Fig. 4. MK2 phosphorylates JAB1 at Ser177 and Ser177 of JAB1 is essential for robust AP1 activity in TNBC cells.

a Alignment of JAB1 protein sequences from different species. The conserved MK2 phosphorylation target motif is underlined. Amino acid residue numbers are to the left and right of the sequences. b Coomassie blue staining of recombinant JAB1 and JAB1A. c In vitro kinase assay with recombinant active MK2 to determine the ability of MK2 to phosphorylate JAB1. Data are the representative of two independent experiments. d Western blot analysis to detect Ser¹⁷⁷-phosphorylated JAB1 (p-JAB1) in breast cancer cell lines. e BT549 or MDA-MB-231 cells were under UV exposure for 1 min, treated with 5 µM SB203580 for 1 days, 30 nM MK2 siRNA (siMK2-1) for 3 days or left untreated followed by western blot analysis to detect Ser¹⁷⁷-phosphorylated JAB1 and JAB1. Data are the representative of two independent experiments. All blots derived from the same experiment and were processed in parallel. f MK2−/− MEFs were transduced with JAB1D, JAB1A or empty vector for 3 days followed by analyzing AP1 activity. Wild-type MEF was included for comparison. Data are means ± SD from three experiments. *P < 0.01 vs wild-type MEF (MEF WT). g, h BT549 (g) and MDA-MB-231 cells (h) were first transduced with JAB1, JAB1D, JAB1A, or empty vector (Control) for 3 days and then treated with 5 µM SB203580, vehicle, or 30 nM MK2 siRNA for 3 days followed by analyzing AP1 activity. Data are means ± SD from three experiments. *P < 0.01 vs Vehicle.