Amino Acid Activation of mTORC1 by a PB1-Domain-Driven Kinase Complex Cascade

Abstract

The mTORC1 complex is central to the cellular response to changes in nutrient availability. The signaling adaptor p62 contributes to mTORC1 activation in response to amino acids and interacts with TRAF6, which is required for the translocation of mTORC1 to the lysosome and the subsequent K63 polyubiquitination and activation of mTOR. However, the signal initiating these p62-driven processes was previously unknown. Here, we show that p62 is phosphorylated via a cascade that includes MEK3/6 and p38δ and is driven by the PB1-containing kinase MEKK3. This phosphorylation results in the recruitment of TRAF6 to p62, the ubiquitination and activation of mTOR, and the regulation of autophagy and cell proliferation. Genetic inactivation of MEKK3 or p38δ mimics that of p62 in that it leads to inhibited growth of PTEN-deficient prostate organoids. Analysis of human prostate cancer samples showed upregulation of these three components of the pathway, which correlated with enhanced mTORC1 activation.

Figure 1. p62 Phosphorylation at T269/S272 Was Required for mTORC1 Activation in Response to Amino Acids

(A) p62 domain organization and phosphorylation sites identified by mass spec. Phosphorylation of T269 and S272 was induced by amino acids (upper panel). Alignment of the amino acid sequence of human p62 (264–276) with orthologs in other species is shown (lower panel). (B) p62 phosphorylation at T269 and S272 is induced by amino acids. HEK293T cells were starved of amino acids for 50 min and restimulated with amino acids for the indicated durations. Cell lysates were analyzed by immunoblotting. (C) p62 phosphorylation was determined in response to different amino acids. HEK293T cells were starved of amino acids and restimulated with the indicated amino acids for 30 min. (D) Amino acid starvation inhibits p62 phosphorylation and mTORC1 activation. HEK293T cells were starved of amino acids for the indicated durations. Total cell lysates were analyzed by immunoblotting. (E) Mutation of p62-T269/S272 sites to alanine (p62^T269/S272AA) abolished p62 phosphorylation in response to amino acids. HEK293T cells stably expressing FLAG-p62^WT or FLAG-p62^T269/S272AA were treated and analyzed as in (B). (F) The p62^T269/S272AA mutant was not able to reconstitute mTOR activation in p62KO MEFs. WT and p62KO MEFs, reconstituted with p62^WT or p62^T269/S272AA, were treated as in (B). Cell lysates were analyzed by western blot. (G) RagB^GTP overexpression rescued the defects in mTOR activation by amino acids in cells stably expressing the p62^T269/S272AA mutant. HEK293T cells stably expressing FLAG-p62^WT, FLAG-p62^T269/S272AA, or FLAG-p62^T269/S272AA and FLAG-RagB^GTP were treated as in (B), and immunoblotted for the specified proteins. (H) p62 phosphorylation was not required for mTOR activation by insulin. HEK293T cells stably expressing FLAG-p62^WT or FLAG-p62^T269/S272AA were deprived of serum for 24 hr and stimulated with insulin for the indicated durations. Cell lysates were analyzed by western blot. (I) The p62^T269/S272AA mutant eliminated the interaction of p62 with different components of the mTORC1 complex. HEK293T cells stably expressing FLAG-p62^WT or FLAG-p62^T269/S272AA were treated as in (B). Cell lysates and FLAG-tagged immunoprecipitates were immunoblotted to detect the indicated proteins. Results are representative of three experiments. See also Figure S1

Figure 2. MEKK3 Was a Critical Kinase for p62 Phosphorylation and mTORC1 Activation in Response to Amino Acids

(A) Schematic of the different types of PB1 domains based on the presence of an acid cluster (Type I), basic cluster (Type II), or both in the same domain (Type I/II). (B) p62 and MEKK3 domain architecture and schematic of the interaction between the acidic cluster of the PB1 domain of p62 and the basic domain of MEKK3. (C) Mutation of p62-D69/73 sites to alanine (p62^D69/73AA) abolished p62 phosphorylation. HEK293T cells transfected with the indicated plasmids were starved of amino acids for 50 min and restimulated with amino acids for the indicated durations. Myc-tagged immunoprecipitates were analyzed by western blot. (D) MEKK3 promoted p62 phosphorylation at T269/S272. HEK293T cells were transfected with the indicated plasmids and cell lysates were analyzed by western blot. (E) MEKK3-induced p62 phosphorylation required the PB1 domain of p62. HEK293T cells were transfected with the indicated plasmids and cell lysates were immunoblotted to detect the specified proteins. (F) Overexpression of MEKK3, but not that of MEKK3 kinase-dead mutant, induced p62 phosphorylation and mTORC1 activation by amino acids. HEK293T cells transfected with the indicated plasmids, were deprived of amino acids for 50 min and stimulated with amino acids for 15 min. Cells were analyzed by western blot. (G) MEKK3 expression rescued p62 phosphorylation and mTOR activation in MEKK3-deficient cells. MEKK3-deficient HEK293T cells generated with the CRISPR/CAS9 system were reconstituted with MEKK3. Cells were deprived of amino acids for 50 min and then stimulated with amino acids for the indicated durations. Cell lysates were immunoblotted for the specified proteins. (H) RagB^GTP overexpression rescued mTOR activation by amino acids in MEKK3-deficient cells. Control and MEKK3-deficient HEK293T cells expressing FLAG-RagB^GTP were treated as in (G) and immunoblotted to detect the specified proteins. (I) MEKK3 was not required for mTOR activation by insulin. Control and MEKK3-deficient HEK293T cells were deprived of serum for 24 hr and stimulated with insulin for the indicated durations. Cell lysates were analyzed by western blot. (J) MEKK3 is a component of the mTORC1 complex. mTOR immunoprecipitates and cell lysates from HEK293T cells, treated as in (F), were immunoblotted for the indicated proteins. (K) MEKK3 kinase activity was activated upon amino acid stimulation. HEK293T cells transfected with the indicated plasmids were treated as in (F). HA-tagged immunoprecipitates were used in an in vitro phosphorylation with ATPγS, with myelin basic protein (MyBP) as the substrate, followed by PNBM alkylation and immunoblotting to detect the indicated proteins. Results are representative of three experiments. See also Figure S2.

Figure 3. MEKK3/MEK3/6-p38δ Induced p62 Phosphorylation in Response to Amino Acids

(A) p38δ was required for mTORC1 activation in response to amino acids. Results of siRNA screening of MAPKs in mTORC1 activation. (B) p38δ was required for p62 phosphorylation at T269/S272 in response to amino acids. p38δ-deficient HEK293T cells generated with the CRISPR/CAS9 system. Cells were deprived of amino acids for 50 min and then stimulated with amino acids for 15 min. Cell lysates were immunoblotted for the indicated proteins. (C) RagB^GTP overexpression rescued amino acid-induced mTOR activation in p38δ-deficient cells. Control and p38δ-deficient HEK293T cells, expressing FLAG-RagB^GTP, were treated as in (B). Cell lysates were immunoblotted for the indicated proteins. (D) p38δ was not required for insulin-induced mTORC1 activation. Control and p38δ-deficient HEK293T cells were deprived of serum for 24 hr and then stimulated with insulin for the indicated durations. Cell lysates were immunoblotted to detect the indicated proteins. (E) p38δ overexpression promotes p62 phosphorylation at T269/S272. HEK293T cells were transfected with the indicated plasmids and immunoblotted for the specified proteins. (F) p38δ kinase activity was required for p62 phosphorylation. HEK293T cells were transfected with the indicated plasmids and immunoblotted for the specified proteins. (G) p38δ directly phosphorylated p62 at T269/S272 in vitro. An in vitro phosphorylation assay using recombinant p62 and recombinant p38δ is shown. (H) T269/S272 sites accounted for p62 phosphorylation by p38δ. FLAG-tagged immunoprecipitates from HEK293T cells were phosphorylated in vitro by recombinant p38δ with ATPγS, followed by PNBM alkylation and immunoblotting for the indicated proteins. (I) p38δ kinase activity was activated by amino acids. HEK293T cells transfected with the indicated plasmids were treated as in (B). In vitro phosphorylation was carried out with the FLAG-tagged immunoprecipitates and MBP-p62 recombinant protein as a substrate. (J) MEKK3 was required for p38δ-induced p62 phosphorylation by amino acids. shNT or shMEKK3 HEK293T cells transfected with the indicated plasmids were treated as in (B). In vitro phosphorylation was carried out with the FLAG-tagged immunoprecipitates and MBP-p62 recombinant protein was used as the substrate. (K) MEK3/6 was required for p62 phosphorylation and mTORC1 activation in response to amino acids. HEK293T cells transfected with scramble siRNA or MEK3 and MEK6 siRNAs and FLAG-RagB^GTP were treated as in (B). Cell lysates were immunoblotted for the indicated proteins. (L) MEK3 is activated in response to amino acids. HEK293T cells transfected with the indicated plasmids were treated as in (B). FLAG-tagged MEK3 immunoprecipitates were used in an in vitro phosphorylation, using MyBP as substrate, with ATPγS followed by PNBM alkylation and immunoblotting for the indicated proteins. (M) Schematic showing that MEK3/6-p38δ channels MEKK3-induced phosphorylation of p62 and mTORC1 activation by amino acids. Results are representative of three experiments. See also Figure S3.

Figure 4. MEKK3/p38δ Cascade Is Required for the Lysosomal Translocation of mTOR

(A–B) MEKK3 and p38δ localize at the lysosome in an amino acid-independent manner. Images of HeLA cells co-immunostained for MEKK3 and LAMP2 (A) or p38δ and LAMP2 (B). Cells were starved for 50 min and then stimulated with amino acids for 10 min before processing. In all images, graphs show the areas of staining overlap (merge). Scale bars=10μm. The quantification of colocalization was carried out on at least 15 cells per condition from 2 independent experiments. Results are shown as means ± SEM. (C) MEKK3, p38δ and p62 were present in the lysosomal fraction. HEK293T cells were treated as in (A) and lysates were separated into heavy membrane and light/cytosolic fractions. (D) MEKK3 and p38δ deficiency prevented amino acid-induced translocation of mTOR to lysosomes. Images of Control, MEKK3-deficient, or p38δ-deficient HEK293T cells treated and analyzed as in (A) that were co-immunostained to detect mTOR and LAMP2. Scale bars= 10μm. Results are shown as means ± SEM. ***p<0.001. Images are representative of two independent experiments. See also Figure S4.

Figure 5. The MEKK3/p38δ Cascade Was Required for TRAF6-Catalyzed K63-Polyubiquitination of mTOR in Response to Amino Acids

(A–B) Knockdown of MEKK3 or p38δ impaired the interaction of TRAF6 with p62 in response to amino acids. shNT, shMEKK3, or shp38δ HEK293T cells were deprived of amino acids for 50 min and then restimulated with amino acids for 30 min. Cell lysates and p62 immunoprecipitates were analyzed by western blot for the indicated proteins. (C–E) MEKK3, p38δ, and p62 phosphorylation were required for polyubiquitination of mTOR in response to amino acids. shNT, shMEKK3, and shp38δ HEK293T cells or cells stably expressing FLAG-p62^WT or FLAG-p62^T269/S272AA were treated as in (A). Cell lysates and mTOR immunoprecipitates were immunoblotted for the indicated proteins. Results are representative of three experiments.

Figure 6. The MEKK3/p38δ Cascade Controlled Cell Proliferation and Autophagy through mTORC1 Activation

(A) MEKK3, p62, or p38δ deficiencies reduced cell size. Results are shown as means ± SEM (n=3) *p< 0.05, **p<0.01. (B–C) Knock-down of MEKK3 or p38δ reduced cell proliferation under normal growing conditions. shNT, shMEKK3, or shp38δ PC3 cells were cultured under normal growing conditions, and cell viability was determined by trypan blue exclusion assay. Results are shown as means ± SEM (n=3). *p< 0.05, **p<0.01. (D–E) RagB^GTP overexpression rescued the defects in cell proliferation in MEKK3- or p38δ-knockdown cells. PC3 cells stably expressing FLAG-RagB^GTP were infected with shNT, shMEKK3, or shp38δ lentiviral vectors. Cell lysates were analyzed by western blot, and cell viability was determined as in (C). Results are shown as means ± SEM (n=3). *p< 0.05, **p<0.01. (F–I) Knockdown of MEKK3 or p38δ promoted autophagy in response to nutrient deprivation. shNT, shMEKK3, or shp38δ PC3 cells were deprived of amino acids and serum for 4 hr in the absence or presence of bafilomycin A1. Cell lysates were immunoblotted for the indicated proteins. Graphs represents LC3-II/actin ratio as measured by densitometry. (J–K) Knockdown of MEKK3 or p38δ promoted increased autophagic flux. Images of shNT, shMEKK3, or shp38δ cells stably expressing GFP-mCherry-LC3 and treated as in (G). Scale bars=10μm. Quantification of the number of autophagosomes and autolysosomes per cell is shown. Results are shown as means ± SEM (n=20). *p< 0.05 **p< 0.01, ***p<0.001. Results are representative of three experiments.

Figure 7. MEKK3/p38δ/p62/mTOR Is Relevant to Prostate Cancer

(A) Knockdown of MEKK3, p38δ, or p62 led to a reduction in the efficiency of organoid formation, size, and hyperplastic phenotype of PTEN-null prostate organoids. Representative images of organoids and H&E staining are shown. Prostate organoids were prepared from PTEN^fl/fl-PBcre mice and infected with lentiviral vectors for shNT, shp62, shMEKK3, and shp38δ. Organoids were analyzed after 7 days in culture. Scale bars = 100 μm. (B) Quantification of the efficiency of organoid formation and size in the experiment shown in (A). Scale bars =100 μm. Results are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Knockdown of MEKK3, p38δ, or p62 in PTEN-null prostate organoids led to decreased mTORC1 activation. Cell lysates from (A) were immunoblotted for the indicated proteins. (D) Increased expression of MEKK3, p38δ, p62, and S6 phosphorylation in PIN areas (red arrows) of PTEN^+/− prostates, compared with normal prostate glands (asterisks). Representative images of MEKK3, p38δ, p62, and phospho-S6 staining of primary PCa samples from PTEN+/− mice are shown. Scale bars = 25 μm. (E) Human prostate shows increased expression of p38δ, p62, and S6 phosphorylation in tumor tissue, as compared with normal tissue. Images of human prostate co-immunostained for p38δ and p62, or and p38δ and p-S6 are shown. Scale bars=100 μm. (F) Increased expression of MEKK3, p38δ, p62, and S6 phosphorylation in human prostate tissue microarray (TMA). Box plot graphs showing a statistical analysis of MEKK3 (p=0.006), p38δ (p=0.004), p62 (p=0.003), or phospho-S6 (p=0.049) expression in prostate tumors with GS 7-10 compared to prostate tissue with GS 2-6. Results are presented as mean ± SEM. *p < 0.05, **p < 0.01. (G) MEKK3 and p38δ expression in human prostate tumors was correlated with p62 and phospho-S6. Correlation plots showing the relationship between MEKK3/p62, MEKK3/phosho-S6, p38δ/p62, and p38δ/phospho-S6 (arbitrary units). The coefficient of correlation (r) and the p value (p) are indicated.