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. 2006 Jul 10;203(7):1665-70.
doi: 10.1084/jem.20052469. Epub 2006 Jul 3.

Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes

Peter Tamás  1 , Simon A Hawley, Rosemary G Clarke, Kirsty J Mustard, Kevin Green, D Grahame Hardie, Doreen A Cantrell
Affiliations

Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes

Peter Tamás et al. J Exp Med. .

Abstract

The adenosine monophosphate (AMP)-activated protein kinase (AMPK) has a crucial role in maintaining cellular energy homeostasis. This study shows that human and mouse T lymphocytes express AMPKalpha1 and that this is rapidly activated in response to triggering of the T cell antigen receptor (TCR). TCR stimulation of AMPK was dependent on the adaptors LAT and SLP76 and could be mimicked by the elevation of intracellular Ca(2+) with Ca(2+) ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca(2+) stimulation of AMPK required the activity of Ca(2+)-calmodulin-dependent protein kinase kinases (CaMKKs), whereas AMPK activation induced by increased AMP/ATP ratios did not. These experiments reveal two distinct pathways for the regulation of AMPK in T lymphocytes. The role of AMPK is to promote ATP conservation and production. The rapid activation of AMPK in response to Ca(2+) signaling in T lymphocytes thus reveals that TCR triggering is linked to an evolutionally conserved serine kinase that regulates energy metabolism. Moreover, AMPK does not just react to cellular energy depletion but also anticipates it.

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Figures

Figure 1.
Figure 1.
Ca2+ activation of AMPKα1 in T cells. (A) Mouse and human T cells express the AMPKα1 isoform. Data show Western blot (WB) analysis of cell lysates from mouse thymocytes or human and mouse T lymphoblasts probed with antisera specific for AMPKα1 and α2. Protein extracts corresponding to 10 and 20 million mouse thymocytes or 5, 10, and 20 million mouse and human T blast cells were loaded successively on the gel. Cell lysates prepared from mouse muscle extracts were used as a positive control for AMPKα2 expression. (B) Ionomycin but not phorbol ester induces AMPK Thr-172 phosphorylation in human T lymphocytes. Human T cells were unstimulated or treated with 20 ng/ml PdBu or 0.5 μg/ml ionomycin for the indicated time periods (given in minutes). (C) Ionomycin induces AMPK Thr-172 phosphorylation in mouse thymocytes. Mouse thymocytes were unstimulated or treated with 0.5 μg/ml ionomycin in duplicate. (B and C) The data show Western blot analyses of cell lysates prepared from these cells with pThr-172–AMPK or AMPKα1 antisera. (D) Ionomycin and thapsigargin induce AMPK Thr-172 phosphorylation in mouse and human T cells. Mouse and human T lymphocytes were unstimulated (in duplicate for mouse T cells and in triplicate for human cells) or treated with 0.5 μg/ml ionomycin or 500 nM thapsigargin (in duplicate in the case of human cells). Data show Western blot analyses with pThr-172–AMPK or AMPKα1 antisera. (E) Iomomycin but not phorbol ester induces phosphorylation of the AMPK substrate ACC. Human T cells were unstimulated or treated with 20 ng/ml PdBu or 0.5 μg/ml ionomycin in a duplicate experiment. Proteins were separated by SDS-PAGE and Western blotted using a pSer-79–ACC antibody. The quantity of ACC loaded on the gel was measured by its ability to bind streptavidin. Western blots were analyzed by the Odyssey Infrared Imaging System (LI-COR Biosciences). Data show the ratio of phospho-ACC to the total ACC signal.
Figure 2.
Figure 2.
TCR activation of AMPK in human T cells. (A) TCR stimulation induces AMPK Thr-172 phosphorylation in human peripheral blood-derived T cells. Human T cells were unstimulated or treated with 10 μg/ml of the CD3 antibody UCHT1 to cross-link the TCR for the indicated time periods (given in minutes). The data show Western blot (WB) analyses of cell lysates prepared from these T cells with pThr-172–AMPK or AMPKα1 antisera. (B) TCR-stimulated phosphorylation of the AMPK substrate ACC. Human T cells were unstimulated or treated with 10 μg/ml UCHT1, a CD3 cross-linking antibody, for the indicated time periods (given in minutes). Proteins were separated by SDS-PAGE and Western blotted using the pSer79-ACC antibody. The quantity of ACC loaded on the gel was measured by its ability to bind streptavidin. Western blots were analyzed by the Odyssey Infrared Imaging System. Data show the ratio of phospho-ACC to the total ACC signal.
Figure 3.
Figure 3.
Ca2+ and TCR activation of AMPK in Jurkat cells. (A) Ionomycin and thapsigargin induce AMPK Thr-172 phosphorylation in Jurkat cells. Jurkat cells were unstimulated or treated with 0.5 μg/ml ionomycin or 500 nM thapsigargin for the indicated time periods (given in minutes). (B) TCR stimulation of AMPK Thr-172 phosphorylation in Jurkat cells. Jurkat T cells were unstimulated or treated with 10 μg/ml of the CD3 antibody UCHT1 to cross-link the TCR for the indicated time periods (given in minutes). (C) TCR stimulation of AMPK Thr-172 phosphorylation is dependent on LAT and SLP76. Wild-type, Slp76-, or LAT-negative Jurkat cells were unstimulated or treated with 10 μg/ml of the CD3 antibody UCHT1 or 0.5 μg/ml ionomycin for 5 min. (A–C) The data show Western blot (WB) analyses of cell lysates prepared from these T cells with pThr-172–AMPK or AMPKα1 antisera. Black lines indicate that intervening lanes have been spliced out.
Figure 4.
Figure 4.
The CaMKK inhibitor STO-609 inhibits TCR activation of AMPK. (A) AMP/ATP ratios in TCR-triggered T cells are comparable with those in unstimulated cells. Jurkat cells were unstimulated or treated with 10 μg/ml of the CD3 antibody UCHT1 or 50 mM 2-deoxyglucose for 1 and 5 min. The data show the ratios of AMP and ATP intracellular concentrations over the indicated time periods (given in minutes) under different stimulation conditions. (B and C) The CaMKK inhibitor STO-609 inhibits TCR-induced AMPK Thr-172 phosphorylation in human peripheral blood-derived T cells (B) and in Jurkat cells (C). Human T cells (B) and Jurkat T cells (C) were pretreated with STO-609 at the indicated concentrations and were stimulated with the CD3 antibody UCHT1 for 5 min. The data show Western blot (WB) analyses of cell lysates prepared from these T cells with pThr-172–AMPK, AMPKα1 antisera, or pSer-916–PKD antibodies. (D) The CaMKK inhibitor STO-609 inhibits TCR-dependent AMPK activation in human T cells. Human T cells were pretreated with 2.5 μM STO-609 and were stimulated with the CD3 antibody UCHT1 for 5 min. Cells were lysed, and the AMPKα1 subunits were immunoprecipitated both from stimulated and unstimulated cells. Data show the catalytic activity of the immunoprecipitated AMPK in nanomoles/minute/nanogram protein units (mean ± SEM [error bars]; n = 3). (E) The CaMKK inhibitor STO-609 does not inhibit TCR or ionomycin-induced increases in intracellular Ca2+ concentration. Jurkat T cells were labeled with 4.5 μM Indo-1 and treated with 2.5 μM STO-609 as indicated and were stimulated with 10 μg/ml of the CD3 antibody UCHT1 or 0.5 μg/ml ionomycin for the indicated times. [Ca2+]i was then analyzed by flow cytometry. Data show [Ca2+]i over the indicated time periods under different stimulation conditions. (F) 2-deoxyglucose–induced AMPK Thr-172 phosphorylation is resistant to STO-609. Untreated and STO-609–pretreated Jurkat cells were stimulated with 50 mM 2-deoxyglucose or 500 nM thapsigargin for the indicated time periods (given in minutes). Data show Western blot analyses of cell lysates prepared from these T cells with pThr-172–AMPK or AMPKα1 antisera.
Figure 5.
Figure 5.
TCR-induced phosphorylation of AMPK Thr-172 is not prevented by Ly294002. Human T cells were pretreated with either 2.5 μM STO-609 or 10 μM Ly294002 and were stimulated with the CD3 antibody UCHT1 for 5 min. Data show Western blot analyses of cell lysates prepared from these T cells with pThr-172–AMPK, AMPKα1, or phospho-Thr24/Thr32-FOXO1/3 antisera.
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