Figure 6.

SRC-mediated tyrosine phosphorylation of MEF2D is required for transcriptional activity of MEF2D and in regulation of MTORC1 activity. (A) His-tagged MEF2D-WT or MEF2D mutant constructs (mutation of Tyr33, 57, 69, 72, 117, 131, 225, 333, 337 and 478 residues) were co-transfected with or without Flag-tagged SRC in HEK293T cells for 24 h. Cells were lysed and subjected to immunoprecipitation against His-tag, followed by immunoblotting with p-tyr antibody. (B) HEK293T cells transfected His-tagged MEF2D-WT or MEF2D-3YF mutants with or without Flag-tagged SRC were lysed and subjected to immunoprecipitation against His-tag, followed by immunoblotting with indicated antibodies. (C) Flag-tagged MEF2D-WT or its 3YF mutant protein purified from HEK293T cells was incubated with commercial active GST-tagged SRC kinase in a kinase assay buffer, followed by immunoblotting with p-tyr antibody. (D) sequence alignment of the residues flanking across different species. Black arrowheads point to the tyrosine residues corresponding to the Tyr333 and tyr 337 residues in human MEF2D. (E) HeLa cells that transfected with Flag-tagged MEF2D-WT or MEF2D-3YF were maintained in a serum free medium for 4 h, followed with or without EGF treatment. Cell lysates were prepared and immunoprecipitation were analyzed by immunoblotting. (F) luciferase assay was performed in depletion of both MEF2A and MEF2D HeLa cells after co-transfection of indicated expression plasmids and wild-type (MEF2 reporter-WT) or mutated (MEF2 reporter-mt) luciferase reporter plasmids for 24 h. (G) qRT-PCR analysis was performed in MEF2A and MEF2D double-knockdown HeLa cells that reconstructed with MEF2D-WT or MEF2D-3YF. The mRNA levels of FNIP1, FNIP2, FLCN, NR4A1, ZMAT4 and DAAM1 were shown. (H) MEF2A and MEF2D double-knockdown HeLa cells were starved with serum for 4 h and then treated with EGF for 3 h before immunoblotting analysis of the activation of MTORC1 with indicated antibodies. Right plots show phosphorylated p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (bottom) ratios. (I) MEF2A and MEF2D double-knockdown HeLa cells that transfected with indicated plasmids were subjected to serum starvation for 4 h and restimulated with EGF for 3 h. MTORC1 activity was analyzed similarly to (H). Right plots show phosphorylated p-RPS6KB1:RPS6KB1 (top), p-EIF4EBP1:ACTB (bottom) ratios. Data are presented as the mean ± S.E.M. (n = 3 independent experiments. two-sided Student’s t-test for H, one-way ANOVA for I, *P < 0.05, **P < 0.01, ***P < 0.001).