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CRP2 is an autonomous actin-binding protein
- Source :
- FEBS letters. 557(1-3)
- Publication Year :
- 2004
-
Abstract
- Cysteine-rich proteins (CRPs) have been shown to be involved in cell differentiation, transcriptional regulation and the organisation of the actin cytoskeleton. Thus far, the latter function has been inferred solely from the in vitro interaction of CRP1, CRP2, and CRP3 with alpha-actinin and zyxin. We show here that purified, recombinant CRP2 binds directly to F-actin in vitro in co-sedimentation assays. Using a green fluorescent protein (GFP)-tagged construct of CRP2 we analysed its localisation and dynamics in A7r5 rat smooth muscle cells. CRP2 was associated with the actin cytoskeleton and decorated actin stress fibres in a continuous fashion, unlike the periodic labelling pattern observed for alpha-actinin and zyxin, which also accumulate in focal adhesions. Using live video fluorescence microscopy we observed the behaviour of GFP-CRP2 during the dynamic rearrangement of the actin cytoskeleton in phorbol 12,13-dibutyrate-treated A7r5 cells. In contrast to the actin-binding proteins SM22alpha and alpha-actinin, GFP-CRP2 did not translocate into the podosomes induced by this treatment, but remained preferentially bound to the stress fibres, suggesting an actin filament-stabilising role for CRP2. When fused to the mitochondrial targeting sequence from the Listeria protein ActA, GFP-CRP2 was almost completely localised to mitochondria, but no significant recruitment of either alpha-actinin or zyxin could be observed. Taken together, our results demonstrate that CRP2 can bind to F-actin directly and that the association with the actin cytoskeleton is independent of alpha-actinin or zyxin localisation in the cell, thus questioning the role of CRP2 as a regulator of alpha-actinin function in vivo.
- Subjects :
- Actin binding
Recombinant Fusion Proteins
Biophysics
Arp2/3 complex
macromolecular substances
Transfection
Biochemistry
Zyxin
Actin remodeling of neurons
Structural Biology
Genetics
Escherichia coli
Animals
Green fluorescent protein
Actinin
Actin-binding protein
Cloning, Molecular
Molecular Biology
Cytoskeleton
Cells, Cultured
DNA Primers
Cysteine-rich protein
Binding Sites
Microscopy, Video
biology
Base Sequence
CCAAT-Enhancer-Binding Protein-beta
Actin remodeling
α-Actinin
Muscle, Smooth
Cell Biology
Actin cytoskeleton
Actins
Recombinant Proteins
Cell biology
Rats
Profilin
Paracytophagy
biology.protein
Subjects
Details
- ISSN :
- 00145793
- Volume :
- 557
- Issue :
- 1-3
- Database :
- OpenAIRE
- Journal :
- FEBS letters
- Accession number :
- edsair.doi.dedup.....ae3d719ecee88976403a61db85ae7f03