STAP-2 facilitates insulin signaling through binding to CAP/c-Cbl and regulates adipocyte differentiation.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor molecule involved in several cellular signaling cascades. Here, we attempted to identify novel STAP-2 interacting molecules, and identified c-Cbl associated protein (CAP) as a binding protein through the C-terminal proline-rich region of STAP-2. Expression of STAP-2 increased the interaction between CAP and c-Cbl, suggesting that STAP-2 bridges these proteins and enhances complex formation. CAP/c-Cbl complex is known to regulate GLUT4 translocation in insulin signaling. STAP-2 overexpressed human hepatocyte Hep3B cells showed enhanced GLUT4 translocation after insulin treatment. Elevated levels of Stap2 mRNA have been observed in 3T3-L1 cells and mouse embryonic fibroblasts (MEFs) during adipocyte differentiation. The differentiation of 3T3-L1 cells into adipocytes was highly promoted by retroviral overexpression of STAP-2. In contrast, STAP-2 knockout (KO) MEFs exhibited suppressed adipogenesis. The increase in body weight with high-fat diet feeding was significantly decreased in STAP-2 KO mice compared to WT animals. These data suggest that the expression of STAP-2 correlates with adipogenesis. Thus, STAP-2 is a novel regulatory molecule that controls insulin signal transduction by forming a c-Cbl/STAP-2/CAP ternary complex.

Tyrosinase regulates the motility of human melanoma cell line A375 through its hydroxylase enzymatic activity.

Melanoma, originating from melanocytes, is a highly aggressive tumor. Tyrosinase is involved in melanin production in melanocytes, and its overexpression is noted in malignant melanomas. However, the role of tyrosinase in melanomas remains unclear. Therefore, this study aimed to evaluate the potential functions of tyrosinase in the human melanoma cell line A375. The expression level of tyrosinase in A375 cells was undetectable. However, markedly increased expression level was observed in the mouse melanoma cell line B16F10 and the human melanoma cell line WM266-4. Subsequently, we investigated the effect of ectopic tyrosinase expression on A375 cell motility using wound-healing assay. The overexpression of tyrosinase resulted in enhanced cell migration in both stable and transient tyrosinase expression cells. The levels of filamentous actin were decreased in tyrosinase-expressing A375 cells, suggesting that tyrosinase regulates cell motility by modulating actin polymerization. Histidine residues in tyrosinase are important for its enzymatic activity for synthesizing melanin. Substitution of these histidine residues to alanine residues mitigated the promotion of tyrosinase-induced A375 cell metastasis. Furthermore, melanin treatment enhanced A375 cell metastasis and phosphorylation of Cofilin. Thus, our findings suggest that tyrosinase increases the migration of A375 cells by regulating actin polymerization through its enzymatic activity.

Role of Signal-Transducing Adaptor Protein-1 for T Cell Activation and Pathogenesis of Autoimmune Demyelination and Airway Inflammation.

Signal-transducing adaptor protein (STAP)-1 is an adaptor protein that is widely expressed in T cells. In this article, we show that STAP-1 upregulates TCR-mediated T cell activation and T cell-mediated airway inflammation. Using STAP-1 knockout mice and STAP-1-overexpressing Jurkat cells, we found that STAP-1 enhanced TCR signaling, resulting in increased calcium mobilization, NFAT activity, and IL-2 production. Upon TCR engagement, STAP-1 binding to ITK promoted formation of ITK-LCK and ITK-phospholipase Cγ1 complexes to induce downstream signaling. Consistent with the results, STAP-1 deficiency reduced the severity of symptoms in experimental autoimmune encephalomyelitis. Single-cell RNA-sequencing analysis revealed that STAP-1 is essential for accumulation of T cells and Ifng and Il17 expression in spinal cords after experimental autoimmune encephalomyelitis induction. Th1 and Th17 development was also attenuated in STAP-1 knockout naive T cells. Taken together, STAP-1 enhances TCR signaling and plays a role in T cell-mediated immune disorders.

STAP-2 negatively regulates BCR-mediated B cell activation by recruiting tyrosine-protein kinase CSK to LYN.

Although signal-transducing adaptor protein-2 (STAP-2) acts in certain immune responses, its role in B cell receptor (BCR)-mediated signals remains unknown. In this study, we have revealed that BCR-mediated signals, cytokine production and antibody production were increased in STAP-2 knockout (KO) mice compared with wild-type (WT) mice. Phosphorylation of tyrosine-protein kinase LYN Y508 was reduced in STAP-2 KO B cells after BCR stimulation. Mechanistic analysis revealed that STAP-2 directly binds to LYN, dependently of STAP-2 Y250 phosphorylation by LYN. Furthermore, phosphorylation of STAP-2 enhanced interactions between LYN and tyrosine-protein kinase CSK, resulting in enhanced CSK-mediated LYN Y508 phosphorylation. These results suggest that STAP-2 is crucial for controlling BCR-mediated signals and antibody production by enhanced CSK-mediated feedback regulation of LYN.

STAP-2-Derived Peptide Suppresses TCR-Mediated Signals to Initiate Immune Responses.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a proline-rich region in its C-terminal region. Our previous study demonstrated that STAP-2 positively regulates TCR signaling by associating with TCR-proximal CD3ζ ITAMs and the lymphocyte-specific protein tyrosine kinase. In this study, we identify the STAP-2 interacting regions of CD3ζ ITAMs and show that the STAP-2-derived synthetic peptide (iSP2) directly interacts with the ITAM sequence and blocks the interactions between STAP-2 and CD3ζ ITAMs. Cell-penetrating iSP2 was delivered into human and murine T cells. iSP2 suppressed cell proliferation and TCR-induced IL-2 production. Importantly, iSP2 treatment suppressed TCR-mediated activation of naive CD4+ T cells and decreased immune responses in CD4+ T cell-mediated experimental autoimmune encephalomyelitis. It is likely that iSP2 is a novel immunomodulatory tool that modulates STAP-2-mediated activation of TCR signaling and represses the progression of autoimmune diseases.

Evaluation of sexual function after dutasteride treatment in patients with once-negative prostate biopsy and benign prostate hyperplasia.

INTRODUCTION: Patients with benign prostatic hyperplasia are usually treated with 5α-reduced inhibitors (5ARIs) such as finasteride and dutasteride. However, studies on the influence of 5ARIs on sexual function have been controversial. In this study, we evaluated the impact of dutasteride treatment for erectile function in patients with once-negative prostate biopsy and benign prostate hyperplasia. PATIENTS AND METHODS: 81 patients with benign prostate hyperplasia were enrolled in a one-armed prospective study. They were administrated 0.5 mg/day of dutasteride for 12 months. Patient characteristics and changes of International Prostate Symptom Score (IPSS) and International Index of Erectile Function (IIEF)-15 scores at baseline and 12 months after dutasteride administration were examined. RESULTS: The mean ± standard deviation (SD) age of the patients was 69.4 ± 4.9 years and the prostate volume was 56.6 ± 21.3 mL, respectively. The mean ± SD prostate volume and PSA levels were decreased 25.0 and 50.9%, respectively, after 12 months of dutasteride administration. IPSS total, voiding subscore, storage subscore, and quality of life score significantly improved after 12 months of dutasteride administration. No statistically significant change in IIEF-total score from 16.3 ± 13.5 to 18.8 ± 16.0 (p = 0.14), IIEF-EF score from 5.1 ± 6.9 to 6.4 ± 8.3 (p = 0.13) were observed. There was no decrease in erectile function severity. CONCLUSION: Twelve months administration of dutasteride for patients with BPH improved urinary function and did not increase the risk of sexual dysfunction.

The Physiological and Pathophysiological Role of IL-6/STAT3-Mediated Signal Transduction and STAT3 Binding Partners in Therapeutic Applications.

The interleukin 6 (IL-6) family of cytokines is defined by the usage of gp130, a common ß-receptor signaling subunit, which promotes a variety of signals. They induce many biological functions on many cell types, including immune and inflammatory cells. They also exhibit hormone-like features, which are involved in homeostatic processes. Signal transducer and activator of transcription 3 (STAT3) is a significant signaling molecule fundamental in regulating IL-6/gp130 and is highly implicated in pathological conditions; therefore, STAT3 activation is tightly regulated through various mechanisms and at multiple levels. There is a large amount of information about STAT3-interacting proteins, which positively or negatively regulate STAT3 activity. This review is focused on IL-6-mediated signal transduction and the introduction of novel STAT3-binding partners. The review will help develop new strategies for clinically controlling the functions of IL-6/STAT3.

A peptide derived from adaptor protein STAP-2 inhibits tumor progression by downregulating epidermal growth factor receptor signaling.

Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signals. We previously demonstrated that STAP-2 binds to epidermal growth factor receptor (EGFR) and facilitates its stability and activation of EGFR signaling in prostate cancer cells. Inhibition of this interaction may be a promising direction for cancer treatment. Here, we found that 2D5 peptide, a STAP-2-derived peptide, blocked STAP-2-EGFR interactions and suppressed EGFR-mediated proliferation in several cancer cell lines. 2D5 peptide inhibited tumor growth of human prostate cancer cell line DU145 and human lung cancer cell line A549 in murine xenograft models. Additionally, we determined that EGFR signaling and its stability were decreased by 2D5 peptide treatment during EGF stimulation. In conclusion, our study shows that 2D5 peptide is a novel anticancer peptide that inhibits STAP-2-mediated activation of EGFR signaling and suppresses prostate and lung cancer progression.

The Functional Properties and Physiological Roles of Signal-Transducing Adaptor Protein-2 in the Pathogenesis of Inflammatory and Immune Disorders.

Adaptor molecules play a crucial role in signal transduction in immune cells. Several adaptor molecules, such as the linker for the activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), are essential for T cell development and activation following T cell receptor (TCR) aggregation, suggesting that adaptor molecules are good therapeutic targets for T cell-mediated immune disorders, such as autoimmune diseases and allergies. Signal-transducing adaptor protein (STAP)-2 is a member of the STAP family of adaptor proteins. STAP-2 functions as a scaffold for various intracellular proteins, including BRK, signal transducer, and activator of transcription (STAT)3, STAT5, and myeloid differentiation primary response protein (MyD88). In T cells, STAP-2 is involved in stromal cell-derived factor (SDF)-1α-induced migration, integrin-dependent cell adhesion, and Fas-mediated apoptosis. We previously reported the critical function of STAP-2 in TCR-mediated T cell activation and T cell-mediated autoimmune diseases. Here, we review how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases in order to develop novel STAP-2-targeting therapeutic strategies.

Psychological Stress Induced by Prone Positioning among Adults with Severe Cerebral Palsy.

The purpose of this study was to investigate the psychological impact of various positionings in subjects with cerebral palsy (CP). The participants were 17 individuals with severe motor and intellectual disability due to CP. They began in a sitting position in their wheelchair, and were placed consecutively in prone or supine positions, with no intervals between placements. Physiological observations were made in each position, and included salivary α-amylase activity, pulse, percutaneous oxygen saturation, respiratory rate, learance or not of airway secretions, and occurrence or not of adverse events. Salivary α-amylase activity values were higher in the prone position than in the baseline and supine positions (p<0.05). Clearance of airway secretions was significantly more prevalent in the prone position than in the baseline and supine positions (p <0.05). The participants' pulse was significantly lower in the supine and prone positions than in the baseline position (p<0.05). Greater prevalence of airway secretion clearance and significantly higher stress levels as indicated by saliva amylase were observed in the prone position than in the other two positions. Therefore, when such patients are placed in a prone position, close attention to airway management and the potential for psychological stress may be necessary.

CARD11 mutation and HBZ expression induce lymphoproliferative disease and adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). In addition to HTLV-1 bZIP factor (HBZ), a leukemogenic antisense transcript of HTLV-1, abnormalities of genes involved in TCR-NF-κB signaling, such as CARD11, are detected in about 90% of patients. Utilizing mice expressing CD4+ T cell-specific CARD11(E626K) and/or CD4+ T cell-specific HBZ, namely CARD11(E626K)CD4-Cre mice, HBZ transgenic (Tg) mice, and CARD11(E626K)CD4-Cre;HBZ Tg double transgenic mice, we clarify these genes' pathogenetic effects. CARD11(E626K)CD4-Cre and HBZ Tg mice exhibit lymphocytic invasion to many organs, including the lungs, and double transgenic mice develop lymphoproliferative disease and increase CD4+ T cells in vivo. CARD11(E626K) and HBZ cooperatively activate the non-canonical NF-κB pathway, IRF4 targets, BATF3/IRF4/HBZ transcriptional network, MYC targets, and E2F targets. Most KEGG and HALLMARK gene sets enriched in acute-type ATL are also enriched in double transgenic mice, indicating that these genes cooperatively contribute to ATL development.

Central Roles of STAT3-Mediated Signals in Onset and Development of Cancers: Tumorigenesis and Immunosurveillance.

Since the time of Rudolf Virchow in the 19th century, it has been well-known that cancer-associated inflammation contributes to tumor initiation and progression. However, it remains unclear whether a collapse of the balance between the antitumor immune response via the immunological surveillance system and protumor immunity due to cancer-related inflammation is responsible for cancer malignancy. The majority of inflammatory signals affect tumorigenesis by activating signal transducer and activation of transcription 3 (STAT3) and nuclear factor-κB. Persistent STAT3 activation in malignant cancer cells mediates extremely widespread functions, including cell growth, survival, angiogenesis, and invasion and contributes to an increase in inflammation-associated tumorigenesis. In addition, intracellular STAT3 activation in immune cells causes suppressive effects on antitumor immunity and leads to the differentiation and mobilization of immature myeloid-derived cells and tumor-associated macrophages. In many cancer types, STAT3 does not directly rely on its activation by oncogenic mutations but has important oncogenic and malignant transformation-associated functions in both cancer and stromal cells in the tumor microenvironment (TME). We have reported a series of studies aiming towards understanding the molecular mechanisms underlying the proliferation of various types of tumors involving signal-transducing adaptor protein-2 as an adaptor molecule that modulates STAT3 activity, and we recently found that AT-rich interactive domain-containing protein 5a functions as an mRNA stabilizer that orchestrates an immunosuppressive TME in malignant mesenchymal tumors. In this review, we summarize recent advances in our understanding of the functional role of STAT3 in tumor progression and introduce novel molecular mechanisms of cancer development and malignant transformation involving STAT3 activation that we have identified to date. Finally, we discuss potential therapeutic strategies for cancer that target the signaling pathway to augment STAT3 activity.

Site-specific Risk Stratification Models for Postoperative Recurrence and Survival Prediction in Patients with Upper Tract Urothelial Carcinoma Undergoing Radical Nephroureterectomy: Better Stratification for Adjuvant Therapy.

Background: Site-specific postoperative risk models for localized upper tract urothelial carcinoma (UTUC) are unavailable. Objective: To create specific risk models for renal pelvic urothelial carcinoma (RPUC) and ureteral urothelial carcinoma (UUC), and to compare the predictive accuracy with the overall UTUC risk model. Design setting and participants: A multi-institutional database retrospective study of 1917 UTUC patients who underwent radical nephroureterectomy (RNU) between 2000 and 2018 was conducted. Outcome measurements and statistical analysis: A multivariate hazard model was used to identify the prognostic factors for extraurinary tract recurrence (EUTR), cancer-specific death (CSD), and intravesical recurrence (IVR) after RNU. Patients were stratified into low-, intermediate-, high-, and highest-risk groups. External validation was performed to estimate a concordance index of the created risk models. We investigated whether our risk models could aid decision-making regarding adjuvant chemotherapy (AC) after RNU. Results and limitations: The UTUC risk models could stratify the risk of cumulative incidence of three endpoints. The RPUC- and UUC-specific risk models showed better stratification than the overall UTUC risk model for all the three endpoints, EUTR, CSD, and IVR (RPUC: concordance index, 0.719 vs 0.770, 0.714 vs 0.794, and 0.538 vs 0.569, respectively; UUC: 0.716 vs 0.767, 0.766 vs 0.809, and 0.553 vs 0.594, respectively). The UUC-specific risk model can identify the high- and highest-risk patients likely to benefit from AC after RNU. A major limitation was the potential selection bias owing to the retrospective nature of this study. Conclusions: We recommend using site-specific risk models instead of the overall UTUC risk model for better risk stratification and decision-making for AC after RNU. Patient summary: Upper tract urothelial carcinoma comprises renal pelvic and ureteral carcinomas. We recommend using site-specific risk models instead of the overall upper tract urothelial carcinoma risk model in risk prediction and decision-making for adjuvant therapy after radical surgery.

Identification of RPL15 60S Ribosomal Protein as a Novel Topotecan Target Protein That Correlates with DAMP Secretion and Antitumor Immune Activation.

Damage-associated molecular patterns (DAMPs) contribute to antitumor immunity during cancer chemotherapy. We previously demonstrated that topotecan (TPT), a topoisomerase I inhibitor, induces DAMP secretion from cancer cells, which activates STING-mediated antitumor immune responses. However, how TPT induces DAMP secretion in cancer cells is yet to be elucidated. Here, we identified RPL15, a 60S ribosomal protein, as a novel TPT target and showed that TPT inhibited preribosomal subunit formation via its binding to RPL15, resulting in the induction of DAMP-mediated antitumor immune activation independent of TOP1. TPT inhibits RPL15-RPL4 interactions and decreases RPL4 stability, which is recovered by CDK12 activity. RPL15 knockdown induced DAMP secretion and increased the CTL population but decreased the regulatory T cell population in a B16-F10 murine melanoma model, which sensitized B16-F10 tumors against PD-1 blockade. Our study identified a novel TPT target protein and showed that ribosomal stress is a trigger of DAMP secretion, which contributes to antitumor immunotherapy.

STAP-2 Is a Novel Positive Regulator of TCR-Proximal Signals.

TCR ligation with an Ag presented on MHC molecules promotes T cell activation, leading to the selection, differentiation, and proliferation of T cells and cytokine production. These immunological events are optimally arranged to provide appropriate responses against a variety of pathogens. We here propose signal-transducing adaptor protein-2 (STAP-2) as a new positive regulator of TCR signaling. STAP-2-deficient T cells showed reduced, whereas STAP-2-overexpressing T cells showed enhanced, TCR-mediated signaling and downstream IL-2 production. For the mechanisms, STAP-2 associated with TCR-proximal CD3ζ immunoreceptor tyrosine activation motifs and phosphorylated LCK, resulting in enhancement of their binding after TCR stimulation. In parallel, STAP-2 expression is required for full activation of downstream TCR signaling. Importantly, STAP-2-deficient mice exhibited slight phenotypes of CD4+ T-cell-mediated inflammatory diseases, such as experimental autoimmune encephalomyelitis, whereas STAP-2-overexpressing transgenic mice showed severe phenotypes of these diseases. Together, STAP-2 is an adaptor protein to enhance TCR signaling; therefore, manipulating STAP-2 will have an ability to improve the treatment of patients with autoimmune diseases as well as the chimeric Ag receptor T cell therapy.

Regulation of NFKBIZ gene promoter activity by STAT3, C/EBPß, and STAT1.

Interleukin-17A (IL-17A) is a cytokine that affects the functions of non-immune cells, including keratinocytes, and thereby amplifies immune responses. An IκB family protein IκB-ζ, encoded by the NFKBIZ gene, mediates IL-17A-induced inflammatory cellular responses. Previously we reported that a transcription factor STAT3 mediates the transcriptional induction of NFKBIZ through its binding to the specific binding site existing in the NFKBIZ promoter. However, it remains unclear how other transcription factors regulate NFKBIZ transcription. Here, we investigated the NFKBIZ promoter regulation by transcription factors C/EBPß and STAT1 and revealed opposing roles of C/EBPß and STAT1 in NFKBIZ transcription. We found that siRNA-mediated knockdown of C/EBPß attenuates IL-17A-induced upregulation of NFKBIZ in the HaCaT cell line. A putative C/EBP-binding site is located adjacent to the STAT-binding site in the NFKBIZ promoter, the deletion of which abolished C/EBPß-driven promoter activation in transient NFKBIZ promoter-luciferase assay. Deleting the STAT-binding site also led to a reduction in C/EBPß-driven promoter activation, suggesting a cooperative action between C/EBP- and STAT-binding sites. Furthermore, Co-overexpression of STAT1 suppressed both C/EBPß- and STAT3-driven NFKBIZ promoter activation independently of its tyrosine 701 phosphorylation. siRNA-mediated STAT1 knockdown augmented IκB-ζ induction in IL-17A-treated HaCaT cells, with enhanced expression of an IκB-ζ target gene DEFB4A. Together, these results indicate that both C/EBPß and STAT3 are transcription factors that coordinately induce NFKBIZ promoter activation, indicating that STAT1 has an inhibitory role. Thus, these could be a fine-tuning mechanism of IL-17A-IκB-ζ-mediated cellular responses.

Clinical Utility of Germline Genetic Testing in Japanese Men Undergoing Prostate Biopsy.

Background: Multiple common variants and also rare variants in monogenic risk genes such as BRCA2 and HOXB13 have been reported to be associated with risk of prostate cancer (PCa); however, the clinical setting in which germline genetic testing could be used for PCa diagnosis remains obscure. Herein, we tested the clinical utility of a 16 common variant-based polygenic risk score (PRS) that has been developed previously for Japanese men and also evaluated the frequency of PCa-associated rare variants in a prospective cohort of Japanese men undergoing prostate biopsy. Methods: A total of 1336 patients undergoing first prostate biopsy were included. PRS was calculated based on the genotype of 16 common variants, and sequencing of 8 prostate cancer-associated genes was performed by multiplex polymerase chain reaction based target sequencing. PRS was combined with clinical factors in logistic regression models to assess whether addition of PRS improves the prediction of biopsy positivity. Results: The top PRS decile was associated with an odds ratio of 4.10 (95% confidence interval = 2.46 to 6.86) with reference to the patients at average risk, and the estimated lifetime absolute risk approached 20%. Among the patients with prostate specific antigen 2-10 ng/mL who had prebiopsy magnetic resonance imaging, high PRS had an equivalent impact on biopsy positivity as a positive magnetic resonance imaging finding. Rare variants were detected in 19 (2.37%) and 7 (1.31%) patients with positive and negative biopsies, respectively, with BRCA2 variants being the most prevalent. There was no association between PRS and high-risk rare variants. Conclusions: Germline genetic testing could be clinically useful in both pre- and post-PSA screening settings.

Current understanding of the role of tyrosine kinase 2 signaling in immune responses.

Immune system is a complex network that clears pathogens, toxic substrates, and cancer cells. Distinguishing self-antigens from non-self-antigens is critical for the immune cell-mediated response against foreign antigens. The innate immune system elicits an early-phase response to various stimuli, whereas the adaptive immune response is tailored to previously encountered antigens. During immune responses, B cells differentiate into antibody-secreting cells, while naïve T cells differentiate into functionally specific effector cells [T helper 1 (Th1), Th2, Th17, and regulatory T cells]. However, enhanced or prolonged immune responses can result in autoimmune disorders, which are characterized by lymphocyte-mediated immune responses against self-antigens. Signal transduction of cytokines, which regulate the inflammatory cascades, is dependent on the members of the Janus family of protein kinases. Tyrosine kinase 2 (Tyk2) is associated with receptor subunits of immune-related cytokines, such as type I interferon, interleukin (IL)-6, IL-10, IL-12, and IL-23. Clinical studies on the therapeutic effects and the underlying mechanisms of Tyk2 inhibitors in autoimmune or chronic inflammatory diseases are currently ongoing. This review summarizes the findings of studies examining the role of Tyk2 in immune and/or inflammatory responses using Tyk2-deficient cells and mice.

A novel intramolecular negative regulation of mouse Jak3 activity by tyrosine 820.

Jak3, a member of the Janus kinase family, is essential for the cytokine receptor common gamma chain (γc)-mediated signaling. During activation of Jak3, tyrosine residues are phosphorylated and potentially regulate its kinase activity. We identified a novel tyrosine phosphorylation site within mouse Jak3, Y820, which is conserved in human Jak3, Y824. IL-2-induced tyrosine phosphorylation of Jak3 Y824 in human T cell line HuT78 cells was detected by using a phosphospecific, pY824, antibody. Mutation of mouse Jak3 Y820 to alanine (Y820A) showed increased autophosphorylation of Jak3 and enhanced signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and transcriptional activation. Stably expressed Jak3 Y820A in F7 cells, an IL-2 responsive mouse pro-B cell line Ba/F3, exhibited enhanced IL-2-dependent cell growth. Mechanistic studies demonstrated that interaction between Jak3 and STAT5 increased in Jak3 Y820A compared to wild-type Jak3. These data suggest that Jak3 Y820 plays a role in negative regulation of Jak3-mediated STAT5 signaling cascade upon IL-2-stimulation. We speculate that this occurs through an interaction promoted by the tyrosine phosphorylated Y820 or a conformational change by Y820 mutation with either the STAT directly or with the recruitment of molecules such as phosphatases via a SH2 interaction. Additional studies will focus on these interactions as Jak3 plays a crucial role in disease and health.