Interleukin 4 Regulates Phosphorylation of Serine 756 in the Transactivation Domain of Stat6: ROLES FOR MULTIPLE PHOSPHORYLATION SITES AND Stat6 FUNCTION

Lymphokines interleukin-4 (IL4) and IL13 exert overlapping biological activities via the shared use of the IL4 receptor α-chain and signal transducer and activator of transcription 6 (Stat6). Stat6 is critical for T-helper 2 cell differentiation, B-cell Ig class switch, and allergic diseases; thus, understanding its regulation is of central importance. Phosphorylation is crucial for Stat activity. Whereas Stat6 is phosphorylated on Tyr641, less is known about serine or threonine. We demonstrate in primary human T-cells (>95% CD3+) that IL4 and for the first time IL13 induce Stat6 serine but not threonine phosphorylation that closely paralleled early IL4 receptor α-chain activation (10 min). Stat6 uniquely fails to share a positionally conserved Stat serine phosphorylation sequence; however, known phosphoacceptor sites are proline-flanked. Alanine substitutions of these conserved residues revealed that the transactivation domain, which localized Ser756 but not Ser827 or Ser176, is the IL4-regulated site based on phosphoamino acid analysis. Tyr641 was dispensable for IL4-mediated serine phosphorylation, suggesting that dimerization is not preconditional. Only Stat6 Y641F variant showed a significant effect on IL4-inducible Cϵ DNA-binding and reporter gene expression. Lastly, recent work has shown that protein phosphatase 2A negatively regulates Stat6 (Woetmann, A., Brockdorff, J., Lovato, P., Nielsen, M., Leick, V., Rieneck, K., Svejgaard, A., Geisler, C., and Odum, N. (2003) J. Biol. Chem. 278, 2787–2791). We propose this target residue(s) is distinct from Ser756 and may be proximal to Tyr641 at Thr645, a residue conserved only among Stat6 members. The phosphomimic variants T645E or T645D ablated Stat6 activation, whereas polar uncharged substitutions (Gln or Asn) and additional mutants (Ala, Val, or Phe) showed no effect. These findings suggest that Stat6 has mechanisms of regulation distinct from other Stats

Transcription factor signal transducer and activator of transcription 5 promotes growth of human prostate cancer cells in vivo

Purpose: Stat5a/b is the key mediator of prolactin (Prl) effects in prostate cancer cells via activation of Jak2. Prl is locally produced growth factor in human prostate cancer. Prl protein expression and constitutive activation of Stat5a/b are associated with high histological grade of clinical prostate cancer. Moreover, activation of Stat5a/b in primary prostate cancer predicts early disease recurrence. Here, we inhibited Stat5a/b by several different methodological approaches. Our goal was to establish a proof-of-principle that Stat5a/b is critical for prostate cancer cell viability in vitro and for prostate tumor growth in vivo. Experimental Design: We inhibited Stat5a/b protein expression by antisense oligonucleotides or RNA interference and transcriptional activity of Stat5a/b by adenoviral expression of a dominant-negative mutant of Stat5a/b in prostate cancer cells in culture. Moreover, Stat5a/b activity was suppressed in human prostate cancer xenograft tumors in nude mice. Stat5a/b regulation of BclXL and Cyclin-D1 protein levels was demonstrated by antisense suppression of Stat5a/b protein expression followed by Western blotting. Results and Conclusions: We show here that inhibition of Stat5a/b by antisense oligonuleotides, RNA interference, or adenoviral expression of DNStat5a/b all effectively kill prostate cancer cells. Moreover, we demonstrate that Stat5a/b is critical for human prostate cancer xenograft growth in nude mice. Stat5a/b effects on the viability of on prostate cancer cells involve Stat5a/b-regulation of BclXL and Cyclin-D1 protein levels, but not the expression or activation of Stat3. This work establishes Stat5a/b as a therapeutic target protein for prostate cancer. Pharmacological inhibition of Stat5a/b in prostate cancer can be achieved by small-molecule inhibitors of transactivation, dimerization or DNA-binding of Stat5a/b

Uncoupling JAK3 activation induces apoptosis in human lymphoid cancer cells via regulating critical survival pathways

AbstractIn the current work, we report that specific inhibition of Janus tyrosine kinase (JAK3) via NC1153 induces apoptosis of certain leukemia/lymphoma cell lines. Affymetrix microarray profiling following NC1153 treatment unveiled JAK3 dependent survival modulating pathways (p53, TGF-β, TNFR and ER stress) in Kit225 cells. IL-2 responsive NC1153 target genes were regulated in human JAK3 positive, but not in JAK3 negative lymphoid tumor cells. Moreover, primary lymphoma samples revealed that a number of these genes were reciprocally regulated during disease progression and JAK3 inhibition suggesting that downstream targets of JAK3 could be exploited in the development of novel cancer treatment regimes

STAT5 regulation of BCL10 parallels constitutive NFkappaB activation in lymphoid tumor cells.

BACKGROUND: Signal Transducer and Activator of Transcription 5 A and B (STAT5) are key survival factors in cells of the lymphoid lineage. Identification of novel, tissue-specific STAT5 regulated genes would advance the ability to combat diseases due to aberrant STAT5 signaling. In the present work a library of human STAT5 bound genomic elements was created and validated. RESULTS: Of several STAT5 responsive genomic regulatory elements identified, one was located within the first intron of the human BCL10 gene. Chromatin immuno-precipitation reactions confirmed constitutive in vivo STAT5 binding to this intronic fragment in various human lymphoid tumor cell lines. Interestingly, non-phosphorylated STAT5 was found in the nuclei of Kit225 and YT cells in the absence of cytokine stimulation that paralleled constitutive NFkappaB activation. Inhibition of the hyperactive JAK3/STAT5 pathway in MT-2 cells via the Mannich-base, NC1153, diminished the constitutive in vivo occupancy of BCL10-SBR by STAT5, reduced NFkappaB activity and BCL10 protein expression in a dose dependent manner. Moreover, depletion of STAT5 via selective antisense oligonucleotide treatment similarly resulted in decreased BCL10 mRNA and protein expression, cellular viability and impaired NFkappaB activity independent of IL-2. CONCLUSION: These results suggest that the NFkappaB regulator BCL10 is an IL-2-independent STAT5 target gene. These findings proffer a model in which un-activated STAT5 can regulate pathways critical for lymphoid cell survival and inhibitors that disrupt STAT5 function independent of tyrosine phosphorylation may be therapeutically effective in treating certain leukemias/lymphomas

STAT5 regulation of BCL10 parallels constitutive NFκB activation in lymphoid tumor cells

<p>Abstract</p> <p>Background</p> <p>Signal Transducer and Activator of Transcription 5 A and B (STAT5) are key survival factors in cells of the lymphoid lineage. Identification of novel, tissue-specific STAT5 regulated genes would advance the ability to combat diseases due to aberrant STAT5 signaling. In the present work a library of human STAT5 bound genomic elements was created and validated.</p> <p>Results</p> <p>Of several STAT5 responsive genomic regulatory elements identified, one was located within the first intron of the human <it>BCL10 </it>gene. Chromatin immuno-precipitation reactions confirmed constitutive <it>in vivo </it>STAT5 binding to this intronic fragment in various human lymphoid tumor cell lines. Interestingly, non-phosphorylated STAT5 was found in the nuclei of Kit225 and YT cells in the absence of cytokine stimulation that paralleled constitutive NFκB activation. Inhibition of the hyperactive JAK3/STAT5 pathway in MT-2 cells via the Mannich-base, NC1153, diminished the constitutive <it>in vivo </it>occupancy of BCL10-SBR by STAT5, reduced NFκB activity and BCL10 protein expression in a dose dependent manner. Moreover, depletion of STAT5 via selective antisense oligonucleotide treatment similarly resulted in decreased BCL10 mRNA and protein expression, cellular viability and impaired NFκB activity independent of IL-2.</p> <p>Conclusion</p> <p>These results suggest that the NFκB regulator BCL10 is an IL-2-independent STAT5 target gene. These findings proffer a model in which un-activated STAT5 can regulate pathways critical for lymphoid cell survival and inhibitors that disrupt STAT5 function independent of tyrosine phosphorylation may be therapeutically effective in treating certain leukemias/lymphomas.</p

Genome wide mapping reveals PDE4B as an IL-2 induced STAT5 target gene in activated human PBMCs and lymphoid cancer cells

IL-2 is the primary growth factor for promoting survival and proliferation of activated T cells that occurs following engagement of the Janus Kinase (JAK)1-3/and Signal Transducer and Activator of Transcription (STAT) 5 signaling pathway. STAT5 has two isoforms: STAT5A and STAT5B ( commonly referred to as STAT5) which, in T cells, play redundant roles transcribing cell cycle and survival genes. As such, inhibition of STAT5 by a variety of mechanisms can rapidly induce apoptosis in certain lymphoid tumor cells, suggesting that it and its target genes represent therapeutic targets to control certain lymphoid diseases. To search for these molecules we aligned IL-2 regulated genes detected by Affymetrix gene expression microarrays with the STAT5 cistrome identified by chip-on-ChIP analysis in an IL-2-dependent human leukemia cell line, Kit225. Select overlapping genes were then validated using qRT(2)PCR medium-throughput arrays in human PHA-activated PBMCs. Of 19 putative genes, one key regulator of T cell receptor signaling, PDE4B, was identified as a novel target, which was readily up-regulated at the protein level (3 h) in IL-2 stimulated, activated human PBMCs. Surprisingly, only purified CD8+ primary T-cells expressed PDE4B, but not CD4+ cells. Moreover, PDE4B was found to be highly expressed in CD4+ lymphoid cancer cells, which suggests that it may represent a physiological role unique to the CD8+ and lymphoid cancer cells and thus might represent a target for pharmaceutical intervention for certain lymphoid diseases

Forskolin-inducible cAMP Pathway Negatively Regulates T-cell Proliferation by Uncoupling the Interleukin-2 Receptor Complex

Cytokine-mediated regulation of T-cell activity involves a complex interplay between key signal transduction pathways. Determining how these signaling pathways cross-talk is essential to understanding T-cell function and dysfunction. In this work, we provide evidence that cross-talk exists between at least two signaling pathways: the Jak3/Stat5 and cAMP-mediated cascades. The adenylate cyclase activator forskolin (Fsk) significantly increased intracellular cAMP levels and reduced proliferation of the human T-cells via inhibition of cell cycle regulatory genes but did not induce apoptosis. To determine this inhibitory mechanism, effects of Fsk on IL-2 signaling was investigated. Fsk treatment of MT-2 and Kit 225 T-cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also uncoupled IL-2 induced association of the IL-2Rβ and γc chain, consequently blocking Jak3 activation. Interestingly, phosphoamino acid analysis revealed that Fsk-treated cells resulted in elevated serine phosphorylation of Jak3 but not Stat5, suggesting that Fsk can negatively regulate Jak3 activity possibly mediated through PKA. Indeed, in vitro kinase assays and small molecule inhibition studies indicated that PKA can directly serine phosphorylate and functionally inactivate Jak3. Taken together, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate IL-2 signaling at multiple levels that include IL-2R complex formation and Jak3/Stat5 activation

Párhuzamos jelátviteli útvonalak szerepe limfoid sejtek túlélésében

T lymphocytes are critical for a functional immune system. However, their uncontrolled activation and growth can be manifested as a number of lymphoid derived diseases such as autoimmunity, allograft rejection, and lymphoma. Therefore, understanding the role of key signaling molecules and the ability to modify their activity in mature cells may provide new therapeutic strategies to treat T cell mediated disorders. The purpose of this study was to dissect the role of key signaling pathways activated by interleukin-2 (IL2) and other T cell growth factors that share the IL2 receptor gamma chain (γc) (IL2, 4, 7, 9, 13, 15, 21) in mediating T cell growth and survival. To accomplish this objective, antisense oligonucleotides targeted against signaling molecules including γc, Raf isoforms, Stat3, traditional pharmaceuticals to disrupt Janus tyrosine kinase 3, PI3K (wortmannin), mTor (rapamycin) and MEK kinase (PD98059) activity were utilized and their effects characterized in lymphoid cells. Antisense oligonucleotide targeted against γc inhibited protein expression (40%) in a lymphoid tumor cell line inducing apoptotic death and cell cycle arrest in G2-M phase, as measured by FACS analysis of Annexin V and Propidium Iodide stained cells. Inhibition of Jak3 (PNU156804) showed similar effects and disrupted Stat5 and MAPK activation. Novel evidence is shown here that IL2 can activate multiple isoforms of Raf, and inhibition of these isoforms by antisense oligonucleotides failed to affect cell survival or Stat5 activation. Similar results were found for inhibition of PI3K, mTor and MEK kinases. Lastly, we show that Stat3 antisense oligonucleotide treatment of a human tumor cell line bearing constitutively activated Stat3 induces cell death (40%, measured with TUNEL assay) but that IL2 can rescue these cells possibly mediated via Stat5 that becomes hyperactivated in Stat3 deleted cells. Evidence is provided that suggests Bcl2 plays key role in this cell survival process. We conclude from our findings that the Mapk, Stat and PI3K pathways likely function as separate entities in lymphoid cells, suggesting that evolutionarily these pathways might have evolved to perform specialized cell functions that may be redundant for other T cell activities. Moreover, our results suggest that Stat molecules are critical to the survival and growth of T cells and represent a therapeutically relevant target to combat T cell derived disorders.PhDEgyetemi doktori (Ph.D.) értekezés ; University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Department of Biochemistry and Molecular Biology and University of Texas at Houston Medical School, Department of Integrative Biology and Pharmacology, 200

Phosphorylation of Human Jak3 at Tyrosines 904 and 939 Positively Regulates Its Activity▿

Janus tyrosine kinase 3 (Jak3) is essential for signaling by interleukin-2 (IL-2) family cytokines and proper immune function. Dysfunctional regulation of Jak3 may result in certain disease states. However, the molecular mechanisms governing Jak3 activation are not fully understood. In this study, we used a functional-proteomics approach to identify two novel tyrosine phosphorylation sites within Jak3, Y904 and Y939, which are conserved among Jak family proteins. By using phosphospecific antibodies, both residues were observed to be rapidly induced by stimulation of cells with IL-2 or other γc cytokines. Mechanistic studies indicated that Y904 and Y939 regulate Jak3 activities. A phenylalanine substitution at either site greatly reduced Jak3 kinase activity in vitro and its ability to phosphorylate signal transducer and activator of transcription 5 (Stat5) in vivo, suggesting that phosphorylation of these previously unrecognized residues positively regulates Jak3 activity. Y904 and Y939 were required for optimal ATP usage by Jak3, while phosphorylation of Y939 preferentially promoted Stat5 activity in intact cells. Together, these findings demonstrate positive functional roles for two novel Jak3 phosphoregulatory sites which may be similarly important for other Jak family members. Identification of these sites also provides new therapeutic opportunities to modulate Jak3 function