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Published on: March 10, 2010
Analysis of the adaptor function of the LIM domain-containing protein FHL2 using an affinity chromatography approach
Haquima El Mourabit1, Stefan Müller, Lucy Tunggal
1Center for Biochemistry, Faculty of Medicine, University of Cologne, Joseph-Stelzmann-Str 52, 50931 Cologne, Germany.
Insights
The Four and a half LIM domain protein 2 (FHL2) acts as an adaptor protein. FHL2 interacts with cytoskeleton-associated proteins, localizing to cell lamellipodia.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Four and a half LIM domain protein 2 (FHL2) contains multiple LIM domains.
- FHL2 is predicted to function as an adaptor in forming molecular complexes within cellular compartments.
Purpose of the Study:
- To identify FHL2 interaction partners.
- To investigate the cellular localization of FHL2 and its binding partners.
Main Methods:
- Recombinant FHL2 expression in insect cells using a baculovirus system.
- Affinity chromatography to isolate FHL2 interaction partners from fibroblast cytosolic fractions.
- Peptide mass fingerprinting via MALDI-TOF mass spectrometry for protein identification.
- Indirect immunofluorescence staining to determine co-localization in cell lamellipodia.
Main Results:
- Several interaction partners of FHL2 were identified.
- A subset of these partners were identified as cytoskeleton-associated proteins.
- FHL2 and identified cytoskeleton-associated proteins co-localized in cell lamellipodia.
Conclusions:
- FHL2 interacts with cytoskeleton-associated proteins.
- FHL2's localization in lamellipodia suggests a role in cytoskeletal organization or dynamics.
Abstract:
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.
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