Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

LIM domain proteins are found to be important regulators in cell growth, ce ll fate determination, cell differentiation and remodeling of the cell cyto skeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM- only protein that contains four tandemly repeated LIM motifs with an N-term inal single zinc finger (half LIM motif). FHL3 expresses predominantly in h uman skeletal muscle. In this report, FHL3 was shown to be a novel interact ing partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-dir ected mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was u sed to tag FHL3 in order to study its distribution during myogenesis. Our r esult shows that FHL3 was localized in the focal adhesions and nucleus of t he cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreo ver, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at par ticular subcellular locations of eukaryotic cells. To determine whether FHL 2 and FHL3 can interact with one another and to locate the site of this int eraction in a single intact mammalian cell, we fused FHL2 and FHL3 to diffe rent mutants of GFP and studied their interactions using FRET. BFP/GFP fusi on constructs were cotransfected into muscle myoblast C2C12 to verify the c olocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was o bserved by using an epi-fluorescent microscope equipped with an FRET specif ic filter set. (C) 2001 Wiley-Liss, Inc.