CRP2 is an autonomous actin-binding protein.
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.
Grubinger
Markus
M
Department of Cell Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria. markusgeubinger@yahoo.com
Gimona
Mario
M
D000185:Actinin / Q000737:chemistry / Q000378:metabolism; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018715:Microscopy, Video; D009130:Muscle, Smooth; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D000199:Actins; D022782:CCAAT-Enhancer-Binding Protein-beta; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000185:Actinin