Analysis of the adaptor function of the LIM domain-containing protein FHL2 using an affinity chromatography approach.
Containing four LIM domains and an N-terminal half LIM domain, FHL2 has been predicted to have an adaptor function in the formation of higher order molecular complexes in the nucleus and the cytoplasm of cells. We expressed recombinant FHL2 in insect cells using the baculovirus system and used it to isolate direct or indirect interaction partners from the cytosolic fraction of fibroblasts by affinity chromatography. These were identified by their peptide mass fingerprints using MALDI-TOF mass spectrometry. Cytoskeleton-associated proteins present among the bound proteins were shown to co-localise with FHL2 in cell lamellipodia by indirect immunofluorescence staining.
El Mourabit
Haquima
H
Center for Biochemistry, Faculty of Medicine, University of Cologne, Joseph-Stelzmann-Str 52, 50931 Cologne, Germany.
Müller
Stefan
S
Tunggal
Lucy
L
Paulsson
Mats
M
Aumailley
Monique
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007313:Insecta; D060850:LIM-Homeodomain Proteins; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011554:Pseudopodia / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection
C105987:FHL2 protein, human; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors