Quantitative phosphoproteomic analyses identify STK11IP as a lysosome-specific substrate of mTORC1 that regulates lysosomal acidification

The evolutionarily conserved serine/threonine kinase mTORC1 is a central regulator of cell growth and proliferation. mTORC1 is activated on the lysosome surface. However, once mTORC1 is activated, it is unclear whether mTORC1 phosphorylates local lysosomal proteins to regulate specific aspects of lysosomal biology. Through cross-reference analyses of the lysosome proteome with the mTORC1-regulated phosphoproteome, we identify STK11IP as a lysosome-specific substrate of mTORC1. mTORC1 phosphorylates STK11IP at Ser404. Knockout of STK11IP leads to a robust increase of autophagy flux. Dephosphorylation of STK11IP at Ser404 represses the role of STK11IP as an autophagy inhibitor. Mechanistically, STK11IP binds to V-ATPase, and regulates the activity of V-ATPase. Knockout of STK11IP protects mice from fasting or Methionine/Choline-Deficient Diet (MCD)-induced fatty liver. Thus, our study demonstrates that STK11IP phosphorylation represents a mechanism for mTORC1 to regulate lysosomal acidification and autophagy, and points to STK11IP as a promising therapeutic target for the amelioration of diseases with aberrant autophagy signaling.

(h) The extracted ion chromatogram of the light (control, blue) and heavy (rapamycin-treated, yellow) ions of a Stk11ip peptide in the rat protein (TLDPS*PAGWFVQQHR, * indicates the phosphorylation site).
(i) pS404-STK11IP is evolutionarily conserved among the various species. The S404 (in human) site is highlighted in red.
(j) The level of changes of several representative phosphopeptides under the indicated conditions, including pS1859-Cad, pS236/240-Rps6, pS405-Stk11ip (pS404 in the human protein) and pT757-Ulk1. The data were extracted from the hepatic phosphoproteomic results.

Supplementary Fig. 2. STK11IP is a lysosome-specific mTORC1 substrate.
(a) The antibody against to pS404-STK11IP was specific to pS404-STK11IP. When indicated, the cells were treated with DMSO (Ctrl) or Torin1 (2 μM for 4 hours). STK11IP was immunoprecipitated and was analyzed by the indicated antibodies. The level of pS6K, S6K, pS6, S6 and actin (in the whole cell lysates) was also analyzed.  (e) STK11IP is phosphorylated by mTOR in vitro. 4EBP1 was used as the positive control. SE, short exposure; LE, long exposure.
(k) Quantification of the colocalization between STK11IP and LAMP2 ( Fig. 1f), Tom20, EEA1 and Rab7 ( Supplementary Fig. 2j). The quantification was performed using the Image J 1.50i and was determined using the Pearson correlation of the colocalization coefficient. Data are mean ± SEM, n= 4 independent biology samples.
Source data are provided as a Source Data file. (c) STK11IP interacts with ATP6V1B and ATP6V1H. Flag-Metap2 was used as the negative control.
(d) STK11IP interacts with ATP6V1A. Flag-Metap2 was used as the negative control.
(e) STK11IP did not interact with other subunits of the V1 section of the V-ATPase complex. Flag-Metap2 was used as the negative control, Flag-ATP6V1B was used as the positive control. (g) Body weight (in grams) of the WT and STK11IP KO mice (2-month-old) that were fed with the MCD diet for 2 weeks (n=8 mice per group). Results represent mean ± SEM.
Source data are provided as a Source Data file. Supplementary Fig. 7. Gating strategies for the FACS analysis.
Using the FSC/SSC gating to remove the debris or attached cells; using the RFP gating to get cells that have similar RFP signals; then compare the GFP signal.     Supplementary Fig. 1a