , STAT1 Transcription Factor , STAT2 Transcription Factor , Signal Transduction , TYK2 Kinase , Trans-Activators/metabolism , Tumor Cells, Cultured
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