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Am J Physiol Gastrointest Liver Physiol 295: G1159-G1172, 2008. First published October 2, 2008; doi:10.1152/ajpgi.90345.2008
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MUCOSAL BIOLOGY

Calcium/calmodulin-dependent phosphorylation of tumor protein D52 on serine residue 136 may be mediated by CAMK2{delta}6

Catherine S. Chew,1 Xunsheng Chen,1 Hanfang Zhang,1 Eric A. Berg,2 and Han Zhang1,3

1Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia; 221st Century Biochemicals, Marlboro, Massachusetts; 3Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China

Submitted 20 May 2008 ; accepted in final form 25 September 2008

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, S136, that undergoes increased phosphorylation upon elevation of intracellular Ca2+ concentration. A phosphospecific antibody (pS136) 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 (Mr) of ~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 (pT286/287) antibody when activated. Carbachol-stimulated phosphorylation of S136 was inhibited by the CAMK2 inhibitor KN93 (IC50 38 µM) and by the calmodulin antagonist W7 (IC50 3.3 nM). A previously uncharacterized CAMK2 isoform, CAMK2{delta}6, which has the same domain structure and Mr as CAM2{alpha}, 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 CAMK2{delta}6 in the mediation of D52 phosphorylation.

mouse gastric glands; T84 cells; HEK293 cells; protein phosphorylation


Address for reprint requests and other correspondence: C. S. Chew, IMMAG, Medical College of GA, Rm. CB2803, Sanders R&E Bldg., Augusta, GA 30912 (e-mail: cchew{at}mcg.edu)






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