Show simple item record

dc.contributor.authorYu, Kin-Tak
dc.contributor.authorKhalaf, Nazer
dc.contributor.authorCzech, Michael P.
dc.date2022-08-11T08:10:05.000
dc.date.accessioned2022-08-23T16:54:43Z
dc.date.available2022-08-23T16:54:43Z
dc.date.issued1987-12-05
dc.date.submitted2008-08-15
dc.identifier.citationJ Biol Chem. 1987 Dec 5;262(34):16677-85.
dc.identifier.issn0021-9258 (Print)
dc.identifier.pmid2960679
dc.identifier.urihttp://hdl.handle.net/20.500.14038/42567
dc.description.abstractThe cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=2960679&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://www.jbc.org/content/262/34/16677.full.pdf+html
dc.subjectAdipose Tissue
dc.subjectAnimals
dc.subjectChromatography, Ion Exchange
dc.subjectCytosol
dc.subjectDithiothreitol
dc.subjectDose-Response Relationship, Drug
dc.subjectHeparin
dc.subjectHydrogen-Ion Concentration
dc.subjectInsulin
dc.subjectInsulin Antibodies
dc.subjectMale
dc.subjectManganese
dc.subjectOligopeptides
dc.subjectProtein Kinases
dc.subjectProtein-Serine-Threonine Kinases
dc.subjectRats
dc.subjectRats, Inbred Strains
dc.subjectSubstrate Specificity
dc.subjectTemperature
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.titleInsulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes
dc.typeJournal Article
dc.source.journaltitleThe Journal of biological chemistry
dc.source.volume262
dc.source.issue34
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/903
dc.identifier.contextkey579792
html.description.abstract<p>The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.</p>
dc.identifier.submissionpathoapubs/903
dc.contributor.departmentDepartment of Biochemistry
dc.source.pages16677-85


This item appears in the following Collection(s)

Show simple item record