Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling

Smith, Geoffrey A.; Uchida, Kenji; Weiss, Arthur; Taunton, Jack

doi:10.1038/nchembio.2056

Cited by 79 publications

(87 citation statements)

References 34 publications

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“…(2015) reported irreversible JAK3 inhibitors showing good isoform selectivity but also potent off-target activity in the remaining kinome. Merck patented several irreversible acrylamide-based inhibitors (Ahearn et al., 2013), and recently Smith et al. (2016) utilized an inhibitor from this structural class for comprehensive investigation of the time dependency of JAK1/3 signaling in T cells.…”

Section: Introductionmentioning

confidence: 85%

Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket

Förster

Chaikuad

Bauer

et al. 2016

Cell Chemical Biology

107

126

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SummaryJanus kinases (JAKs) are a family of cytoplasmatic tyrosine kinases that are attractive targets for the development of anti-inflammatory drugs given their roles in cytokine signaling. One question regarding JAKs and their inhibitors that remains under intensive debate is whether JAK inhibitors should be isoform selective. Since JAK3 functions are restricted to immune cells, an isoform-selective inhibitor for JAK3 could be especially valuable to achieve clinically more useful and precise effects. However, the high degree of structural conservation makes isoform-selective targeting a challenging task. Here, we present picomolar inhibitors with unprecedented kinome-wide selectivity for JAK3. Selectivity was achieved by concurrent covalent reversible targeting of a JAK3-specific cysteine residue and a ligand-induced binding pocket. We confirmed that in vitro activity and selectivity translate well into the cellular environment and suggest that our inhibitors are powerful tools to elucidate JAK3-specific functions.

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Section: Introductionmentioning

confidence: 85%

Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket

Förster

Chaikuad

Bauer

et al. 2016

Cell Chemical Biology

107

126

View full text Add to dashboard Cite

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“…Inhibitors were used at the following concentrations: Syk inhibitor Bay 61–3606 10µM (19, 22), cyclosporine A 20µM (23, 24), Go-6983 40nM (19), PP2 20µM (19, 25), purchased from Calbiochem and cycloheximide (CHX) 10µg ml −1 (Sigma); pan-PI(3)K inhibitor GDC-0941 500nM and mTOR inhibitor INK128 200nM (26, 27) (gift of K. Shokat, UCSF); pan-Jak inhibitor tofacitinib (CP-690,550) 1µM (26), Go-6976 40nM (28), MEK inhibitor PD0325901 100nM (29) and AKT inhibitor MK2206 1µM (26) were purchased from Selleck Chemicals (Houston, TX) and Rapamycin 10nM (23) from Gemini Bio-Products. DMSO was used at 0.1% as a vehicle control in both unstimulated and stimulated samples without inhibitor treatment.…”

Section: Methodsmentioning

confidence: 99%

Endogenous Nur77 Is a Specific Indicator of Antigen Receptor Signaling in Human T and B Cells

Ashouri

Weiss

2017

The Journal of Immunology

Self Cite

174

178

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Distinguishing true antigen stimulated lymphocytes from bystanders activated by the inflammatory milieu has been difficult. Nur77 is an immediate early gene whose expression is rapidly up-regulated by T cell receptor (TCR) signaling in murine T cells and human thymocytes. Nur77-GFP transgenes serve as specific TCR and B cell receptor (BCR) signaling reporters in murine transgenic models. In this study, we demonstrate that endogenous Nur77 protein expression can serve as a reporter of TCR and BCR specific signaling in human PBMCs. Nur77 protein levels were assessed by immunofluorescence and flow cytometry in T and B cells isolated from human PBMCs obtained from healthy donors that had been stimulated by their respective Ag receptors. We demonstrate that endogenous Nur77 is a more specific reporter of antigen-specific signaling events than the commonly used CD69 activation marker in both human T and B cells. This is reflective of the disparity in signaling pathways that regulate the expression of Nur77 and CD69. Assessing endogenous Nur77 protein expression has great potential to identify antigen-activated lymphocytes in human disease.

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“…Peptides: OTII-specific ovalbumin (OVA 323–339 ) peptide; OTI-specific ovalbumin (OVA257-264) peptide (aka SIINFEKL/“N4”), as well as altered peptide ligands “Q4R7”, and “G4” have been previously described (13), as have MCC and altered peptide ligands “K99E” and “T102L”(14) (Genscript). Inhibitor: Jak3i (15). …”

Section: Methodsmentioning

confidence: 99%

IL-2 Modulates the TCR Signaling Threshold for CD8 but Not CD4 T Cell Proliferation on a Single-Cell Level

Au-Yeung

Smith

Mueller

et al. 2017

The Journal of Immunology

Self Cite

101

106

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Lymphocytes integrate antigen and cytokine receptor signals to make cell fate decisions. Using a specific reporter of TCR signaling that is insensitive to cytokine signaling, Nur77-eGFP, we identify a sharp, minimal threshold of cumulative TCR signaling required for proliferation in CD4 and CD8 T cells that is independent of both antigen concentration and affinity. Unexpectedly, IL-2 reduces this threshold in CD8 but not CD4 T cells, suggesting that integration of multiple mitogenic inputs may alter the minimal requirement for TCR signaling in CD8 T cells. Neither naïve CD4 nor naïve CD8 T cells are responsive to low doses of IL-2. We show that activated CD8 T cells become responsive to low doses of IL-2 more quickly than CD4 T cells, and propose that this relative delay in turn accounts for differential effects of IL-2 on the minimal TCR signaling threshold for proliferation in these populations. In contrast to Nur77-eGFP, c-Myc protein expression integrates mitogenic signals downstream of both IL-2 and the TCR, yet marks an invariant minimal threshold of cumulative mitogenic stimulation required for cell division. Our work provides a conceptual framework for understanding regulation of clonal expansion of CD8 T cells by sub-threshold TCR signaling in the context of mitogenic IL-2 signals, thereby rendering CD8 T cells exquisitely dependent upon environmental cues. Conversely, CD4 T cell proliferation requires an invariant minimal intensity of TCR signaling that is not modulated by IL-2, thereby restricting responses to low affinity or low abundance self-antigens even in the context of an inflammatory milieu.

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Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling

Cited by 79 publications

References 34 publications

Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket

Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket

Endogenous Nur77 Is a Specific Indicator of Antigen Receptor Signaling in Human T and B Cells

IL-2 Modulates the TCR Signaling Threshold for CD8 but Not CD4 T Cell Proliferation on a Single-Cell Level

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