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

Abstract

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. 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-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, 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 particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.

Seeing the Light

“…Construction of Vectors to Measure FRET in Cells and Transfections-As noted above, GFP and BFP were discovered and developed by a number of groups (19,20) who enabled these fluorescent proteins to be seen and localized in cells (19 -35), and several groups have begun to use FRET to study the interactions of proteins in cells (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). In our studies, the IFN-␥ receptor complex ( Fig.…”

“…The association of Bcl-2 and Beclin fused to various mutants of GFP was determined by FRET (22). Additional reports in the last few years have begun to determine the association of proteins in cells with the use of GFP and BFP or cyan fluorescent protein and yellow fluorescent protein pairs (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). The use of fluorescence lifetime imaging microscopy can eliminate the need for spectral separation and problems in FRET (36) as we found previously by pure spectral methods (13) because fluorescence lifetime measurements permit calculation of distances between donor and acceptor fluorophores totally independent of FRET (11,12).…”

The multifunctional roles of the four-and-a-half-LIM only protein FHL2

“…It has been suggested that FHL2 may act as a transducer of apoptotic signals from the extracellular matrix at the focal adhesion plaques to the mitochondria. Interestingly, FHL2-GFP fusion protein was detected in the mitochondria [41]. FHL2 may also be implicated in radiation-induced apoptosis, as irradiation increases expression of p53.…”

“…5 GST pull down failed to show interaction between FHL2 and type IIa Na/P i cotransporter. cation of FHL2 in a ROCK/Rho GTPases-dependent way [15,27,40,41]. UV treatment of NIH3T3 cells also induced nuclear translocation of ectopic Myc-FHL2, but the mechanism was not investigated [15].…”

“…BFP in C2C12 cells demonstrated that both proteins localized in the mitochondria[41]. However, studies with ectopic expressed FHL2 may have some pitfalls.…”