Abstract
Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co- factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM- PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.
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- Ahmad Z., DePaoli-Roach A. A., Roach P. J. Purification and characterization of a rabbit liver calmodulin-dependent protein kinase able to phosphorylate glycogen synthase. J Biol Chem. 1982 Jul 25;257(14):8348–8355. [PubMed] [Google Scholar]
- Burnham D. B., Williams J. A. Effects of carbachol, cholecystokinin, and insulin on protein phosphorylation in isolated pancreatic acini. J Biol Chem. 1982 Sep 10;257(17):10523–10528. [PubMed] [Google Scholar]
- Carlin R. K., Grab D. J., Siekevitz P. Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density. J Cell Biol. 1981 Jun;89(3):449–455. doi: 10.1083/jcb.89.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chafouleas J. G., Dedman J. R., Munjaal R. P., Means A. R. Calmodulin. Development and application of a sensitive radioimmunoassay. J Biol Chem. 1979 Oct 25;254(20):10262–10267. [PubMed] [Google Scholar]
- Freedman S. D., Jamieson J. D. Hormone-induced protein phosphorylation. I. Relationship between secretagogue action and endogenous protein phosphorylation in intact cells from the exocrine pancreas and parotid. J Cell Biol. 1982 Dec;95(3):903–908. doi: 10.1083/jcb.95.3.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Freedman S. D., Jamieson J. D. Hormone-induced protein phosphorylation. II. Localization to the ribosomal fraction from rat exocrine pancreas and parotid of a 29,000-dalton protein phosphorylated in situ in response to secretagogues. J Cell Biol. 1982 Dec;95(3):909–917. doi: 10.1083/jcb.95.3.909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Freedman S. D., Jamieson J. D. Hormone-induced protein phosphorylation. III. regulation of the phosphorylation of the secretagogue-responsive 29,000-dalton protein by both Ca2+ and cAMP in vitro. J Cell Biol. 1982 Dec;95(3):918–923. doi: 10.1083/jcb.95.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gershoni J. M., Palade G. E. Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal Biochem. 1982 Aug;124(2):396–405. doi: 10.1016/0003-2697(82)90056-2. [DOI] [PubMed] [Google Scholar]
- Gershoni J. M., Palade G. E. Protein blotting: principles and applications. Anal Biochem. 1983 May;131(1):1–15. doi: 10.1016/0003-2697(83)90128-8. [DOI] [PubMed] [Google Scholar]
- Goldenring J. R., Gonzalez B., DeLorenzo R. J. Isolation of brain Ca2+-calmodulin tubulin kinase containing calmodulin binding proteins. Biochem Biophys Res Commun. 1982 Sep 16;108(1):421–428. doi: 10.1016/0006-291x(82)91883-6. [DOI] [PubMed] [Google Scholar]
- Issinger O. G., Beier H., Speichermann N., Flokerzi V., Hofmann F. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Biochem J. 1980 Jan 1;185(1):89–99. doi: 10.1042/bj1850089. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kennedy M. B., McGuinness T., Greengard P. A calcium/calmodulin-dependent protein kinase from mammalian brain that phosphorylates Synapsin I: partial purification and characterization. J Neurosci. 1983 Apr;3(4):818–831. doi: 10.1523/JNEUROSCI.03-04-00818.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
- Payne M. E., Schworer C. M., Soderling T. R. Purification and characterization of rabbit liver calmodulin-dependent glycogen synthase kinase. J Biol Chem. 1983 Feb 25;258(4):2376–2382. [PubMed] [Google Scholar]
- Schulz I. Messenger role of calcium in function of pancreatic acinar cells. Am J Physiol. 1980 Nov;239(5):G335–G347. doi: 10.1152/ajpgi.1980.239.5.G335. [DOI] [PubMed] [Google Scholar]
- Traugh J. A., Porter G. G. A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro. Biochemistry. 1976 Feb 10;15(3):610–616. doi: 10.1021/bi00648a025. [DOI] [PubMed] [Google Scholar]
- Udenfriend S., Stein S., Böhlen P., Dairman W., Leimgruber W., Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972 Nov 24;178(4063):871–872. doi: 10.1126/science.178.4063.871. [DOI] [PubMed] [Google Scholar]
- Wrenn R. W., Katoh N., Kuo J. F. Stimulation by phospholipid of calcium-dependent phosphorylation of endogenous proteins from mammalian tissues. Biochim Biophys Acta. 1981 Aug 17;676(2):266–269. doi: 10.1016/0304-4165(81)90195-1. [DOI] [PubMed] [Google Scholar]