Raufman J P, Malhotra R, Xie Q, Raffaniello R D
Division of Gastroenterology, University of Arkansas for Medical Sciences, Little Rock 72205-7199, USA.
J Cell Biochem. 1997 Mar 1;64(3):514-23.
In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and 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 resulted in staining of a myristoylated 72-kDA protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 microM), on pepsinogen secretion and phosphorylation of the 72-KDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 microM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 microM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium, PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 microM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS "phosphorylation/calmodulin binding domain peptide" indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion.
在胃主细胞中，激活蛋白激酶C（PKC）的物质可刺激胃蛋白酶原分泌以及一种72 kDa酸性蛋白的磷酸化。该蛋白的等电点和分子量与常见的PKC底物——豆蔻酰化富含丙氨酸的C激酶底物（MARCKS）蛋白相似。我们检测了豚鼠胃主细胞近乎均一的悬浮液中类MARCKS蛋白的表达和磷酸化情况。用特异性MARCKS抗体对主细胞裂解物的各组分进行蛋白质印迹分析，结果显示一种豆蔻酰化的72 kDa蛋白（pp72）被染色，该蛋白主要与膜组分相关。使用透化处理的主细胞，我们检测了在基础钙浓度（100 nM）或细胞钙浓度升高（1 μM）的情况下，PKC激活剂（佛波酯PMA）对胃蛋白酶原分泌以及72 kDa类MARCKS蛋白磷酸化的影响。分别用100 nM PMA、1 μM钙以及PMA加钙孵育后，分泌量分别增加了2.3倍、2.6倍和4.5倍。一种PKC抑制剂（1 μM CGP 41 251）消除了PMA诱导的分泌，但未改变钙诱导的分泌。这表明钙诱导的分泌独立于PKC激活。用32P-正磷酸盐标记主细胞蛋白，并通过二维聚丙烯酰胺凝胶放射自显影检测pp72的磷酸化情况。在基础钙浓度存在的情况下，PMA（100 nM）使pp72的磷酸化增加了两倍以上。在没有PMA的情况下，钙不会改变pp72的磷酸化。然而，1 μM钙使PMA诱导的pp72磷酸化大约减弱了50%。用MARCKS“磷酸化/钙调蛋白结合域肽”进行的实验表明，钙/钙调蛋白通过结合磷酸化/钙调蛋白结合域而非抑制PKC活性来抑制pp72的磷酸化。这些观察结果支持以下假说：在胃主细胞中，钙/钙调蛋白结合与72 kDa类MARCKS蛋白上一个共同结构域的磷酸化之间的相互作用在调节胃蛋白酶原分泌中起作用。
