Negative Regulation of Zap70 by Lck Forms the Mechanistic Basis of Differential Expression in CD4 and CD8 T Cells

Abstract

Lck and Zap70, two non-receptor tyrosine kinases, play a crucial role in the regulation of membrane proximal TCR signaling critical for thymic selection, CD4/CD8 lineage choice and mature T cell function. Signal initiation upon TCR/CD3 and peptide/MHC interaction induces Lck-mediated phosphorylation of CD3 ITAMs. This is necessary for Zap70 recruitment and its phosphorylation by Lck leading to full Zap70 activation. In its native state Zap70 maintains a closed conformation creating an auto-inhibitory loop, which is relieved by Lck-mediated phosphorylation of Y315/Y319. Zap70 is differentially expressed in thymic subsets and mature T cells with CD8 T cells expressing the highest amount compared to CD4 T cells. However, the mechanistic basis of differential Zap70 expression in thymic subsets and mature T cells is not well understood. Here, we show that Zap70 is degraded relatively faster in DP and mature CD4 T cells compared to CD8 T cells, and inversely correlated with relative level of activated Zap70. Importantly, we found that Zap70 expression is negatively regulated by Lck activity: augmented Lck activity resulting in severe diminution in total Zap70. Moreover, Lck-mediated phosphorylation of Y315/Y319 was essential for Zap70 degradation. Together, these data shed light on the underlying mechanism of Lck-mediated differential modulation of Zap70 expression in thymic subsets and mature T cells.

Keywords: CD4/CD8 T cells; Lck kinase; TCR signaling; Zap70 kinase; negative regulation.

Figure 1

Differential Zap70 stability in thymic and splenic T cells. (A) shows a representative example of CD4/CD8 staining and Zap70 histogram of splenic CD4 and CD8 T cells from WT mice and bar graph comparing Zap70 mean fluorescent intensity (MFI) normalized to Zap70 expression in CD8 T cells (n=16 mice in 6 or more independent experiments). Staining in TCR^-ve cells serves as control for Zap70 specific staining. (B) QPCR analysis of Zap70 transcript in purified WT splenic CD4 and CD8 T cells. Average of triplicate for two biological replicate and SD is shown. Data normalized to Zap70 value in CD4. (C) Zap70 staining for DP and CD4⁺CD8^lo (gated for total thymocytes) and CD4⁺ and CD8⁺ SP thymocytes (gated for CD24^loTCRβ^hi) thymocytes is shown. Bar graph shows compilation of Zap70 MFI for various thymic subsets normalized to Zap70 MFI in CD8 SP thymocytes (n ≥ 10 in 6 or more independent experiments). (D) Zap70 transcription in purified thymic subsets was determined by QPCR and normalized to that in DN thymocytes. An average of triplicate for two biological replicate and SD is shown. (E) Kinetics of Zap70 expression in thymocytes was determined by flow cytometry. Thymocytes were cultured in the presence of cyclohexamide for the indicated time point, surface stained followed Zap70 intracellular stain. MFI for each time point was normalized to 0 hr time point. Kinetics of Zap70 protein degradation in unactivated (F) and activated (G) purified CD4 and CD8 T cells in the presence of cyclohexamide was determined by Western blot. Graph shows ratio of Zap70/actin band intensity for each time point normalized to 0 hr time point. Data representative of two independent experiments (B, D, E, G) or three experiments (F). Frequency of cells and MFI values are shown in FACS plots and histograms (A, C) *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005, *****p < 3.9 × 10-6 (10 to power -6).

Figure 2

Zap70 stability inversely correlates with relative phosphorylation of the protein in thymocytes and mature T cell subsets. Histogram shows a representative example of phospho staining of thymic (A) and splenic (B) T cell subsets from WT mice. Bar graph shows compilation of relative amount of total Zap70 and pY493 and pY319 Zap70 (expressed as ratio of phospho MFI over total Zap70 MFI) normalized to that in CD8 SP thymocytes or CD8 splenic T cells. MFI values are shown in the histograms (6 mice from 3 independent experiments) *p < 0.05, **p < 0.005, and ***p < 0.0005.

Figure 3

Elevated Lck activity negatively affects Zap70 expression. (A) 293T cells stably expressing hZap70-GFP fusion were transfected with plasmid expressing control, WT-Lck, constitutively active Lck^Y505F or kinase-dead Lck^K273R fused to mCherry. Histograms show GFP expression in mCherry-ve and mCherry+ve cells. Bar graph shows average of ratio of GFP expression in mCherry+/mCherry- cells normalized to that in control transfectants for three independent experiments. (B) Histogram shows a representative GFP expression and pY319 Zap70 staining in NGFR+ve 293T cells transfected with control, Lck^WT or Lck^Y505F plasmids. Bar graph shows an average of ratio of MFI for pY319 to GFP normalized to that for control transfectants in NGFR+ve and NGFR-ve cells from two independent experiments. (C) CD4 SP thymocyte cell line RLM was transduced with lentivirus expressing control, WT-Lck, constitutively active Lck^Y505F or kinase-dead Lck^K273R fused to mCherry. Zap70 expression in RLM cells expressing or not mCherry was determined. A representative histogram for Zap70 staining in mCherry- and mCherry+ cells is shown. Bar graph shows average of ratio of mCherry+/mCherry- cells normalized to control for three independent experiments. Error bar represents S.D *p < 0.05, ***p < 0.0005, ****p < 0.00005.

Figure 4

Constitutively active Lck results in Zap70 diminution in vivo. (A) shows CD24/TCR staining of total thymocytes and CD4/CD8 profile of mature thymocytes (CD24^loTCR⁺) from the indicated mice. Histograms show Zap70 staining and bar graph shows comparison of relative Zap70 expression in DP, CD4⁺CD8^lo, CD4⁺ and CD8⁺ thymocytes from OTI mice expressing or not Lck^Y505F transgene. Zap70 expression was normalized to that in CD8⁺ thymocytes from OTI control mice in each experiment. (B) CD4/CD8 profile and Zap70 staining of CD4 and CD8 T cells from OTI mice expressing or not Lck^Y505F is shown. Bar graph shows compilation of Zap70 MFI in the indicated T cell subsets from OTI and OTI⁺Lck^Y505F mice normalized to that in CD8 T cells from OTI mice in each experiment. (C) Western blot shows a representative example of Zap70 and actin expression in total lysates of purified T cells from control (lane 1) and Lck^Y505F (lane 2) expressing OTI mice. Middle panel shows anti-Ubiquitin Western blot of Zap70 immunoprecipiates of purifies splenic T cells from OTI (lane 2) and OTI⁺Lck^Y505F (lane 3) mice. Lane 1 is anti-Zap70 antibody control. Total lysates was probed for actin and serves as control for cell numbers. (n=2 independent experiments). (D) Histograms for total, pY319 and pY493 Zap70 staining in CD4 and CD8 T cells from the indicated mice is shown. Bar graph shows relative pY319 and pY493 value expressed as ratio of MFI for phospho staining to total Zap70 staining for CD4 and CD8 T cells from OTI⁺Lck^Y505F mice (filled bars) and normalized to that in CD8 T cells from OTI mice (open bar). Data are representative example of 6 (A, B) or 3 (D) independent experiments. Numbers in FACS plots and histograms represent frequency of cells and MFI values *p < 0.05, **p < 0.005, and ****p < 0.00005.

Figure 5

Diminution of Zap70 expression in thymocytes and mature T cells in MHCII^-/- mice expressing Lck^Y505F transgene. (A) CD24/TCR and CD4/CD8 staining of, respectively, total and mature (CD24^loTCR^hi) thymocytes is shown. Histograms show Zap70 staining and bar graph compares relative Zap70 expression in the indicated thymic subsets from MHCII^-/- mice expressing or not Lck^Y505F. (B) shows TCR and CD4/CD8 staining of splenic T cells, histograms for Zap70 staining and data compilation for relative Zap70 expression in CD4 and CD8 T cells from MHCII^-/- mice expressing or not Lck^Y505F transgene. Data are representative example of 4 or more independent experiments (n > 6 mice). *p < 0.05, **p < 0.005, ***p < 0.0005.

Figure 6

Constitutive Thpok expression does not alter Zap70 expression. (A) CD24/TCR and CD4/CD8 staining of, respectively, total and mature thymocytes and Zap70 histograms for the indicated thymic subsets from OTI and OTI⁺Th-163⁺ mice is shown. Bar graph shows comparison of relative Zap70 MFI in the indicated thymic subsets from OTI mice expressing or not Th-163 transgene. (B) shows TCR and CD4/CD8 staining of splenic T cells and Zap70 expression in CD4 and CD8 T cells from OTI and OTI⁺Th-163⁺ mice. Bar graph shows relative Zap70 expression in CD4 and CD8 T cells from the indicated mice (n>4 mice in three independent experiments). Frequency of cells and MFI values are shown in FACS plots and histograms.

Figure 7

Phosphorylation of Y315/Y319 in Zap70 is required for Lck-induced Zap70 degradation. 293T cells were co-transfected with control, Lck^WT or Lck^Y505F (expressing NGFR reporter) and Zap70^WT, Zap70^Y319F or Zap70^YYFF (expressing ZsGreen reporter) plasmids. Histograms show Zap70 expression in NGFR+ZsGreen+ 293T cells. Bar graph shows compilation of relative Zap70 expression in the indicated 293T transfectants from three independent experiments. *p < 0.05, **p < 0.005, ***p < 0.0005.

Figure 8

Model of Lck-mediated regulation of Zap70 expression/activation in T cell activation. In DP thymocytes higher amount of Zap70 is associated with surface TCR complex compared with mature T cells (ref 39) likely due to higher Lck activity resulting from CD4 and MHCII interaction (ref 15). We propose that the closed auto-inhibited conformation of Zap70 is stable and upon its recruitment to phosphor-CD3 chains Lck-mediated phosphorylation of Y315/Y319 and Y493 results in partially or fully active Zap70 essential for signal transduction during thymic maturation and mature T cell activation. However, partially or fully active Zap70 must be rapidly degraded to prevent potentially dominant negative effect of partially active Zap70 or over-activation during thymic development or mature T cell response to ligand. Partially or fully open but free (not associated with phospho-CD3) Zap70 may be rapidly degraded as well thereby preventing undesired antigen independent signaling during thymic development. It is quite possible that association of partially open Zap70 with phospho-CD3 may protect it from degradation.