Purification and cloning of the novel phosphoprotein copine III and characterization of its associated kinase activity

Eva Gisela Caudell, The University of Texas Graduate School of Biomedical Sciences at Houston

Abstract

The copines, named and first described by Creutz et al. (1998), comprise a two C2 domain-containing protein family that can aggregate phosphatidylserine membranes in a calcium-dependent manner. Although no enzymatic function has been attributed to copines, their carboxyl terminus shows homology to the A domain found in integrins that allows binding of magnesium ions. The secondary structure of A domains resembles a Rossmann fold, which can bind dinucleotides and is present in a number of intracellular enzymes. Due to a crossreacting activity of Mik[special characters omitted]1, an antibody to the IL-2R[special characters omitted] chain, we were able to serendipitously clone human copine III (CIII). CIII is 65% identical to copine I (CI) and the 5 kb CIII transcript is expressed ubiquitously as determined by a multitissue Northern blot. A polyclonal antibody generated against the carboxyl terminus of CIII recognized CIII in immunoblots and immunoprecipitations. Phosphorylation of CIII was observed on serine and threonine residues, as determined by phosphoamino acid analysis. Experiments were designed to determine whether or not any enzymatic activity, specifically kinase activity, was intrinsic to or associated with CIII. In vitro and in gel kinase assays were performed using transfected HA-tagged CI and CIII, immunoprecipitated endogenous CIII and purified endogenous CIII. The exogenous substrate MBP was phosphorylated in all in vitro kinase assays containing CIII protein purification and column chromatography expertise with me.

Subject Area

Cellular biology|Molecular biology

Recommended Citation

Caudell, Eva Gisela, "Purification and cloning of the novel phosphoprotein copine III and characterization of its associated kinase activity" (1999). Texas Medical Center Dissertations (via ProQuest). AAI9951894.
https://digitalcommons.library.tmc.edu/dissertations/AAI9951894

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