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

TitleProtein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)
Authors
KeywordsFHL2
FHL3
FRET
GFP
LIM domain protein
Protein-protein interaction
Issue Date2000
PublisherJohn Wiley & Sons, Inc. The Journal's web site is located at http://www3.interscience.wiley.com/cgi-bin/jhome/35503
Citation
Journal Of Cellular Biochemistry, 2000, v. 80 n. 3, p. 293-303 How to Cite?
AbstractLIM 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. © 2001 Wiley-Liss, Inc.
Persistent Identifierhttp://hdl.handle.net/10722/92221
ISSN
2015 Impact Factor: 3.446
2015 SCImago Journal Rankings: 1.520
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLi, HYen_HK
dc.contributor.authorNg, EKOen_HK
dc.contributor.authorLee, SMYen_HK
dc.contributor.authorKotaka, Men_HK
dc.contributor.authorTsui, SKWen_HK
dc.contributor.authorLee, CYen_HK
dc.contributor.authorFung, KPen_HK
dc.contributor.authorWaye, MMYen_HK
dc.date.accessioned2010-09-17T10:39:38Z-
dc.date.available2010-09-17T10:39:38Z-
dc.date.issued2000en_HK
dc.identifier.citationJournal Of Cellular Biochemistry, 2000, v. 80 n. 3, p. 293-303en_HK
dc.identifier.issn0730-2312en_HK
dc.identifier.urihttp://hdl.handle.net/10722/92221-
dc.description.abstractLIM 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. © 2001 Wiley-Liss, Inc.en_HK
dc.languageengen_HK
dc.publisherJohn Wiley & Sons, Inc. The Journal's web site is located at http://www3.interscience.wiley.com/cgi-bin/jhome/35503en_HK
dc.relation.ispartofJournal of Cellular Biochemistryen_HK
dc.subjectFHL2en_HK
dc.subjectFHL3en_HK
dc.subjectFRETen_HK
dc.subjectGFPen_HK
dc.subjectLIM domain proteinen_HK
dc.subjectProtein-protein interactionen_HK
dc.titleProtein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)en_HK
dc.typeArticleen_HK
dc.identifier.emailNg, EKO: ngko@hku.hken_HK
dc.identifier.emailKotaka, M: masayo@hku.hken_HK
dc.identifier.authorityNg, EKO=rp01364en_HK
dc.identifier.authorityKotaka, M=rp00293en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1002/1097-4644(20010301)80:3<293::AID-JCB10>3.0.CO;2-Uen_HK
dc.identifier.pmid11135358-
dc.identifier.scopuseid_2-s2.0-0034484478en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0034484478&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume80en_HK
dc.identifier.issue3en_HK
dc.identifier.spage293en_HK
dc.identifier.epage303en_HK
dc.identifier.isiWOS:000166756000001-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridLi, HY=12762326800en_HK
dc.identifier.scopusauthoridNg, EKO=21135553700en_HK
dc.identifier.scopusauthoridLee, SMY=35233892600en_HK
dc.identifier.scopusauthoridKotaka, M=6604073578en_HK
dc.identifier.scopusauthoridTsui, SKW=7004961364en_HK
dc.identifier.scopusauthoridLee, CY=7410142857en_HK
dc.identifier.scopusauthoridFung, KP=35271657800en_HK
dc.identifier.scopusauthoridWaye, MMY=7006687733en_HK

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