Protein kinase C inhibitor proteins. Purification from sheep brain and sequence similarity to lipocortins and 14-3-3 protein.
Potent inhibitors of protein kinase C have been isolated from sheep brain by DEAE-cellulose, phenyl-Sepharose CL-4B and Mono Q anion-exchange chromatography. Analysis by one- and two-dimensional SDS/polyacrylamide gel electrophoresis showed the purified preparation to contain three bands ranging over 29-33 kDa in molecular mass, each consisting of several charge isomers with similar pI values (5.4-5.7). Peptide mapping, amino acid analysis and sequencing suggested that the proteins are related, with the possibility that some species are distinct gene products. The concentration of inhibitor proteins required for half-maximal inhibition of protein kinase C activity is 1.7 microM. Inhibitory activity could not be affected by increasing the substrate, cofactor or ATP concentration in the standard protein kinase C assay, but was abolished by heat treatment. The inhibitor preparation did not affect the binding of phorbol dibutyrate to protein kinase C and could inhibit phosphorylation over a wide range of calcium concentrations. Inhibitory activity could be removed by immunoprecipitation of the purified inhibitor proteins with polyclonal antibodies raised against synthetic peptides, the sequences corresponding to those of peptide fragments obtained from protein digests. Amino acid sequence analysis of the inhibitors confirms they are novel proteins although similarities exist with a neuronal specific protein termed 14-3-3 and the carboxy terminus of the calcium-lipid binding series (endonexin/calpactin/lipocortin).
Toker
A
A
Laboratory of Protein Structure, National Institute for Medical Research, Mill Hill, London, England.
Ellis
C A
CA
Sellers
L A
LA
Aitken
A
A
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D017302:Annexins; D001921:Brain / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002352:Carrier Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000302:isolation & purification*; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D012756:Sheep; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D017302:Annexins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; C015482:protein kinase modulator; D014446:Tyrosine 3-Monooxygenase; D011493:Protein Kinase C