Abstract
In order to understand the regulatory role of protein kinase C (PKC) in secretory epithelia, it is necessary to identify and characterize specific downstream targets. We previously identified one such protein in studies of gastric parietal cells. This protein was referred to as pp66 because it migrated with an apparent molecular mass of 66 kDa on SDS-polyacrylamide gels. The phosphorylation of pp66 is increased by the cholinergic agonist, carbachol, and by the PKC activator, phorbol-12-myristate-13-acetate, in a calcium-independent manner. In this study, we have purified pp66 to homogeneity and cloned the complete open reading frame. GenBankTM searches revealed a 45% homology with the Dictyostelium actin-binding protein, coronin, and approximately 67% homology with the previously cloned human and bovine coronin-like homologue, p57. pp66 appears to be most highly expressed in the gastrointestinal mucosa and in kidney and lung. Confocal microscopic studies of an enhanced green fluorescent protein fusion construct of pp66 in cultured parietal cells and in Madin-Darby canine kidney cells indicate that pp66 preferentially localizes in F-actin-rich regions. On the basis of our findings, we propose that pp66 may play an important, PKC-dependent role in regulating membrane/cytoskeletal rearrangements in epithelial cells. We have tentatively named this protein coroninse, because it appears to be highly expressed in secretory epithelia.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF056312 and AF100414
We report the isolation, cloning, and characterization of a new mammalian coronin family member, which we have named coroninse because, in contrast to p57, it is highly expressed in a variety of secretory-type epithelial tissues including stomach, intestine, kidney, and lung
Characterization of pp66 Phosphorylation Response in Vivo and in Vitro—We previously demonstrated that carbachol increases pp66 phosphorylation in parietal cells and that this response is 1) mimicked by the protein kinase C (PKC) activator, phorbol-12myristate-13-acetate; 2) calcium-independent; and 3) inhibited by the bisindolylmaleimide PKC inhibitor, Ro 318220 [11, 12]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF056312 and AF100414. We report the isolation, cloning, and characterization of a new mammalian coronin family member, which we have named coroninse because, in contrast to p57, it is highly expressed in a variety of secretory-type epithelial tissues including stomach, intestine, kidney, and lung. Coroninse contains numerous PKC phosphorylation consensus sites as well as a putative membrane-spanning region, characteristic of type 1b membrane proteins, near the amino terminus. This latter region was not previously identified in either coronin or p57, it is highly conserved in all three protein sequences. We present evidence that pp66/coroninse is primarily located in an intracellular canalicular region of parietal cells, which is the site of active HCl secretion. The onset of HCl secretion is correlated with dramatic morphological transformations in which internal tubulovesicles containing the proton-
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