An alternative mode of CD43 signal transduction activates pro-survival pathways of T lymphocytes

Abstract

CD43 is one of the most abundant co-stimulatory molecules on a T-cell surface; it transduces activation signals through its cytoplasmic domain, contributing to modulation of the outcome of T-cell responses. The aim of this study was to uncover new signalling pathways regulated by this sialomucin. Analysis of changes in protein abundance allowed us to identify pyruvate kinase isozyme M2 (PKM2), an enzyme of the glycolytic pathway, as an element potentially participating in the signalling cascade resulting from the engagement of CD43 and the T-cell receptor (TCR). We found that the glycolytic activity of this enzyme was not significantly increased in response to TCR+CD43 co-stimulation, but that PKM2 was tyrosine phosphorylated, suggesting that it was performing moonlight functions. We report that phosphorylation of both Y¹⁰⁵ of PKM2 and of Y⁷⁰⁵ of signal transducer and activator of transcription 3 was induced in response to TCR+CD43 co-stimulation, resulting in activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway. ERK5 and the cAMP response element binding protein (CREB) were activated, and c-Myc and nuclear factor-κB (p65) nuclear localization, as well as Bad phosphorylation, were augmented. Consistent with this, expression of human CD43 in a murine T-cell hybridoma favoured cell survival. Altogether, our data highlight novel signalling pathways for the CD43 molecule in T lymphocytes, and underscore a role for CD43 in promoting cell survival through non-glycolytic functions of metabolic enzymes.

Keywords: CD43; T-cell survival; cAMP response element binding protein signalling; extracellular signal-regulated kinase 5; pyruvate kinase isoform M2.

Figure 1

CD43 co‐stimulatory signals induce pyruvate kinase isoform M2 (PKM2) moonlight functions. (a) Quantitative proteomic analysis results were validated in cell lysates from CD4⁺ T cells activated for 6 hr as described in the Experimental Procedures. PKM2 and PKM2 Y¹⁰⁵ phosphorylation was assessed by immunoblot of two‐dimensional gels with samples from three different donors. (b) CD4⁺ T cells (1·5 × 10⁷) were left unstimulated or stimulated through the T‐cell receptor (TCR), CD43, TCR+CD43, or TCR+CD28 for 3 hr and nuclear and cytosolic extracts were prepared. Phosphorylation of PKM2 on Y¹⁰⁵ (pPKM2) and that of signal transducer and activator of transcription (STAT) on Y⁷⁰⁵ was assessed by immunoblot in cytosolic and nuclear extracts. Total extracellular signal‐regulated kinase 2 (ERK2) and histone 3 levels in the cytoplasm and nuclei, respectively, are shown as loading controls. The data shown are representative of three independent experiments. Numbers represent fold increase values. (c) The levels of pERK5 were evaluated in total cell extracts from unstimulated or TCR, CD43 or TCR+CD43 co‐stimulated CD4⁺ T cells for 3 hr. Total tubulin levels are shown as loading control. The data shown are representative of three independent experiments. Numbers represent fold increase values. (d) cAMP response element binding protein (CREB) phosphorylation was assessed by phosphoflow analysis of T cells co‐stimulated for 3 hr with TCR+CD43, TCR+CD28, TCR, or CD43 alone and compared with unstimulated cells. The data shown are representative of three independent experiments. Fold change analysis was based on MFI. (e) The activation of downstream targets of the PKM2/STAT3/ERK5 pathway was assessed after 3 hr stimulation in cytoplasmic or nuclear extracts from unstimulated, TCR, CD43, TCR+CD28, or TCR+CD43 co‐stimulated CD4⁺ T cells, using specific antibodies for phosphorylated Bad (pBad), cyclin D1 and c‐Myc; ERK 2 and histone 3 protein levels are shown as loading control for cytoplasmic and nuclear fractions, respectively. The data shown are representative of three independent experiments. Numbers represent fold increase values. Graphs show fold change in protein phosphorylation or abundance in three donors. P values were considered significant if P ≤ 0·05 (*P <0.05, **P <0.01, and ***P <0.001).

Figure 2

The T‐cell receptor (TCR)+CD43‐induced pyruvate kinase isoform M2 (PKM2)/ signal transducer and activator of transcription (STAT3)/extracellular signal‐regulated kinase 5 (ERK5) pathway targets Bad, nuclear factor‐κB (NF‐κB) and c‐Myc. (a) Cells were pre‐incubated with the ERK5 inhibitor XMD8‐92 (5 μm) 30 min before stimulation for 3 hr with TCR+CD43. Phosphorylation levels of pSTAT3, cAMP response element binding protein (pCREB) and pBad, as well as nuclear c‐Myc and p65 levels, were evaluated by immunoblot; total tubulin and histone 3 levels are shown as loading controls for cytoplasmic and nuclear fractions, respectively. The data shown are representative of three independent experiments. Numbers represent fold increase values. (b) CD4⁺ T lymphocytes were pre‐incubated with the Src inhibitor PP2 (10 μm) 30 min before stimulation with TCR+CD43 or TCR+CD28. ERK5 and Bad phosphorylation was evaluated by immunoblot; ERK2 levels are shown as loading control. Numbers represent fold increase values. (c) Jurkat and JCAM‐1 cells were stimulated with TCR, CD43, or TCR+CD43, for 3 hr and pPKM2, pBad, and pERK5 phosphorylation levels were evaluated by immunoblot; total actin levels are shown as loading controls. The data shown are representative of three independent experiments. Numbers represent fold increase values. Graphs show fold change in protein phosphorylation or abundance in three donors. P values were considered significant if P ≤ 0·05 (*P <0.05, **P <0.01, and ***P <0.001).

Figure 3

T‐cell receptor (TCR)+CD43 signals induce protein kinase A (PKA) ‐dependent cAMP response element binding protein (CREB) activation. (a) Jurkat cells were stimulated with anti‐TCR, and/or anti‐CD43 antibodies for 10 min and pCREB and total CREB levels were measured in whole cell lysates by immunoblot. The data shown are representative of three independent experiments. (b) Jurkat cells were left untreated or pre‐incubated with enzyme inhibitors for MEK 1/2 (PD98059, 8·3 μm), p38 (SB203580, 0·83 μm), protein kinase A (PKA; H89, 8·3 μm), and protein kinase C (PKC; Gö6976, 3·3 nm) before TCR+CD43 co‐stimulation. pCREB levels were evaluated by immunoblot in whole cell lysates after 10 min of stimulation; CREB and Sp1 levels are shown as loading controls. Numbers represent fold change values. The data shown are representative of two independent experiments. (*P <0.05, **P <0.01, and ***P <0.001).

Figure 4

CD43 expression promotes cell survival of activated cells. (a) The murine T‐cell hybridoma BY155.16 was transfected with empty vector (pFneo), full‐length human CD43 (CD43 WT), a mutant lacking the intracellular domain (CD43 ΔIC), or human CD4. The surface expression of each co‐stimulatory molecule was evaluated by FACS analysis (left panel). The molecular weight of the full‐length CD43 (CD43 WT) and truncated CD43 (CD43 ΔIC) was determined by immunoblot (right panel) using the L10 anti‐CD43 monoclonal antibody. Actin levels are shown as loading control. (b) Cells were left unstimulated (ᴓ) or stimulated with PMA (50 ng/ml)/ionomycin (1 μg/ml) (P/I) for 48 hr, and cell viability was measured by quantification of propidium‐iodide‐negative cells. The data shown are representative of four independent experiments. (c) Alternatively, cells were left unstimulated (ᴓ) or stimulated with 4 μg/ml plate‐bound anti‐TCR monoclonal antibody (F23.1) for 48 hr, and cell viability was measured by quantification of propidium iodide‐negative cells. The data shown are representative of three independent experiments. P values were considered significant if P ≤ 0.05, * indicates P values from 0.01 to 0.05.