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

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 AMPKα1 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 Ca2+ with Ca2+ ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca2+ stimulation of AMPK required the activity of Ca2+–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 Ca2+ 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.

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 AMPK 1 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.
[Ca 2+ ] i -CaMKK pathways regulate AMPK in T cells has not been examined, but it is an important question because of the key role for AMPK as a regulator of cellular energy balance. AMPK activation by the TCR would be a mechanism to stimulate the conservation and production of ATP in anticipation of the demand for ATP that is invariably initiated by Ca 2+ -mediated signaling pathways. In this context, a previous study has identifi ed phosphatidylinositol-3,4,5-bisphosphate (PIP 3 ), the product of phosphatidylinositol 3-kinases (PI3Ks), and PIM serine kinases as important regulators of T lymphocyte metabolism (18). However, it is likely that T cells will need to use diverse mechanisms to cope with the energy demands of an immune response. This study shows that triggering of the TCR results in the rapid activation of AMPK via a Ca 2+ -CaMKK-dependent pathway. The data provide novel insight that Ca 2+ signaling in T cells regulates an evolutionally conserved kinase that controls the conservation and production of ATP. analysis of cell lysates from mouse thymocytes or human and mouse T lymphoblasts probed with antisera specifi c 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. Fig. 1 A shows that mouse and human T cells express the AMPKα1 catalytic domain but do not express detectable levels of AMPKα2. Pharmacological agents that elevate [Ca 2+ ] i (e.g., Ca 2+ ionophores) can mimic many aspects of antigen receptor triggering and are widely used to probe the Ca 2+ signaling pathways in lymphocytes. Thus, to investigate the possible Ca 2+ regulation of AMPK in T cells, primary human T lymphoblasts were stimulated with the Ca 2+ ionophore ionomycin and monitored for the phosphorylation of Thr-172 in the activation loop of AMPKα1, a marker of AMPK activation. The data show that ionomycin induced the rapid and sustained phosphorylation of AMPK Thr-172 ( Fig. 1 B). Phosphorylation of Thr-172 was not induced when T cells were stimulated with the phorbol ester (phorbol 12,13-dibutyrate [PdBu]), which mimics the action of DAG. Ionomycininduced Thr-172 phosphorylation was also seen in mouse thymocytes ( Fig. 1 C) and peripheral T cells ( Fig. 1 D). Thapsigargin, which inhibits the ER Ca 2+ -ATPase pump and, thereby, promotes Ca 2+ release from the ER, also induced Thr-172 AMPK phosphorylation (Fig. 1 D). Further experiments examined the impact of elevating intracellular Ca 2+ levels on the phosphorylation of the AMPK substrate Ser-79 in acetyl-CoA carboxylase (ACC). Fig. 1 E shows that ionomycin but not phorbol ester induced ACC phosphorylation. Phorbol esters induce a wide range of energy-consuming processes in lymphocytes, including changes in cell adhesion, motility, and gene expression. Accordingly, the failure of phorbol esters to induce AMPK or ACC phosphorylation indicates that this is a selective response to the elevation of [Ca 2+ ] i and is not a response to T cell activation per se.

TCR activation of AMPK
Increased [Ca 2+ ] i is an immediate response to TCR triggering. Fig. 2 A shows that TCR triggering of human T cells rapidly increased AMPK Thr-172 phosphorylation and induced phosphorylation of the AMPK substrate ACC on Ser-79 (Fig. 2 B). The human T leukemic cell line Jurkat has low basal levels of AMPK Thr-172 phosphorylation, but this is

BRIEF DEFINITIVE REPORT
readily induced when cells are stimulated with ionomycin, thapsigargin, or via the TCR (Fig. 3, A and B). To probe the signaling pathways used by the TCR to regulate AMPK, we examined AMPK activity in Jurkat cells that lack expression of the adapters LAT or SLP76. These molecules coordinate the assembly of the PLCγ1 signaling complex and are required for the TCR to increase [Ca 2+ ] i (19,20). Fig. 3 C shows that TCR ligation does not induce the phosphorylation of AMPK on Thr-172 in SLP76-or LAT-defi cient Jurkat cells, whereas ionomycin activated AMPK in both cell clones. The failure of TCR activation of AMPK in Jurkat cells with defective TCR-mediated Ca 2+ fl ux is consistent with a model whereby the TCR regulates AMPK via a Ca 2+regulated mechanism. An alternative possibility is that TCR activates AMPK because it increases cellular AMP/ATP ratios. To address this possibility, we directly measured cellular ATP and AMP concentrations in T cells stimulated via TCR. Fig. 4 A shows that 2-deoxyglucose, which is known to deplete cellular ATP, rapidly increases the cellular AMP/ATP ratio in T cells, whereas triggering of the TCR complex did not. AMPK can thus be activated by energy stress that causes ATP depletion, but the ability of TCR to activate AMPK is not secondary to ATP depletion.

The CaMKK inhibitor STO-609 inhibits TCR activation of AMPK
To examine the role of CaMKK in AMPK regulation in T cells, experiments with the CaMKK selective inhibitor STO-609 were performed. Fig. 4 (B-D) shows that STO-609 prevents TCR-mediated increases in AMPK activity. STO-609 did not prevent the TCR-induced activation of protein kinase D (PKD; Fig. 4 B). PKD activation is absolutely dependent on PLCγ-mediated increases in intracellular DAG, and an intact PKD response demonstrates that STO-609 does not aff ect PLC regulation. Similarly, the data in Fig. 4 E show that STO-609 did not prevent TCR-mediated increases in [Ca 2+ ] i . STO-609 also had no eff ect on the activation of AMPK induced by 2-deoxyglucose, which regulates AMPK via LKB1 (Fig. 4 F).

TCR activation of AMPK is not PI3K dependent
Jurkat cells do not express PTEN (phosphatase and tensin homologue deleted on chromosome 10) and have high basal levels of PIP 3 and constitutive activation of PKB-Akt. PI3Kmediated signaling pathways, specifi cally PKB-Akt, regulate T cell metabolism (21,22), but the low basal activity of AMPK in Jurkat cells argues that the elevation of PIP 3 is not suffi cient to activate this kinase (Fig. 3, A and B). Fig. 5 also shows that TCR-induced phosphorylation of AMPK Thr-172 was not sensitive to the PI3K inhibitor LY294002, whereas this inhibitor did suppress the TCR-induced phosphorylation of FOXO3A on the PKB-Akt site Thr-32. Conversely, STO-609 treatment, which inhibited the TCRinduced phosphorylation of AMPK, did not block the TCRmediated phosphorylation of FOXO3A. The PI3K insensitivity of AMPK activation in T cells argues that the activation of AMPK via TCR is a primary response to T cell activation and is not an indirect response to TCR-induced energy depletion.
In summary, in T lymphocytes, triggering of TCR activates AMPK, a key regulator of cellular energy homeostasis. TCR regulation of AMPK is mediated via a [Ca 2+ ] i -CaMKK pathway and is not a response to energy stress. Thus, in T cells, AMPK does not function solely to restore energy balance after the depletion of energy stores. Rather, increases in [Ca 2+ ] i that activate CaMKKs stimulate AMPK to promote the conservation and accelerated production of ATP in anticipation of energy supplies becoming depleted. The ability to anticipate energy-intensive processes would be advantageous for cells that need to rapidly respond to an increased demand for ATP. In this context, triggering of the TCR initiates an energy-demanding program that is only successful when cellular energy production satisfi es the biosynthetic demands of an immune response. T cells will need to use diverse mechanisms to cope with the energy demands of an immune stimulus, and Ca 2+ activation of AMPK would allow a rapid activation of ATP production before the onset of cell proliferation and diff erentiation.