Figure 3

MMGL acts as a dual-specific AKAP by anchoring PKA regulatory isoforms R1A and R2A. A. Activation of nutritional reporter genes by PKA-MMGL interaction during direct protein-protein interaction assays. Small-scale Y2H matings between pGBKT7-MMGL and pACT2-PRKAR1A (i) and pACT2-PRKAR2A (ii) on solid medium lacking Leu, Trp, His and Ade. Growth of yeast yells indicate interaction of MMGL with PRKAR1A as well as PRKAR2A. B. Representative images of live cell fluorescence microscopy showing co-localization of MMGL with PRKAR1A and PRKAR2A in differentiated H9C2 cardiomyocytes. (i) GFP-tagged PRKAR1A and PRKAR2A is seen in the cytosol as green fluorescence. (ii) dsRed-tagged MMGL expression is seen as red fluorescence. (iii) Yellow fluorescence indicates the co-localization of PRKAR1A and PRKAR2A with MMGL. (iv) Overlay of images A-C with Hoechst H-33342 labelling of the nuclei (blue fluorescence), for orientation purposes. Scale bar: 0.02 mm. C. Western blots of in vivo co-immunoprecipitations of PKA regulatory subunits and MMGL; the antibodies used in immunoprecipitation (IP) and Western Blot (WB) are shown above each lane. Endogenous PRKAR1A and PRKAR2A immunoprecipitated exogenous dsRed-MMGL in lysates of dsRed-MMGL-transfected, differentiated H9C2 cardiomyocytes. Conversely, PRKAR1A and PRKAR2A also immunoprecipitated exogenous dsRed-MMGL in these lysates. The negative control lanes included lysates from cells not transfected with dsRed-MMGL, showing that these precipitations are not spurious, but are the result of physical association between the relevant proteins. Abbreviations: Prot G = protein G control; R1A = PRKAR1A; R2A = PRKAR2A, UT/- = negative control lanes.