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.