Intracellular calcium signaling regulates autophagy via calcineurin-mediated TFEB dephosphorylation

Abstract

The transcription-regulating activity of TFEB is dependent on its phosphorylation modification, but the phosphatase(s) involved in TFEB dephosphorylation have remained elusive. It has now become clear that lysosomal calcium signaling activates calcineurin, an endogenous serine/threonine phosphatase, which dephosphorylate TFEB leading to upregulation of autophagy.

Keywords:

• calcineurin
• calcium signaling
• transcription factor EB

Autophagy is a conserved cellular process critical for maintaining cellular homeostasis through the targeting of cytosolic proteins and damaged organelles to the lysosomes for degradation. As a major cellular catabolic system, the autophagic process is tightly controlled. Normally, autophagy occurs constitutively at basal levels but is dramatically stimulated by various extra- and intracellular stresses, such as nutrient or growth factor limitation, oxidative stress, and accumulation of damaged organelles or misfolded proteins.¹ In eukaryotes, the underlying mechanisms for regulating autophagy exist at various levels, from transcriptional activation to post-translational protein modification.² Recent advances in understanding the molecular mechanisms of autophagy have highlighted the importance of transcription machinery, for example, via the regulation of TFEB (transcription factor EB), on autophagy-related gene expression. TFEB is a member of the basic-helix-loop-helix leucine-zipper transcription factor MiTF/TFE family. In the past 5 y, TFEB has been identified as a master regulator of antophagic and lysosomal biogenesis.^3,4 In response to starvation or lysosomal dysfunction, TFEB positively regulates the expression of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network, including ATG9, LC3, SQSTM1, and LAMP1. These genes are involved in the sequential steps of autophagy, from cargo recognition and autophagosome formation to vesicle fusion and substrate degradation.^3,5-7

The transcriptional activity of TFEB is closely related to phosphorylation on specific amino acid residues. Under normal conditions, TFEB is located in the cytoplasm in an inactive, phosphorylated form. However, under specific conditions, when cells are stimulated, such as hunger, drugs, and oxidative stress, TFEB is dephosphorylated and translocated into the nucleus, thus leading to a robust transactivation of its target genes.^6,8 To date, at least 3 different kinases have been shown to phosphorylate TFEB: MTORC1, MAPK1/ERK2, and PRKCB/protein kinase Cβ.⁹ These kinases can phosphorylate multiple serine residues of TFEB, which have distinct effects in regulating its activity. For instance, phosphorylation of Ser142 and Ser211 by MTORC1 and/or MAPK1 is crucial in determining the subcellular localization of TFEB.^3,5,8 In contrast, PRKCB-induced phosphorylation of Ser461, Ser466, and Ser468 is suggested to stabilize TFEB and increases its activity.¹⁰ Among these kinases, MTORC1 has been extensively investigated because it is a major kinase complex that controls cell growth and negatively regulates autophagy. Under conditions of nutrient sufficiency, MTORC1 is active and inhibits autophagy by phosphorylating ATG13 and ULK1, thus repressing ULK1 kinase activity. More importantly, MTORC1 has recently been identified as a key regulator of TFEB function.^5,11,12 In the presence of nutrients, MTORC1 phosphorylates TFEB at the lysosome surface, which promotes the binding of TFEB to YWHA/14-3-3 proteins and inhibits its transport into the nucleus. Conversely, conditions that impair MTORC1 activity reduce TFEB phosphorylation, and increase the accumulation of TFEB in the nucleus, where it orchestrates the expansion of lysosomal and autophagic compartments.¹³

The phosphorylation status of TFEB is not only influenced by the above-mentioned kinases, but also by protein phosphatases. For instance, Torin1, an MTOR inhibitor, can promote TFEB dephosphorylation, and leads to TFEB nuclear localization in human pancreatic cancer cells. In contrast, pre-treatment with calyculin A, a serine/threonine phosphatase inhibitor, prevents Torin1-induced TFEB mobility shift, supporting a role for endogenous serine/threonine phosphatases in the dephosphorylation of TFEB.¹⁴ However, until recently the phosphatase responsible for TFEB dephosphorylation has remained elusive; now it has become clear that calcineurin, an endogenous Ca²⁺-calmodulin-dependent protein phosphatase, is involved in the process of TFEB dephosphorylation. Medina et al. report that lysosomal calcium signaling can induce autophagy and lysosomal biogenesis through calcineurin-mediated TFEB activation (see the punctum in issue 11–6 of Autophagy).¹⁵ To identify the phosphatase that dephosphorylate TFEB, Medina and colleagues tested the effects of the specific inhibition of 231 phosphatases on TFEB translocation, and identified calcineurin as a candidate phosphatase responsible for TFEB dephosphorylation. The authors next analyzed the possible interaction between lysosomal calcium signaling, calcineurin, and TFEB in mammalian cells using these genetic and pharmacological tools. Finally, they found that lysosomal Ca²⁺ release through MCOLN1 (mucolipin 1) activates calcineurin, which dephosphorylates TFEB at Ser211 and Ser142, thus promoting its nuclear translocation.

Ca²⁺ is one of the most important regulators of cell survival and death processes. As a second messenger, Ca²⁺ is able to activate various regulatory proteins such as enzymes and transcriptional factors. Physiological roles of calcium signaling range widely, including the regulation of muscle contraction, neuronal transmission, cellular motility, fertilization, and cell growth and proliferation.¹⁶ The 10,000-fold difference between extracellular and cytosolic Ca²⁺ concentration is maintained by the impermeability of the plasma membrane to Ca²⁺ and by transport mechanisms that remove Ca²⁺ from the cytoplasm. Furthermore, intracellular Ca²⁺ is actively pumped from the cytosol across the plasma membrane and is sequestered in the endoplasmic reticulum, mitochondria, and lysosomes.¹⁷

It is now well established that intracellular Ca²⁺ is one of the regulators of autophagy. Although it is still controversial as to whether calcium is an activator or inhibitor for cell autophagy, accumulating evidence suggests that it has dual roles in autophagy regulation. In healthy cells, a constitutive Ca²⁺ release from the ER through ITPR/inositol-1,4,5-trisphosphate receptor in response to basal levels of inositol-1,4,5-trisphosphate is present. Due to the presence of ER-mitochondria microdomains, this Ca²⁺ will be taken up by mitochondria, where it stimulates mitochondrial bioenergetics, suppressing basal autophagy. However, stress conditions may induce elevation of cytoplasmic Ca²⁺ levels by promoting Ca²⁺ influx into, or inhibiting Ca²⁺ efflux from, the cytoplasm. Subsequently, sustained Ca²⁺ signaling in the cytosol will result in autophagy activation through many effector molecules.^18,19 In this respect, a mechanism for activation of autophagy by inhibition of the MTOR pathway has been proposed. Ca²⁺ released from intracellular stores or imported from the extracellular space via distinct calcium channels activates CAMKK2/CaMKKβ, which phosphorylates and activates AMPK. Activated AMPK inactivates MTORC1, releasing its break on autophagy (Fig. 1).²⁰ In contrast to TFEB regulation mediated by protein kinases, Medina et al. propose a new signaling pathway of calcium–activated transcriptional regulation by protein phosphatases. Calcineurin, also known as protein phosphatase 2B, is a serine/threonine-specific protein phosphatase that is active only after binding calmodulin in response to elevated cytosolic Ca²⁺. Calcineurin is ubiquitously distributed in eukaryotes, with high levels in most types of neurons and lymphocytes. Calcineurin participates in a number of cellular processes and Ca²⁺-dependent signal transduction pathways.

Figure 1. Regulation of TFEB activity by intracellular calcium signaling. Extra- and intracellular stimuli induce elevation of cytoplasmic Ca²⁺ levels by releasing Ca²⁺ from the lysosome. Elevated cytosol Ca²⁺ can suppress MTORC1-dependent pathways, and (or) activate calcineurin-dependent pathways, subsequently inducing dephosphorylation of TFEB, and resulting in the activation of autophagy.

Like autophagy, disturbance of intracellular calcium homeostasis is a key molecular event that determines cell survival and cell death. Increasing evidence supports the idea that both dysfunction of autophagy and deregulation of calcineurin signaling have been implicated in the pathogenesis of several human diseases, including cancer, neurodegeneration, and infectious diseases.^21–25 The study by Medina et al. has demonstrated an interplay between calcium and autophagy, in which calcium can indirectly drive the transcription of autophagy-related genes through the calcineurin-dependent dephosphorylation and nuclear translocation of TFEB (Fig. 1).

Medina et al.'s work reveals an important mechanism that regulates autophagy. Meanwhile, their study also sets the stage for further investigation. Open questions include the following: This research suggests that calcium derived from lysosomes can activate TFEB under starvation conditions and during physical exercise. However, some pathological conditions, especially oxidative stress and toxic stimuli can induce a calcium increase from the extracellular space and other organelles. Whether the nonlysosomal calcium signaling can also activate TFEB still remains unclear. In addition to the aforementioned dephosphorylation modification, there is the possibility of other machinery, through which calcium activates TFEB transcription. A recent study confirmed that both NR1H4/FXR and CREB can regulate expression of TFEB.²⁶ Moreover, Singh et al. found that CRE activation involves Ca²⁺-calcineurin-dependent nuclear translocation of transducers of regulated CREBBP (CREB binding protein).²⁷ These studies provide some new clues with regard to the function of calcium-mediated TFEB regulation via transcription factor networks. Studies addressing these questions will help to clarify the role of calcium in autophagy.

Collectively, Medina and colleagues have identified a novel mechanism that results in a prolonged and sustained activation of autophagy. Autophagy dysfunction has been linked to several human disorders and enhancement of autophagy can have a therapeutic effect in certain animal models. This opens the possibility of using Ca²⁺ channel modulators in control of autophagy for therapeutic purposes.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by The Fundamental Research Funds of Shandong University (No. 2014JC018).