Calcium/calmodulin-dependent phosphorylation of tumor protein D52 on serine residue 136 may be mediated by CAMK2delta6.
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.
Chew
Catherine S
CS
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA. cchew@mcg.edu
Chen
Xunsheng
X
Zhang
Hanfang
H
Berg
Eric A
EA
Zhang
Han
H
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*
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