title: Calcium/calmodulin-dependent phosphorylation of tumor protein D52 on serine residue 136 may be mediated by CAMK2delta6.
abstract: Tumor protein D52 is expressed at relatively high levels in cells within the gastrointestinal tract that undergo classical exocytosis and is overexpressed in several cancers. Current evidence supports a role for D52 in the regulation of vesicular trafficking. D52 function(s) are regulated by calcium-dependent phosphorylation; however, the intracellular mechanisms that mediate this process are not well characterized. The goal of this study was to identify the calcium-dependent phosphorylation site(s) in D52 and to characterize the protein kinase(s) that mediate this phosphorylation. Using mass spectrometry and site-directed mutagenesis, we identified a single amino acid residue, S(136), that undergoes increased phosphorylation upon elevation of intracellular Ca(2+) concentration. A phosphospecific antibody (pS(136)) was produced and used to characterize D52 kinase activity in gastric mucosal, colonic T84, and HEK293 cells. By using D52 as a substrate, a protein kinase with a molecular weight (M(r)) of approximately 50 kDa was identified with "in gel" assays. This kinase comigrated with rat brain calcium/calmodulin-dependent protein kinase (CAMK2)alpha cross-reacted with pan-specific CAMK2 antibodies as well as with anti-active CAMK2 (pT(286/287)) antibody when activated. Carbachol-stimulated phosphorylation of S(136) was inhibited by the CAMK2 inhibitor KN93 (IC(50) 38 microM) and by the calmodulin antagonist W7 (IC(50) 3.3 nM). A previously uncharacterized CAMK2 isoform, CAMK2delta6, which has the same domain structure and M(r) as CAM2alpha, was identified in gastric mucosa by RT-PCR. The cloned, expressed protein comigrated with D52 kinase and colocalized with D52 protein in T84 and HEK293 cells. These findings support a role for CAMK2delta6 in the mediation of D52 phosphorylation.
authors.affiliation: Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA. cchew@mcg.edu
authors.forename: Catherine S
authors.initials: CS
authors.lastname: Chew
authors.forename: Xunsheng
authors.initials: X
authors.lastname: Chen
authors.forename: Hanfang
authors.initials: H
authors.lastname: Zhang
authors.forename: Eric A
authors.initials: EA
authors.lastname: Berg
authors.forename: Han
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D005753:Gastric Mucosa / Q000166:cytology; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011817:Rabbits; D012694:Serine / Q000378:metabolism*
chemical_list: D009363:Neoplasm Proteins; D047428:Protein Kinase Inhibitors; C114751:TPD52 protein, human; D012694:Serine; D002217:Carbachol; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium