A scaffold protein, AHNAK1, is required for calcium signaling during T cell activation

Abstract

Engagement of the T cell antigen receptor (TCR) during antigen presentation initiates a coordinated action of a large number of signaling proteins and ion channels. AHNAK1 is a scaffold protein, highly expressed by CD4+ T cells, and is a critical component for calcium signaling. We showed that AHNAK1-deficient mice were highly susceptible to Leishmania major infection. AHNAK1-deficient CD4+ T cells responded poorly to TCR stimulation in vitro with low proliferation and low Interleukin-2 production. Furthermore, AHNAK1 deficiency resulted in a reduced calcium influx upon TCR crosslinking and subsequent poor activation of the transcription factor NFAT. AHNAK1 was required for plasma membrane expression of L-type calcium channels alpha 1S (Cav1.1), probably through its interaction with the beta regulatory subunit. Thus, AHNAK1 plays an essential role in T cell Ca2+ signaling through Cav1 channels, triggered via TCR activation; therefore, AHNAK1 is a potential target for therapeutic intervention.

Figure 1

Expression of AHNAK1 in T cells. (A) Bone marrow, spleen, thymus and lymph nodes were isolated from wild-type and AHNAK1^−/− mice. The expression of AHNAK1 in tissues and CD4 T cells was examined by western blot analysis using anti-AHNAK1-C2 antibody. β-actin was used for loading control. Results are representative of at least three independent experiments. (B) Normal CD4/CD8 composition of AHNAK1^−/− T cells in thymus and spleen. Thymocytes/splenocytes from wild-type and AHNAK1^−/− mice were prepared, stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Results are representative of at least three independent experiments. (C) Normal memory/naïve population of AHNAK1^−/− T cells. Splenocytes from both wild-type and AHNAK1^−/− mice were analyzed by flow cytometry using anti-CD44 and CD62L antibody as in C. Results are representative of at least three independent experiments.

Figure 2

AHNAK1 is required for protection against Leishmania major infection. Wild-type and AHNAK1 mice were challenged in the right hind foot with 10⁶ L. major stationary phase promastigotes. (A) Lesion development (as a ratio of infected foot to non-infected foot) results with time postinfection. Results are representative of at least three independent experiments. (B) At 2 weeks postinfection, four mice per group were sacrificed and parasite burden was determined by limiting dilution assay. Results shown are representative of two independent experiments. (C) wild-type IFNγ and IL-4 levels were measured by ELISA in 96 hrs culture supernatant of purified popliteal lymph node CD4 T cells from infected mice cultured with irradiated wild-type APCs and indicated doses of L.major antigen. Results shown are representative of two independent experiments with 3 infected mice in each group. (D) Macrophages and CD4 T cells were isolated from wild-type mice. The expression of AHNAK1 in these cells was compared by western blot analysis using anti-AHNAK1-C2 antibody. AHNAK1^−/− CD4 T cell extract and β-actin were used as controls for the specificity of the AHNAK1 antibody and loading, respectively. (E) Peritoneal macrophages from wild-type and AHNAK1^−/− mice were infected with L. major in vitro and cultured for 72 hours in the either presence or absence of LPS (200 ng/ml) and IFNγ (300 ng/ml). Internalized parasites were detected by staining with DAPI and counted. Results are representative of at least three independent experiments.

Figure 3

AHNAK1 is required for the proliferation of T cells. (A) Decreased proliferation of AHNAK1^−/− CD4 T cells. CD4 T cells from wild-type and AHNAK1^−/− mice were purified and stimulated in vitro using plate bound anti-CD3 (10 μg/ml), or soluble anti-CD3 (10 μg/ml) and irradiated wild-type splenocytes for indicated periods. [³H] thymidine was added to the culture during the last 8 hours of each time point followed by measurement of the incorporation. Results are representative of at least three independent experiments. (B) Production of IL-2 from wild-type and AHNAK1^−/− CD4 T cells was assessed by ELISA after stimulation with plate bound anti-CD3 antibody (10 μg/ml) for indicated periods. Results are representative of at least three independent experiments. (C) Proliferation of wild-type and AHNAK1^−/− CD4 T cells was assessed by CFSE assay after stimulation with plate bound anti-CD3 antibody (2 μg/ml) with or without addition of exogenous IL-2 into the culture. Results are representative of two independent experiments. (D) [³H] thymidine incorporation assay was performed as A with or without addition of exogenous IL-2. Results are representative of at least three independent experiments..

Figure 4

AHNAK1 is required for calcium signalling and NFAT activation upon TCR stimulation. (A) CD4 T cells from wild-type and AHNAK1^−/− mice were purified and stimulated in vitro using plate bound anti-CD3 (10 μg/ml), anti-CD28 (2 μg/ml) and exogenous IL-2 for 48 hrs. Cells were then washed and incubated with anti-CD3 (10 μg/ml) for 30 min on ice and were subsequently cross-linked by goat anti-hamster Ig antibody (GAH). Calcium concentration was measured by ratiometric method using Fura-2 as a probe. Results are representative of at least three independent experiments. (B) CD4 T cells were stimulated with anti-CD3 (10 μg/ml) and anti-CD28 (2 μg/ml) antibodies for the indicated periods. Nuclear localization of NFATc1 and NFATc2 was examined by western blot analysis using cytoplasmic or nuclear extracts. β-actin was used as internal control. Results are representative of two independent experiments, each with 10 wild-type or AHNAK1^−/− mice. (C) DNA binding of NFAT-c2 was examined using nuclear extracts from wild-type and AHNAK^−/− purified CD4 T cells. (D) [³H] thymidine incorporation assay was performed after stimulation of CD4 cells with plate bound anti-CD3 antibody (2 μg/ml) with or without addition of ionomycin (0.5 μM) into the culture. Results are representative of at least three independent experiments

Figure 5

AHNAK1 is required for membrane expression of Ca_v1 channels. (A) Association of AHNAK1 with the β subunits of Ca_v1 channels. Lysates were prepared from splenocytes of wild-type and AHNAK1^−/− mice and immunoprecipitated with antibodies against β subunit 2, 3 or all βsubunits (β^com). The membrane was blotted with anti-AHNAK1-C2 antibody. Results are representative of two independent experiments. (B) CD4 T cells were stimulated with anti-CD3 (10 μg/ml) and anti-CD28 (2 μg/ml) antibodies and total cell lysates were prepared. The expression of Ca_v1 channels was examined by western blotting with anti-Ca_v1.1 and Ca_v1.2 antibodies. The same membrane was blotted with anti-β-actin antibody for loading control. Results are representative of at least three independent experiments. (C) This experiment was performed as in B with the only difference that IL-2 was added to the culture media. Results are representative of at least three independent experiments. (D) CD4 T Cells were stimulated as in C and Ca_v1.1 and 1.2 α1 subunits expression was examined by real time PCR. Results are representative of at least three independent experiments. p-value represent the difference in expression between wild-type and AHNAK1^−/−(p=0.235 and p=0.366 for Cav1.1 and Ca_v1.2, respectively). (E) Cells were stimulated as in C followed by fractionation of membrane proteins and western blot analysis as described in B. Densitometry represents statistical analysis of three independent experiments (p=0.02 and p=0.25 for membrane and intracellular expression of Ca_v1.1 respectively). Ca_v1.1 membrane protein expression was normalized to pan-cadherin and intracellular expression was normalized to β-actin. A representative western blot used for densitometry is shown.