Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.
Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction.
Provost
P
P
Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institute, Scheeles väg 2, S-171 77 Stockholm, Sweden. patrick.provost@mbb.ki.se
Doucet
J
J
Stock
A
A
Gerisch
G
G
Samuelsson
B
B
Rådmark
O
O
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003432:Cross-Linking Reagents; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014162:Transfection; D020798:Two-Hybrid System Techniques
D000199:Actins; D001704:Biopolymers; C118115:COTL1 protein, human; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; C034037:F-actin-binding proteins; D008840:Microfilament Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008239:Lysine; D002118:Calcium