Characterization of human copine III as a phosphoprotein with associated kinase activity

The copines, first described by Creutz et al. [(1998) J. Biol. Chem. 273, 1 393-1402], comprise a two C2 domain-containing protein family and are known to aggregate phosphatidylserine membranes in a calcium-dependent manner. N o enzymatic function has been attributed to copines yet. Due to a crossreac ting activity of Mik beta1, an antibody to the IL-2R beta chain, we were ab le to serendipitously purify, partially microsequence, and clone human copi ne III. The 5 kb copine III transcript is expressed ubiquitously as determi ned by a multitissue Northern blot analysis. Phosphoamino acid analysis rev ealed phosphorylation of copine III on serine and threonine residues. In vi tro kinase assays were performed with immunoprecipitated endogenous copine III, chromatography-purified endogenous copine III, and recombinant copine III expressed in Saccharomyces cerevisiae. The exogenous substrate myelin b asic protein was phosphorylated in all in vitro kinase assays containing co pine III immunoprecipitate or purified copine III. A 60-kDa band was observ ed in corresponding in gel kinase assays with staurosporine-activated cells . Cell Lines expressing high levels of copine III protein had corresponding ly high kinase activity in copine III antiserum immunoprecipitate. However, the copine amino acid sequences lack the traditional kinase catalytic doma in. Therefore, the data suggest copine III may possess an intrinsic kinase activity and represent a novel unconventional kinase family.