Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220.
Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype.
Ng
Enders Kai On
EK
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China.
Chan
Kwok Keung
KK
Wong
Chi Hang
CH
Tsui
Stephen Kwok Wing
SK
Ngai
Sai Ming
SM
Lee
Simon Ming Yuen
SM
Kotaka
Masayo
M
Lee
Cheuk Yu
CY
Waye
Mary Miu Yee
MM
Fung
Kwok Pui
KP
D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis; D060850:LIM-Homeodomain Proteins; D008164:Luminescent Proteins / Q000032:analysis; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins; D009206:Myocardium / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D016601:RNA-Binding Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis; D017384:Sequence Deletion; D014018:Tissue Distribution; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D004268:DNA-Binding Proteins; C105987:FHL2 protein, human; C491381:Fhl2 protein, rat; D018398:Homeodomain Proteins; D007202:Indicators and Reagents; D060850:LIM-Homeodomain Proteins; D008164:Luminescent Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C099656:ZNF638 protein, human; D049452:Green Fluorescent Proteins