Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^

STAT3 activation by the ErbB2 receptor and development of peptide-based STAT3 inhibitors

Stats (s&barbelow;ignal t&barbelow;ransducer and a&barbelow;ctivator of t&barbelow;ranscription) are latent transcription factors that translocate from the cytoplasm to nucleus. Constitutive activation of Stat3α by upstream oncoproteins and receptor tyrosine kinases has been found in many human tumors and tumor-derived cell lines and it is often correlated with the activation of ErbB-2. In order to explore the involvement of ErbB-2 in the activation of Stat3 and the mechanisms underlying this event, an erbB-2 point mutant was used as a model of a constitutively activated receptor. Phenylalanine mutations (Y-F) were made in the receptor's autophosphorylation sites and their ability to activate Stat3α was evaluated. Our results suggest that Stat3α and Janus tyrosine kinase 2 associates with ErbB-2 prior to tyrosine phosphorylation of the receptor and that full activation of Stat3α by ErbB-2 requires the participation of other non-receptor tyrosine kinases. Both Src and Jak2 kinases contribute to the activation of Stat3α while only Src binds to ErbB-2 only when the receptor is tyrosine phosphorylated. Our results also suggest that tyrosine 1139 may be important for Src SH2 domain association since a mutant lacking this tyrosine reduces the ability of the Src SH2 domain to bind to ErbB-2 and significantly decreases its ability to activate Stat3α. ^ In order to disrupt aberrant STAT3α activation which contributes to tumorigenesis, we sought small molecules which can specifically bind to the STAT3 SH2 domain, thereby abolishing its ability of being recruited into receptors, and also blocking the dimer formation required for STAT3α activation. A phosphopeptide derived from gp130 was found to have a high affinity to STAT3 SH2 domain, and we decided to use this peptide as the base for further modifications. A series of peptide based compounds were designed and tested using electrophoretic mobility shift assay and fluorescence polarization assay to evaluate their affinity to the STAT3 SH2 domain. Two promising compounds, DRIV-73C and BisPOM, were used for blocking STAT3α activity in cell culture. Either can successfully impair STAT3α activation induced by IL-6 stimulation in HepG2 cells. BisPOM proved to be the more effective in blocking STAT3α tyrosine phosphorylation in induced cells and tumor cell lines, and was the more potent in inhibiting STAT3 dependent cell growth. ^

The role and mechanism of supressor of cytokine signaling 1 in brain metastasis

Brain metastasis, which occurs in 40%-60% of patients with advanced melanoma, has led directly to death in the majority of cases. Unfortunately, little is known about the biological and molecular basis of melanoma brain metastases. In our previous study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The increased activation of Stat3 is also responsible for affecting melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affecting the expression of bFGF, VEGF, and MMP-2 in vivo and in vitro. Interestingly, a member of a new family of cytokine-inducible inhibitors of signal transduction, termed suppressors of cytokine signaling 1 (SOCS1) was found to negatively regulate the Janus kinase signal transducer and activator of transcription (Jak/STAT) signaling cascade. Here we report that restoration of SOCS1 expression by transfecting of SOCS1-expressing vector effectively inhibited melanoma brain metastasis through inhibiting Stat3 activation and further affecting melanoma angiogenesis and melanoma cell invasion in vitro, and significantly affected the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in vitro and in vivo. In addition, we used cDNA array to compare mRNA expression in the SOCS1-transfected and vector-transfected cell lines and found some genes are tightly correlated to the restoration of SOCS1. One of them is Caveolin-1 (Cav-1). Cav-1 was reported to function as a tumor suppressor gene by several groups. Finally, the Cav-1 expression is up-regulated in SOCS1-overexpressing cell line. Further study found the regulation of Cav-1 by SOCS1 occurs through inhibiting Stat3 activation. Activated Stat3 binds directly to Cav-1 promoter and the Cav-1 promoter within -575bp is essential for active Stat3 binding. My studies reveal that Stat3 activation and SOCS1 expression play important roles in melanoma metastases. Moreover, the expression between SOCS1, Stat3 and Cav-1 forms a feedback regulation loop. ^

Identification and functional characterization of novel phosphotyrosine and phosphoserine residues in the interleukin-2 receptor complex

Interleukin-2 (IL-2) is a major T cell growth factor and plays an essential role in the development of normal immune responses. The Janus kinases (Jaks) and Signal transducers and activators of transcription (Stats) are critical for transducing signals from the IL-2 receptors (IL2Rs) to the nucleus to control cell growth and differentiation. In recent years there has been increasing evidence to indicate that the IL-2 activated Jak3/Stat5 pathway provides a new molecular target for immune suppression. Thus, understanding the regulation of this effector cascade has important therapeutic potential.^ One objective of this work was to identify and define the role and molecular mechanism of novel phosphorylation sites in Jak3. Using functional proteomics, three novel Jak3 phosphorylation sites, Y904, Y939 and S574 were identified. Phosphospecific antibodies confirmed that phosphorylation of Y904 and Y939 were mediated by IL-2 and other IL-2 family cytokines in distinct cell types. Biochemical analysis demonstrated that phosphorylation of both Y904 and Y939 positively regulated Jak3 enzymatic activity, while phosphorylation of S574 did not affect Jak3 in vitro kinase activity. However, a gain-of-function mutation of S574 in Jak3 abrogated IL-2 mediated Stat5 activation, suggesting that phosphorylation of this residue might serve a negative role to attenuate IL-2 signaling. Furthermore, mechanistic analysis suggested that phosphorylation of Y904 in Jak3 affects the KmATP of Jak3, while phosphorylation of Y939 in Jak3 was required to bind one of its substrates, Stat5.^ The second objective was to determine the role of serine/threonine phosphatases in the regulation of the IL2R complex. Activation of Jak3 and Stat5 by IL-2 is a transient event mediated by phosphorylation. Using a specific PP1/PP2A inhibitor, we observed that inhibition of PP1/PP2A negatively regulated the IL-2 activated Jak3/Stat5 signaling pathway in a human NK cell line (YT) and primary human T cells. More importantly, coimmunoprecipitation assays indicated that inhibition of PP1/PP2A blocked the formation of an active IL2R complex. Pretreatment of cells with the inhibitor also reduced the electrophoretic mobility of the IL2Rβ and IL2Rγ subunits in YT cells, suggesting that inhibition of PP1/PP2A directly or indirectly regulates undefined serine/threonine kinases which phosphorylate these proteins. Based on these observations, a model has emerged that serine/threonine phosphorylation of the IL2Rβ and IL2Rγ subunits causes a conformational change of these proteins, which disrupts IL2R dimerization and association of Jak3 and Stat5 to these receptors.^

cAMP regulates IL-2 receptor signaling in human T cells

Proper immune system function is dependent on positive and negative regulation of T cell signaling pathways. Full T cell activation requires sequential signaling through the T cell receptor (TCR), costimulatory molecules and the IL-2 receptor (IL-2R). The IL-2R associated Janus tyrosine kinase 3 (Jak3), as well as Signal transducer and activator of transcription 5 (Stat5), are required for normal T cell function and survival. Constitutive activation of Jak3 and Stat5 have been linked to cancers of hematopoietic origin, including certain lymphomas and leukemias. ^ The production of cAMP by adenylate cyclase has been shown to negatively regulate human TCR mediated cell proliferation. Since cAMP has been shown to negatively regulate T cell activation, we sought to investigate whether crosstalk exists between cAMP and IL-2R signaling. The first objective of this study was to determine the effect of cAMP on the activation of IL-2R signaling molecules Jak3 and Stat5. We found that the potent adenylate cyclase activator, forskolin, inhibited IL-2 activation of Jak3 and Stat5. Indeed, in vitro kinase assays and electrophoretic mobility shift assays verified a loss of Jak3 enzymatic activity and Stat5 DNA binding ability, respectively. Further analysis of IL-2R signaling showed that forskolin treatment reduced IL-2 induced association of the IL-2Rβ and γc chain. ^ Because cAMP activates protein kinase A (PKA), the second objective was to determine the role for PKA in the cAMP directed regulation of IL-2R signaling intermediates. Interestingly, forskolin induced serine phosphorylation of Jak3, suggesting that cAMP can directly regulate Jak3 via activation of a serine/threonine kinase. Indeed, phosphoamino acid analysis revealed that PKA was able to induce Jak3 serine phosphorylation in the human leukemia cell line MT-2. In addition, in vitro kinase assays established that PKA can directly inhibit Jak3 enzymatic activity. Collectively, these data indicate that cAMP negatively regulates IL-2R signaling via various effector molecules by a previously unrecognized mechanism. This new data suggests that the Jak3/Stat5 pathway may be regulated by various pharmacological agents that stimulate cAMP production and thus can be used to uncouple some types of T cell mediated diseases. ^

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.