Journal
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
Volume 295, Issue 6, Pages G1159-G1172Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpgi.90345.2008
Keywords
mouse gastric glands; T84 cells; HEK293 cells; protein phosphorylation
Categories
Funding
- National Institute of Diabetes and Digestive and Kidney Diseases [DK-31900]
Ask authors/readers for more resources
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 Ca2+ 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 similar to 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 S136 was inhibited by the CAMK2 inhibitor KN93 (IC50 38 mu M) 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 M-r 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available