Abstract
Human calmodulin-like protein (CLP) is an epithelial-specific Ca(2+)-binding protein whose expression is strongly down-regulated in cancers. Like calmodulin, CLP is thought to regulate cellular processes via Ca(2+)-dependent interactions with specific target proteins. Using gel overlays, we identified a approximately 210-kDa protein binding specifically and in a Ca(2+)-dependent manner to CLP, but not to calmodulin. Yeast two-hybrid screening yielded a CLP-interacting clone encoding the three light chain binding IQ motifs of human "unconventional" myosin X. Pull-down experiments showed CLP binding to the IQ domain to be direct and Ca(2+)-dependent. CLP interacted strongly with IQ motif 3 (K(d) approximately 0.5 nm) as determined by surface plasmon resonance. Epitope-tagged myosin X was localized preferentially at the cell periphery in MCF-7 cells, and CLP colocalized with myosin X in these cells. Myosin X was able to coprecipitate CLP and, to a lesser extent, calmodulin from transfected COS-1 cells, indicating that CLP is a specific light chain of myosin X in vivo. Because unconventional myosins participate in cellular processes ranging from membrane trafficking to signaling and cell motility, myosin X is an attractive CLP target. Altered myosin X regulation in (tumor) cells lacking CLP may have as yet unknown consequences for cell growth and differentiation.
Highlights
Human calmodulin-like protein (CLP)1 is encoded by an intronless gene localized on chromosome 10p13-ter [1], a chromo
Initial studies failed to show expression of the CLP gene in a number of tissues and cell types [7]; in an independent study aimed at identifying mRNAs coding for transformation-sensitive proteins, CLP was recloned as a protein named NB1 from a normal primary breast epithelial cell line that had been subtracted with RNA purified from the same cell line after chemical transformation [8]
Identification of a ϳ210-kDa CLP-binding Protein by Gel Overlay—To identify specific CLP targets in total tissue extracts, we used a gel overlay method proven to be successful in the identification of CaM target proteins in various tissues and subcellular fractions (20 –22)
Summary
Human calmodulin-like protein (CLP) is encoded by an intronless gene localized on chromosome 10p13-ter [1], a chromo-. CLP shows a number of characteristics similar to those of CaM; it displays unique features that suggest its functional divergence [11,12,13]. At present there are no known CaM targets that show higher affinity for CLP than for CaM. Knowing the identity of the proteins with which CLP normally interacts will greatly aid in understanding the normal physiological function(s) of CLP as well as the consequences of its down-regulation in transformed cells. We set out to identify such proteins using gel overlays and yeast two-hybrid interaction screening, and report the cloning and characterization of a human unconventional myosin (myosin X) as a specific target of CLP
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