Activation of the cAMP signaling pathway is correlated with increased secretory-related events in a wide variety of cell types including the gastric parietal cell. Within this pathway, as well as in other intracellular signaling pathways, protein phosphorylation serves as a major downstream regulatory mechanism. However, although agonist and cAMP-dependent activation of cAMP-dependent protein kinase (PKA) has been demonstrated, little is currently known about the downstream in vivo phosphoprotein substrates of this enzyme. Here we report the isolation, microsequencing, and cloning of a LIM and SH3 domain-containing, cAMP-responsive, 40-kDa phosphoprotein (pp40) from rabbit gastric parietal cells. The deduced amino acid sequence for pp40 is 93.5%, homologous with the putative protein product of the human gene lasp-1, which was recently identified based on its overexpression in some breast carcinomas. In addition to LIM and SH3 domains, the rabbit homolog contains two highly conserved PKA consensus sequences as well as two conserved SH2 binding motifs and several other putative protein kinase phosphorylation sites, including two for tyrosine kinase(s). Combined Northern and Western blot analyses indicate that pp40/lasp-1 is widely expressed (through a single 3.3-kb message) not only in epithelial tissues but also in muscle and brain. Furthermore, stimulation of isolated parietal cells, distal colonic crypts, and pancreatic cells with the adenylyl cyclase activator forskolin leads to the appearance of a higher molecular weight form of pp40/lasp-1, a finding which is consistent with an increase in protein phosphorylation. Thus pp40/lasp-1 appears to be regulated within the cAMP signaling pathway in a wide range of epithelial cell types. Because the cAMP-dependent increase in pp40 phosphorylation is correlated with secretory responses in the parietal cell and because pp40 appears to be widely distributed among various secretory tissues, this newly defined signaling protein may play an important role in modulating ionic transport or other secretory-related activities in many different cell types.

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. GenBank TM searches revealed a 45% homology with the Dictyostelium actin-binding protein, coronin, and ϳ67% 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 coronin se , because it appears to be highly expressed in secretory epithelia.

Although activation of adenosine 3',5'-cyclic monophosphate by histamine and of Ca2+-dependent signaling pathways by cholinergic agonists is a generally recognized mechanism for increasing parietal cell HCl secretion, the role of protein kinase C (PKC) in this process is controversial. In this study, acid-secretory responses of gastric glands from rabbits [measured as accumulation of aminopyrine (AP)] were found to be relatively resistant to the PKC inhibitors calphostin C, chelerythrine chloride, staurosporine, and the bisindolylmaleimide-like inhibitors Ro 31-8220, Gö 6976, and bisindolylmaleimide I hydrochloride. Western analyses of the PKC isozyme profile in highly enriched parietal cells (98% purity) indicated that this cell type expresses abundant levels of the novel isoforms PKC-epsilon and PKC-mu and abundant levels of the atypical isoforms PKC-iota, PKC-lambda, and PKC-zeta. In contrast, there appeared to be low to undetectable expression of the classical isoforms PKC-alpha and PKC-beta1/beta2, respectively. Relatively high concentrations of Ro 31-8220 potentiated both carbachol- and histamine-stimulated AP accumulation (IC50 857 +/- 100 and 910 +/- 98 nM, respectively). There was a similar dose dependence for Ro 31-8220 inhibition of in situ phosphorylation of a parietal cell phosphoprotein, pp66 (IC50 750 +/- 120 nM). Similar concentrations of Ro 31-8220 also inhibited phosphorylation of the cytoskeletal, actin membrane cross-linking phosphoprotein ezrin, but not other phosphoproteins. Ezrin phosphorylation was increased by carbachol and 12-O-tetradecanoylphorbol 13-acetate (TPA). Because carbachol and TPA stimulate pp66 phosphorylation in a Ca2+-independent manner, our results suggest that one or more novel PKC isoforms may be involved in negative regulation of HCl secretion. In related experiments, PKC-epsilon, but not PKC-mu, was immunolocalized by confocal microscopy to a parietal cell compartment that bore a striking resemblance to that containing filamentous actin. Moreover, pp66 was enriched in a Triton X-100-insoluble parietal cell fraction, suggesting a potential cytoskeletal localization for this unknown protein. Given their location and sensitivity to Ro 31-8220, it is possible that pp66 and ezrin interact in a PKC-dependent manner to regulate the well-known morphological changes that occur in concert with agonist-dependent activation of parietal cell HCl secretion.

Epidermal growth factor (EGF) is a potent mitogen for many cell types; however, the best known effect of EGF on gastric parietal cell HCl secretion is inhibition of this response. Using rabbit parietal cells in primary culture, we recently showed that the effect of EGF is biphasic with acute inhibition followed by sustained enhancement of acid secretory-related responses. We hypothesized that EGF might activate a mitogen-activated protein (MAP) kinase signaling pathway in parietal cells, and this pathway might play a role in mediating sustained and/or acute effects of EGF on parietal cell acid secretory-related functions [C. S. Chew, K. Nakamura, and A. C. Petropolous. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G818-G826, 1994]. We used several methodological approaches to demonstrate the presence of MAP kinase (MAPK) isoforms, extracellular signal-regulated kinases (ERKs) 1 and 2, in parietal cells and to begin to characterize their mechanisms of activation in this highly differentiated cell type. In acutely isolated, 90-98% enriched parietal cells, EGF biphasically activated ERK-1 and ERK-2, with peak response occurring at approximately 5 min followed by a sustained lower level of activation for at least 2 h. The EC50 for EGF (1.2 +/- 0.4 nM) was similar to the previously determined EC50 for the stimulatory effect of EGF on acid secretory responses. In contrast to EGF, the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a sustained activation of ERK-1 and ERK-2 for at least 2 h. Carbachol also activated ERK-1 and ERK-2; however, this response was weaker and monophasic. Neither the Ca2+ ionophore ionomycin nor the adenylyl cyclase activator forskolin altered basal or stimulated ERK activity. Carbachol, but not EGF or TPA, also activated an unidentified 70-kDa protein kinase as detected with in-gel myelin basic protein (MBP) kinase renaturation assays. Parietal cell MAPK activation was not correlated to a shift in apparent relative molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggesting that basal phosphorylation of ERK isoforms may be higher in parietal cells compared with actively proliferating cell lines. Also, in contrast to observations in neutrophils, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor, wortmannin (0.3-3 microM), failed to inhibit ERK activation in response to EGF, carbachol, or TPA. The combined data indicate that 1) EGF, TPA, and carbachol activate overlapping as well as distinct intracellular signaling pathways in gastric parietal cells, 2) EGF activates ERKs and enhances parietal cell acid secretory related functions via receptors with similar affinities, and 3) in contrast to some cell types, the parietal cell ERK-signaling cascade does not appear to be directly modulated by the PtdIns 3-kinase pathway or by elevated intracellular free Ca2+ or adenosine 3',5'-cyclic monophosphate concentrations.

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