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