Figure 4.

MT1-MMP interacts with GelA and facilitates its activation in vivo. (A) Coexpression of MT1-MMP enhances the formation of activated (mature) GelA in vivo. The mRNA encoding FLAG-tagged GelA wild type was injected either alone or together with the mRNA encoding MT1-MMP wild type or inactive mutant (E231A) into one blastomere of stage 2 embryos. Proteins were extracted 1 day after injection and subjected to gelatin zymography with the tail extract from stage 62 tadpoles (the metamorphic climax when tail resorption occurs) used as a positive control. The latent, activation intermediate, and activated forms are indicated with an arrow, dashed arrow, and open arrow, respectively. The percentage of latent, intermediate, and activated form of GelA was calculated by densitometry from four independent experiments and shown at the bottom.

(B) GelA and MT1-MMP interacts in vivo. The mRNA encoding FLAG-tagged GelA wild type and Myc-tagged MT1-MMP wild type were injected either alone or together into one blastomere of stage 2 embryos. Proteins were extracted 1 day after injection and subjected to Western blotting using anti-Myc antibody before (input, top) or after immunoprecipitation with anti-FLAG M2 antibody (FLAG IP, bottom). The arrow indicates the position of Myc-tagged MT1-MMP. The origin of the minor band labeled with a star above the MT1-MMP band is unclear. It might be a form of MT1-MMP modified post-translationally, although we did not detect such a band when the MT1-MMP was labeled with the FLAG tag (Fig. 3). Regardless, it does not affect the conclusion about GelA-MT1-MMP interaction. The arrowheads in A and B indicate the positions of molecular weight markers.