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

Onyenwoke RU, Sexton JZ, Yan F, Díaz MC, Forsberg LJ, Major MB, Brenman JE - Biochem. J. (2015)

Bottom Line: Autophagy is a complex pathway regulated by numerous signalling events that recycles macromolecules and may be perturbed in lysosomal storage disorders (LSDs).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.

View Article: PubMed Central - PubMed

Affiliation: Biomanufacturing Research Institute and Technology Enterprise (BRITE), North Carolina Central University, Durham, NC 27707, U.S.A. ronyenwo@nccu.edu).

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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.
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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.

Mentions: To determine whether TOR could directly phosphorylate TRPML1 in vitro, kinase assays were performed to monitor the incorporation of 32P into modified versions of the TRPML1 C-terminal peptide (refer to the ‘Experimental’ for the exact sequences of these peptides; Figures 1A, 5A and 5B; Supplementary Figure S6) [42,43]. In brief, either Ser572 or Ser576 (or both) was replaced with an unphosphorylatable alanine residue in the C-terminal TRPML1 peptide for the 32P incorporation assays. The kinase assays revealed that the C-terminal peptide is indeed a robust TOR substrate (Figures 5A and 5B), whereas versions of the peptide altered at one or both of the putative serine residues (S572A/S576A) were relatively poor TOR substrates (Supplementary Figure S6).


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

Onyenwoke RU, Sexton JZ, Yan F, Díaz MC, Forsberg LJ, Major MB, Brenman JE - Biochem. J. (2015)

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.
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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.
Mentions: To determine whether TOR could directly phosphorylate TRPML1 in vitro, kinase assays were performed to monitor the incorporation of 32P into modified versions of the TRPML1 C-terminal peptide (refer to the ‘Experimental’ for the exact sequences of these peptides; Figures 1A, 5A and 5B; Supplementary Figure S6) [42,43]. In brief, either Ser572 or Ser576 (or both) was replaced with an unphosphorylatable alanine residue in the C-terminal TRPML1 peptide for the 32P incorporation assays. The kinase assays revealed that the C-terminal peptide is indeed a robust TOR substrate (Figures 5A and 5B), whereas versions of the peptide altered at one or both of the putative serine residues (S572A/S576A) were relatively poor TOR substrates (Supplementary Figure S6).

Bottom Line: Autophagy is a complex pathway regulated by numerous signalling events that recycles macromolecules and may be perturbed in lysosomal storage disorders (LSDs).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.

View Article: PubMed Central - PubMed

Affiliation: Biomanufacturing Research Institute and Technology Enterprise (BRITE), North Carolina Central University, Durham, NC 27707, U.S.A. ronyenwo@nccu.edu).

Show MeSH
Related in: MedlinePlus