Fig 2: 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.

Fig 3: JAB1 is functionally associated with TNBC tumorigenesis.a Box-and-whisker plot was generated to show the expression of JAB1 in breast tumor subtypes. Statistical analysis was performed using GraphPad Prime 7.0 with unpaired t test (two-tailed). b, c JAB1 expression is inversely correlated with breast cancer patient overall (b) and recurrence-free survival (c). d JAB1 expression is inversely correlated with TNBC patient overall survival. e, f BT549 or MDA-MB-231 cells were treated with 30 nM JAB1 siRNAs (siJAB1-1, siJAB1-2), scrambled siRNA control for 3 days or 5 µM SB203580 for 1 day followed by MTT assay to analyze cell growth (e) or Matrigel invasion assay to determine in vitro invasion (f). Data are means ± SD from three experiments. *P < 0.01 vs Control. #P < 0.05.

Fig 4: Ser177 phosphorylation of JAB1 impacts JUN binding to AP1 sites.a BT549 or MDA-MB-231 cells were treated with 5 µM SB203580 for 1 day or 30 nM MK2 siRNA (siMK2-1) for 3 days for left untreated (control) and then subjected to JUN ChIP followed by qPCR to analyze JUN occupancy in cyclin D1, uPA and uPAR promoters. Data are means ± SD from three experiments. *P < 0.01 vs Control. b BT549 or MDA-MB-231 cells were transduced with JAB1D, JAB1A, or empty vector (control) for 3 days and then subjected to JUN ChIP followed by qPCR to analyze JUN occupancy in cyclin D1, uPA and uPAR promoters. Data are means ± SD from three experiments. *P < 0.01 vs Control. c, d BT549 (c) or MDA-MB-231 cells (d) were transduced JAB1D or empty vector for 3 days and then treated with 5 µM SB203580 for 1 day or 30 nM MK2 siRNA (siMK2-1) for 3 days followed by JUN ChIP to detect JUN occupancy in cyclin D1, uPA and uPAR promoters. Data are means ± SD from three experiments. *P < 0.01 vs Control.

Fig 5: JAB1 is an MK2 substrate and is required for robust AP1 activity in TNBC cells.a MDA-MB-231 cells were immunoprecipitated with either JAB1 or MK2 antibody and the immunoprecipitates were subjected to western blot to detect MK2 or JAB1 respectively. All blots derived from the same experiment and were processed in parallel. b MDA-MB-231 cells were subjected to immunofluorescence staining to detect JAB1 and MK2. Data are the representative of four independent experiments. c Diagram of HA-tagged full-length JAB1, N-terminal Jab1 (JAB1-N), and C-terminal JAB1 (JAB1-C). d HEK293 cells were transfected with empty plasmid (Control) or with plasmids encoding HA-tagged full-length JAB1 (FL), JAB1-N or JAB1-C, and cell lysates were immunoprecipitated with MK2 pAb. Immunoprecipitates were subjected to western blot analysis to detect interactions between the JAB1 proteins and MK2 by HA mAb. Data are the representative of two independent experiments. All blots derive from the same experiment and were processed in parallel. e BT549, MDA-MB-231 or MDA-MB-436 cells were treated with 30 nM JAB1 siRNA (siJAB1-1 and siJAB1-2) or scrambled siRNA control for 3 days followed by analyzing AP 1 activity. Data are means ± SD from three experiments. *P < 0.01 vs Control. f Wild-type MEFs were first transduced with MKK3D, MKK6D, MK2-CA, or empty vector (Control) for 3 days and then treated with 30 nM JAB1 siRNAs for 3 days followed by analyzing AP1 activity. Data are means ± SD from three experiments. *P < 0.01 vs Control.