Coactosin-like protein, a human F-actin-binding protein: critical role 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 fu nctional characterization of CLIP as a human filamentous actin (F-actin)-bi nding protein. CLP mRNA shows a wide tissue distribution and is predominant ly expressed in placenta, lung, kidney and peripheral-blood leucocytes. End ogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybri d approach, actin was identified as a CLP-interacting protein. Binding expe riments indicated that CLP associates with F-actin, but does not form a sta ble complex with globular actin. In transfected mammalian cells, CLP co-loc alized with actin stress fibres. CLP bound to actin filaments with a stoich iometry of 1:2 (CLP: actin subunits), but could be crosslinked 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 protein s 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 viv o, and indicate that Lys(75) is essential for this interaction.