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Identification of calcium‐dependent, phospholipid‐ and calmodulin‐activated protein kinases in rat small intestinal epithelium
Author(s) -
WARHURST GEOFFREY,
SMITH GEOFFREY S.,
TONGE ANNE,
LEES MARTIN,
TURNBERG LESLIE A.
Publication year - 1988
Publication title -
journal of gastroenterology and hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.214
H-Index - 130
eISSN - 1440-1746
pISSN - 0815-9319
DOI - 10.1111/j.1440-1746.1988.tb00256.x
Subject(s) - calmodulin , protein kinase c , biochemistry , kinase , calcium , protein kinase a , cytosol , myosin light chain kinase , biology , phosphorylation , phosphatidylserine , diacylglycerol kinase , protein phosphorylation , microbiology and biotechnology , chemistry , phospholipid , enzyme , membrane , organic chemistry
The mechanisms by which increases in cytosolic calcium promote ion‐secretion in the intestinal epithelium may involve the phosphorylation of key transport proteins in the microvillus membrane. Using both direct and indirect methods evidence has been provided for two distinct calcium‐regulated protein kinase activities in rat small intestinal epithelium — one activated by phosphatidylserine (C‐kinase), the other by calmodulin (CDR‐kinase). C‐kinase activity was identified in cytosolic and particulate fractions including purified brush border membranes using a specific histone substrate. In the presence of phosphatidylserine and diacylglycerol, calcium markedly stimulated enzyme activity, half‐maximal effects occurring at 2.8X10 ‐7 mol/1 free calcium. Phorbol esters, known activators of C‐kinase, were also shown to stimulate the phosphorylation of an endogenous peptide (Mr = 94 000) in cell homogenates. The addition of exogenous calmodulin (10–100 μg/ml) stimulated a 50–75% increase in the labelling of peptide (Mr = 50 000–55 000). Trifluoperazine (0.1–10 μmol/l) inhibited basal phosphorylation of this and other peptides suggesting the likely involvement of a protein kinase activated by endogenous levels of calmodulin. Cytosolic fractions were also able to phosphorylate purified myosin light chains, a known substrate for CDR‐kinase, in the presence of submicromolar concentrations of free calcium (ED 50 = 2.4X10 ‐7 mol/l) although no direct stimulation of this activity by calmodulin could be demonstrated. MLC‐phosphorylated activity was not identified in particulate fractions.