Fig 2: Knockdown of MAPK6 sensitizes cancer cells to mTOR kinase blockade.(A) Western blots and (B) proliferation assays on PC3, H1299, and SUM159 cells with siRNA-mediated knockdown of MAPK6 (siMAPK6 #A4, #A6) or luciferase control (siLuc) and treated with PP242 and/or INK128. Cells were first transfected with the indicated siRNA. Seventy-two hours later, cells were treated with PP242 (0.5 µM) or INK128 (25 nM) for 6 hours and processed for Western blots. For proliferation assays, 48 hours after transfection, cells were plated into 24-well plates and treated with PP242 (1 µM for PC3 and SUM159, and 0.5 µM for H1299 cells) or DMSO. (C to E) Soft-agar assays on (C) PC3, (D) SUM159, and (E) H1299 cells with Dox-induced (1 µg/ml) knockdown of MAPK6 (ishRNA #2, #3, and/or #5) or control (iNT) treated with PP242 (concentrations as described above) or DMSO. (F) Soft-agar assays on Dox-induced (1 µg/ml) PC3-iMAPK6 and iCtrl cells treated with 0.5 µM PP242, 25 nM INK128, or DMSO. Data as means ± SD, two-way ANOVA with multiple comparisons corrected with Sidak’s test. *P = 0.05. **P = 0.01. ***P = 0.001. ****P = 0.0001. ns, not significant. Data are representative of at least three independent experiments.

Fig 3: MAPK6 phosphorylates AKT at S473 independent of mTORC2.(A) Coomassie blue staining of the purified MAPK6 protein from HEK293T cells. MW, molecular weight; IgG-H/IgG-L, heavy/light chain of IgG. (B) Western blots on the products from in vitro kinase assays assessing affinity-purified MAPK6 phosphorylation of AKT1. MAPK6 proteins were overexpressed/purified using the EZview Red anti-FLAG M2 affinity gel from the HEK293T cells transiently transfected with pRK5-MAPK6-FLAG/His plasmid (left) or from the engineered PC3 cells overexpressing MAPK6-FLAG (right). Western blots on the products from in vitro kinase assays assessing AKT1 phosphorylation by (C) a commercially available MAPK6 protein, (D) wild-type (WT) MAPK6 versus MAPK62A versus MAPK66A mutants similarly expressed/purified from PC3 cells in (B), and (E) MAPK6-F (MAPK6-FLAG) proteins purified from the engineered Rictor+/+ and Rictor-/- MEF cells with Dox-induced (0.5 µg/ml) expression of MAPK6-F or control (iCtrl). Also shown is the illustration of MAPK62A and MAPK66A mutants with indicated K/R to A mutations at the ATP binding pocket. (F) Western blots on H1299 cells transfected with siRNA targeting MAPK6 (siMAPK6 #A4, #A6) or control (siLuc) for 72 hours followed by 1 µM PP242 or DMSO treatments for 18 hours. Data are representative of at least three independent experiments.

Fig 4: The molecular basis for MAPK6 binding to AKT, part 2.(A) Sequence alignment between MAPK6 and MAPK4 around the potential AKT binding motif of MAPK6. (B and C) Western blots on input and immunoprecipitation products for interaction between AKT1 and FH (2× FLAG and 10× His)–tagged truncated MAPK6. HEK293T cells were similarly transfected/processed, as described in Fig. 7B. MAPK61-379 but not MAPK61-373 can bind AKT (B). Deletion of 363 to 379 [?363–379, SEHDWPVHNNFDIDEVQ in blue/red, (A)] suppressed MAPK61-480 interaction with AKT1 (C). (D) In vitro kinase assays on AKT1 phosphorylation by FLAG-tagged MAPK6, MAPK61-480, and MAPK61-480?363-379 proteins expressed/purified from PC3 cells, as similarly described in Fig. 5B. (E and F) MAPK6?363-379 maintained AKT1 binding but could not phosphorylate AKT1. FLAG-tagged MAPK6?363379 was expressed/purified, and assays were performed as similarly described in (C) and (D). (G and H) Western blots on immunoprecipitation products for AKT1 and MAPK6 mutant interactions. Mutation of DIDE [374 to 377, highlighted in red (A)] into KIKK (D/E3K) blocked MAPK61-384 (G) but not full-length MAPK6 (H) binding to AKT1. (I) In vitro kinase assays on AKT1 phosphorylation by FLAG-tagged WT and D/E3K-mutated MAPK6 proteins expressed/purified from PC3 cells, as similarly described in Fig. 5B. WCL, whole cell lysate. Data are representative of at least three independent experiments.

Fig 5: AKT activation is essential for the oncogenic and tumor-promoting activity of MAPK6.(A) Representative images (whole well and enlarged field) of soft-agar assays on Dox-induced (0.5 µg/ml) PNT1A-iMAPK6 cells treated with 2 µM of MK2206, GSK2141795, or dimethyl sulfoxide (DMSO). Quantification data as means ± SD, one-way ANOVA with multiple comparisons corrected with Dunnett’s test. (B) Western blots on PNT1A-iMAPK6 cells induced with Dox (+) or control (-) for 48 hours followed by treatments of 2 µM MK2206, 2 µM GSK2141795, or DMSO for 18 hours. (C and D) Similar studies as (A) and (B) were performed on the engineered OVCA433 cells. Data as means ± SD, two-way ANOVA with multiple comparisons corrected with Sidak’s test. (E) Representative images (whole well and enlarged field) of soft-agar assays on Dox-induced (1 µg/ml) engineered H1299 cells with overexpression of AKT1T308D/S473D (iAKT-DD) versus control (iCtrl) with knockdown of MAPK6 (ishMAPK6) versus control (iNT). Quantification data as means ± SD, two-way ANOVA analysis as described in (C). (F) Western blots on the engineered H1299 cells in (E). The cells were treated with Dox (1 µg/ml) for 48 hours before Western blots analysis. **P = 0.01. ****P = 0.0001. ns, not significant. Data are representative of at least three independent experiments.