EXPRESSION AND PHOSPHORYLATION OF A MARCKS-LIKE PROTEIN IN GASTRIC CHIEF CELLS - FURTHER EVIDENCE FOR MODULATION OF PEPSINOGEN SECRETION BYINTERACTION OF CA2+ CALMODULIN WITH PROTEIN-KINASE-C/

In gastric chief cells, agents that activate protein kinase C (PKC) st imulate pepsinogen secretion and phosphorylation of an acidic 72-kDa p rotein. The isoelectric point and molecular mass of this protein are s imilar to those for a common PKC substrate; the MARCKS (for Myristoyla ted Alanine-Rich C Kinase Substrate) protein. We examined expression a nd phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody res ulted in staining of a myristoylated 72-kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief c ells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 mu M), on pepsinogen secretion and phosphorylation of the 72-kDa MA RCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 mu M calcium, and PMA plus calcium, res pectively. A PKC inhibitor(1 mu M CGP 41 251) abolished PMA-induced se cretion, but did not alter calcium-induced secretion. This indicates t hat calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with P-32-orthophosphate and phosphorylatio n of pp72 was detected by autoradiography of 2-dimensional polyacrylam ide gels. In the presence of basal calcium, PMA (100 nM) caused a > tw o-fold increase in phosphorylation of pp72. Without PMA, calcium did n ot alter phosphorylation of pp72. However, 1 mu M calcium caused an ap prox. 50% attenuation of PMA-induced phosphorylation of pp72. Experime nts with a MARCKS ''phosphorylation/calmodulin binding domain peptide' ' indicated that calcium/calmodulin inhibits phosphorylation of pp72 b y binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis tha t, in gastric chief cells, interplay between calcium/calmodulin bindin g and phosphorylation of a common domain on the 72-kDa MARCKS-like pro tein plays a role in modulating pepsinogen secretion. (C) 1997 Wiley-L iss, Inc.