The mucolipidosis IV Ca2+ channel TRPML1 (MCOLN1) is regulated by the TOR kinase - PubMed (original) (raw)

. 2015 Sep 15;470(3):331-42.

doi: 10.1042/BJ20150219. Epub 2015 Jul 20.

Affiliations

The mucolipidosis IV Ca2+ channel TRPML1 (MCOLN1) is regulated by the TOR kinase

Rob U Onyenwoke et al. Biochem J. 2015.

Abstract

Autophagy is a complex pathway regulated by numerous signalling events that recycles macromolecules and may be perturbed in lysosomal storage disorders (LSDs). During autophagy, aberrant regulation of the lysosomal Ca(2+) efflux channel TRPML1 [transient receptor potential mucolipin 1 (MCOLN1)], also known as MCOLN1, is solely responsible for the human LSD mucolipidosis type IV (MLIV); however, the exact mechanisms involved in the development of the pathology of this LSD are unknown. In the present study, we provide evidence that the target of rapamycin (TOR), a nutrient-sensitive protein kinase that negatively regulates autophagy, directly targets and inactivates the TRPML1 channel and thereby functional autophagy, through phosphorylation. Further, mutating these phosphorylation sites to unphosphorylatable residues proved to block TOR regulation of the TRPML1 channel. These findings suggest a mechanism for how TOR activity may regulate the TRPML1 channel.

Keywords: adenosine 5′-phosphate (AMP)-activated protein kinase; autophagy; lysosomal storage disease; mammalian target of rapamycin; mucolipidosis type IV; transient receptor potential channels.

© 2015 Authors; published by Portland Press Limited.

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Figures

Figure 1

Figure 1. The phosphorylation state of C-terminal tail serine residues of TRPML1 (Ser572 and Ser576) regulate autophagy

(A) MS identified unique phosphopeptides in the complete sequence of TRPML1 (UniProtKB–Q9GZU1) that are absent after rapamycin treatment. The identified phosphopeptide sequences are in green, phospho-residues are underlined in red and unique peptides absent when rapamycin was used during cell culture are boxed in magenta. The six predicted transmembrane domains of TRPML1 are included underlined in blue. (B) HEK293T cells transfected with either EGFP-tagged WT–MCOLN1; the S51E construct, MCOLN1S51E; or the S572E/S576E construct, MCOLN1S572E/S576E were treated with or without lysosomal PIs [E-64d + Pepstatin A (10 μg/ml of each)] for 12 h. The cells were then lysed and immunoblotted with anti-LC3, anti-GFP [to quantify the GFP–MCOLN1 constructs (∼93 kDa)] and anti-α-tubulin (_n_=4). (C) The quantification of the ratio of LC3BII to LC3BI from (B). A ratio of ‘1’ would indicate equal quantities of LC3BII and LC3BI. (D) The quantification of the relative ratio of GFP to α-tubulin.

Figure 2

Figure 2. The S572E/S576E TRPML1 phosphomimetic is non-responsive to TRPML1 agonists

Intracellular Ca2+ ([Ca2+]i) was investigated using HEK293T cells transfected with either (A) EGFP-tagged WT–MCOLN1; (B) the S572A/S576A construct, MCOLN1S572A/S576A; or (C) the S572E/S576E construct, MCOLN1S572E/S576E and the TRPML1 agonist ML-SA1. TRPML1 protein expression was monitored by the presence of an EGFP signal measured at an excitation of 488 nm (_F_488). [Ca2+]i was monitored with Fura-2 ratios (_F_340/_F_380). The extracellular Ca2+ ([Ca2+]o) was 2 mM and the [ML-SA1] was 10 μM. (D–F) In WT–MCOLN1- and MCOLN1S572A/S576A -transfected cells (D and E), the Fura-2 ratios increased in response to ML-SA1 and gradually recovered. The change in the Fura-2 ratio for MCOLN1S572E/S576E (F) was negligible.

Figure 3

Figure 3. The TOR agonist MHY1485 can deactivate the TRPML1 channel through the phosphorylation of Ser572 and Ser576

Intracellular Ca2+ ([Ca2+]i) was investigated in HEK293T cells transfected with either (A) EGFP-tagged WT–MCOLN1; (B) the S572A/S576A construct, MCOLN1S572A/S576A; or (C) the S572E/S576E construct, MCOLN1S572E/S576E. TRPML1 protein expression was monitored by the presence of an EGFP signal measured at an excitation of 488 nm (_F_488). [Ca2+]i was monitored with Fura-2 ratios (_F_340/_F_380). The extracellular Ca2+ ([Ca2+]o) was 2 mM and the [MHY1485] was 10 μM. (D–F) In the WT–MCOLN1-transfected cells (D), the Fura-2 ratios decreased in response to MHY1485 and gradually recovered whereas the Fura-2 ratios of the MCOLN1S572A/S576A -transfected cells (E) were not affected. The change in the Fura-2 ratios for MCOLN1S572E/S576E (F) was negligible; however, these ratios were well below the MCOLN1S572A/S576A -transfected cells and the recovered WT–MCOLN1-transfected cells.

Figure 4

Figure 4. S572A/S576A TRPML1 and the S572E/S576E TRPML1 phosphomimetic are non-responsive to rapamycin

Intracellular Ca2+ ([Ca2+]i) was investigated using HEK293T cells transfected with either (A) EGFP-tagged WT–MCOLN1; (B) the S572A/S576A construct, MCOLN1S572A/S576A; or (C) the S572E/S576E construct, MCOLN1S572E/S576E and the TOR antagonist rapamycin. TRPML1 protein expression was monitored by the presence of an EGFP signal measured at an excitation of 488 nm (_F_488). [Ca2+]i was monitored with Fura-2 ratios (_F_340/_F_380). The extracellular Ca2+ ([Ca2+]o) was 2 mM and the [rapamycin] was 10 μM. (D–F) In the WT–MCOLN1-transfected cells (D), the Fura-2 ratio increased in response to rapamycin and gradually recovered. The changes in the Fura-2 ratios for MCOLN1S572A/S576A and MCOLN1S572E/S576E (E and F) were negligible.

Figure 5

Figure 5. TRPML1 is phosphorylated by mTOR on Ser572 and Ser576

(A) Kinase assays were performed with purified WT myc-tagged TOR. Synthesized TRPML1 peptides (WT–TRPML1 and TRPML1S572A/S576A; no phosphorylatable residues) were compared as TOR substrates. The kinase assays were performed with varying amounts of the substrate peptides to measure the kinetics (_n_=3). (B) Kinase assays were performed with either purified WT–TOR or KD–TOR and the WT–TRPML1 peptide (_n_=3). The HEK293T cells expressing the TOR constructs were cultured either in the presence or in the absence of 200 nM rapamycin for 12 h before purification. (C) In vitro phosphorylation of WT full-length EGFP–TRPML1 (WT) or full-length EGFP–TRPMLS572A/S576A (S572,576/AA). HEK293T cells transfected with either construct [all cells were additionally transfected with myc-TOR (WT)] were lysed, incubated with or without 200 nM rapamycin (20 min), labelled with [γ-32P]ATP (30 min) and immunoprecipiated with an anti-GFP antibody. Samples were separated by electrophoresis, stained with Coomassie Brilliant Blue (total, lower panel) and subjected to autoradiography (32P, upper panel). Staining with Coomassie Brilliant Blue showed similar levels of fusion protein in each lane.

Figure 6

Figure 6. mTOR inhibits TRPML1 activity during autophagy through phosphorylation

The model in (A) explains how mTOR, AMPK and TRPML1 possibly interact whereas the models presented in (B) and (C) attempt to explain: (1) why loss of AMPK activity alone is lethal in relation to (2) why loss of both AMPK and TRPML1 activity resulted in the limited viability in the presented Drosophila genetic studies that served as the basis for the present work respectively. (A) When active (for example, under starvation conditions), AMPK inhibits mTOR (or its downstream effector molecule S6K1) activity, which in turn modulates TRPML1 activity in a feedback loop (i.e., inactive/less active mTOR leads to increased TRPML1 activity; however, activating TRPML1 increases autophagy, leading to increased amino acid production and activating mTOR; a feedback loop that regulates/modulates mTOR, AMPK and TRPML1 activity). (B) In the absence of only AMPK activity [due to a condition/state where there is a loss of AMPK activity and not simply due to nutrient-rich conditions, which can be simply explained by the feedback loop in (A)], negative TOR regulation is significantly lessened (increased TOR activity), probably contributing to the loss of viability that occurs when AMPK activity is lost, i.e. a complete loss of AMPK activity is known to be developmentally lethal [46,47]. Please note: due to increased mTOR activity, there would also probably be a significant accumulation of amphisomes and lysosomes, possibly leading to the neurodegeneration phenotype often accompanying a loss of functional AMPK [–20] (not pictured). (C) In the absence of both functional AMPK and TRPML1, TOR activity is balanced because (1) the negative regulation of AMPK activity is absent (A), but (2) the positive regulation involving autophagy, i.e. the production of nutrients, such as amino acids, is also absent, leading to a limited viability in the absence of AMPK activity [58,59] (refer to Table 1 for the viability results) and explaining the Drosophila genetic study results described in the present work.

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