Figure 3. Role of MEKK3:CCM2 interaction in signaling and localization.

(a) Flag-tagged full-length CCM2 (Flag–CCM2) was cotransfected with HA–MEKK3^FL, HA–MEKK3^ΔNPB1 or HA–MEKK3^ΔN into 293T cells as indicated. Transfected cells were subjected to immunoprecipitation (IP) and the immunoprecipitates and the whole-cell lysates were further analysed by IB with the indicated antibodies. (b) HA-tagged wild-type MEKK3 (WT) or its derived mutants A6D, L7D, A6D/L7D, I10D, L14D, I10D/L14D and A6D/I10D/L14D were cotransfected and analysed for interaction with Flag–CCM2 by co-IP. (c) HA–MEKK3 was cotransfected with Flag–CCM2, or its derived mutants L299E, M303E, L299E/M303E, A319D, L322D and A319D/L322D. The MEKK3–CCM2 interaction was then analysed by co-IP. (d) MBP–MKK3 was phosphorylated by GST–MEKK3 in the absence or presence of purified WT full-length CCM2 (CCM2^FL) or its derived mutant (CCM2^{FL-A319D/A320D}) as indicated. Loading of GST–MEKK3, HisMBP–MKK3, and CCM2 shown in the top and bottom panels, respectively. Phosphorylated HisMBP-MKK3 shown in the middle panel. Quantification was performed using a LiCor Odyssey Imager and shown below. Error bars indicate s.d. N=3. (e) 293T cells were transfected with HA–MEKK3^FL or HA–MEKK3^ΔNPB1. Cell extracts were prepared and analysed for p-JNK, p-ERK1/2 and p-p38 levels by IB with the indicated antibodies. Relative HA–MEKK3 expression levels shown by IB with an anti-HA antibody. (f) 293T cells were transfected with HA–MEKK3^FL, HA–MEKK3^ΔN, HA–MEKK3^FL-A6D, HA–MEKK3^FL-L7D or HA–MEKK3^FL-A6D/L7D. Cell extracts were analysed for p-JNK and p-ERK1/2 levels by IB. Relative expression levels of MEKK3 and its mutants were determined by IB with an anti-HA antibody. (g) Representative confocal images showing that CCM2 colocalizes with WT MEKK3 (MEKK3^FL), but not its mutants MEKK3^ΔNPB1, MEKK3^ΔN or MEKK3^FL-A6D/L7D. Visualized with a LM8 Leica confocal microscopy. White scale bar, 10 μm; cyan scale bar, 1 μm. Power of objective lens: 63 × . (h) MEKK3^N-peptide disrupts CCM2–MEKK3 interaction. Flag–CCM2 and HA–MEKK3 were cotransfected into 293T cells. After 24 h, the transfected cells were administrated with cell-permeable peptides MEKK3^N-peptide or MEKK3^{mutant-N-peptide}. Flag–CCM2 and HA–MEKK3 interaction was determined by co-IP. (i) MEKK3^N-peptide increases pMLC2 S19 phosphorylation. Mouse brain endothelial cells from WT pups were isolated, cultured for 3 days and treated with either MEKK3^N-peptide or MEKK3^{mutant-N-peptide}.