Fig 1.
Inhibition of Src-family kinases (SFKs) by PP2 enhances distal T cell receptor (TCR) signaling.
(A) Jurkat cell viability was measured following 18 hours treatment with various concentrations of PP2 by trypan blue exclusion method. (B) Activation of SFKs was measured in Jurkat T cells by assessing Lck phosphorylation at tyrosine 394 (Y394) following anti-CD3 stimulation (2 minutes) in presence of various concentrations of PP2, DMSO or culture media (-) for 30 minutes. (C) Quantification of immunoblots from panel B obtained from 3 independent experiments. (D) Immunoblot analysis of phosphorylated Lck (Y394), ZAP-70 (Y319) and LAT (Y226) following 2 minutes of stimulation with isotype control (IgG), anti-CD3 IgG or PMA/Ionomycin. (E) CD25 surface expression was measured in Jurkat T cells following 18 hours of DMSO alone or PP2 treatment and 24 hours of PMA, Ionomycin or PMA/Ionomycin stimulation by flow cytometry. (F) Propidium iodide staining of Jurkat T cells following 18 hours of DMSO or PP2 treatment and 24 hours of PMA/Ionomycin stimulation. US = Unstimulated. Data represent the average of three technical replicates, and the standard deviation is shown. Each experiment was independently performed three times with similar results. *P< 0.01, ns = not significant.
Fig 2.
Inhibition of Src-family kinases (SFKs) enhances distal T cell receptor (TCR) signaling in two different human T cell lines.
Activation of distal TCR signaling was measured in two different human T cell lines by assessing (A, C) IL-2 release and (B, D) CD25 surface expression following DMSO or PP2 (5μg/ml) treatment and PMA/Ionomycin stimulation. Jurkat cells = panels A and B. HuT78 = panels C and D. Data represent the average of three technical replicates, and the standard deviation is shown. Each experiment was independently performed three times with similar results. *P< 0.01.
Fig 3.
Src-family kinases (SFKs) regulate NFAT1 signaling in unstimulated Jurkat T cells.
(A) Immunoblot analysis of Src-kinase Lck and NFAT1 protein expression in Jurkat T cells treated with various concentrations of PP2. (B) Immunoblot analysis of subcellular localization of NFAT1, GAPDH and Lamin in Jurkat T cells treated with DMSO or PP2. GAPDH and Lamin served as a loading control for cytoplasmic and nuclear fractions respectively. (C) Quantification of NFAT1 protein in the nuclear fraction of Jurkat T cells either treated with DMSO or PP2. Amount of NFAT1 protein in the nuclear fraction was normalized by the Lamin protein level. US = unstimulated, P+I = PMA/ Ionomycin. Data represent the average of three technical replicates, and the standard deviation is shown. Each experiment was independently performed three times with similar results. *P< 0.05; **P< 0.01.
Fig 4.
Src-kinase Lck prevents aberrant NFAT1 activation in unstimulated Jurkat T cells.
(A) Immunoblot analysis of Lck expression in Jurkat T cells either expressing (Lck+) or not expressing (Lck-) Src-kinase Lck. GAPDH served as a loading control. (B) NFAT1-dependent firefly luciferase activity (NFAT1-Luc) was measured in Lck+ or Lck- Jurkat T cells. Firefly luciferase activity was normalized to renilla luciferase activity. (C) Immunoblot analysis of subcellular localization of NFAT1, GAPDH and Lamin in Lck+ or Lck- Jurkat T cells. GAPDH and Lamin served as a loading control for cytoplasmic and nuclear fractions respectively. NFAT1 protein levels in the cytoplasmic fraction and nuclear fractions were normalized with GAPDH and Lamin protein levels respectively. (D) Immunoblot analysis of NFAT in nuclear fraction obtained from Lck-deficient (Lck-) and Lck expression rescued (Lck-/Lck) Jurkat T cells. Activation of distal TCR signaling was measured by assessing (E) IL-2 release and (F) CD25 surface expression in Lck+ Jurkat T cells following treatment with DMSO or Lck-specific inhibitor (Lck inhibitor II) and PMA/Ionomycin stimulation. RLU = relative luciferase units. Data represent the average of three technical replicates, and the standard deviation is shown. Each experiment was independently performed three times with similar results. *P< 0.01.
Fig 5.
Active Src-family kinases (SFKs) prevent aberrant NFAT1 nuclear translocation in resting primary human T cells.
(A) Immunoblot analysis of subcellular localization of NFAT1, GAPDH and Lamin in primary human T cells treated with DMSO or PP2. GAPDH and Lamin served as a loading control for cytoplasmic and nuclear fractions respectively. (B) Quantification of the NFAT1 protein in the nuclear fraction of primary human T cells obtained from four healthy blood donors either treated with DMSO or PP2. Amount of NFAT1 protein in the nuclear fraction was normalized by the Lamin protein. Each experiment was independently performed with four different donors with consistent results. *P < 0.01.
Fig 6.
Inhibition of Src-family kinases (SFKs) enhances distal T cell receptor (TCR) signaling in primary human T cells.
Primary human T cells obtained from healthy blood donors were treated with DMSO alone or PP2. Following stimulation with PMA/ Ionomycin, distal TCR signaling was measured by assessing IL-2 (A), IFN-γ (B) release and CD25 surface expression on CD4+ (C) and CD8+ (D) T cells. MFI = mean fluorescent intensity. Data represent the average of three technical replicates, and the standard deviation is shown. Each experiment was independently performed with three different donors with similar results. *P< 0.01.
Fig 7.
Calcineurin inhibitors prevent aberrant NFAT1 nuclear translocation in resting primary human T cells lacking active SFKs.
Primary human T cells obtained from healthy blood donors were treated with DMSO alone or PP2 along with FK506 or Cyclosporin A (CsA). Following subcellular fractionations, NFAT1 protein in the nuclear fraction was quantified and normalized to the nuclear actin levels. Data represent the average obtained from three different donors along with the standard deviation. *P< 0.05, **P< 0.01, Ϯ = average obtained from two donors.
Fig 8.
Proposed model for the role of active SFKs in resting human T cells.
In resting human T cells, a pool of active Src-family kinases (SFKs) phosphorylates the Csk-binding protein (Cbp). Csk bound to the phosphorylated Cbp phosphorylates the majority of SFKs at the inhibitory C-terminal tyrosine. SFKs phosphorylated at the C-terminal inhibitory tyrosine are enzymatically inactive and cannot mediate proximal T cell receptor (TCR) signaling in absence of TCR stimulation. In addition, this active pool of SFKs also prevents aberrant NFAT1 activation and negatively regulates NFAT1-mediated distal TCR signaling in resting human T cells by suppressing calcineurin activity. Together, negative regulation of both proximal and distal TCR signaling by active SFKs contribute to maintain T cells in a quiescent state in absence of TCR stimulation.