Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens

Using an N-bromosuccinimide cleavage fragment of histone H1 as a relatively specific substrate for protein kinase C, we evaluated the partitioning of this kinase activity between soluble and particulate cellular fractions in 3T3-L1 fibroblasts. In confluent, serum-deprived cells, protein kinase C ac...

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Bibliographic Details
Published in:The Journal of biological chemistry 1987-02, Vol.262 (5), p.2234-2243
Main Authors: Halsey, D.L., Girard, P.R., Kuo, J.F., Blackshear, P.J.
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Bombesin - pharmacology
Bromosuccinimide - pharmacology
Dose-Response Relationship, Drug
Enzymes and enzyme inhibitors
Fibroblast Growth Factors - pharmacology
fibroblasts
Fibroblasts - enzymology
Fundamental and applied biological sciences. Psychology
Histocytochemistry
Intracellular Membranes - enzymology
Mice
Mitogens - pharmacology
Molecular Weight
phorbol 12-myristate 13-acetatediester
Phorbol Esters - pharmacology
protein kinase C
Protein Kinase C - metabolism
Tetradecanoylphorbol Acetate - pharmacology
Transferases
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Summary:Using an N-bromosuccinimide cleavage fragment of histone H1 as a relatively specific substrate for protein kinase C, we evaluated the partitioning of this kinase activity between soluble and particulate cellular fractions in 3T3-L1 fibroblasts. In confluent, serum-deprived cells, protein kinase C activity was approximately equally divided between soluble and detergent-extractable particulate fractions; both rapidly growing and transformed cells appeared to contain higher levels of particulate enzyme activity. Soluble protein kinase C activity and immunoreactivity decreased to virtually undetectable levels after exposure of the cells to phorbol 12-myristate 13-acetate (PMA), associated with a commensurate increase in particulate kinase activity and immunoreactivity. In intact cells, PMA appeared to cause a shift of immunoreactive protein kinase C from the cytosol to the perinuclear region, as assessed by immunofluorescent microscopy; however; subcellular fractionation revealed that PMA caused increases in the protein kinase C activity associated primarily with non-nuclear membranes. Exposure of the cells to sn-1,2-dioctanoylglycerol resulted in a modest and transient membrane association of protein kinase C, whereas platelet-derived growth factor, fibroblast growth factor, and bombesin caused no detectable increases in the membrane association of the kinase. Activation of protein kinase C by growth factors in fibroblasts may occur without the gross disturbances in intracellular kinase location which occur in response to phorbol esters.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)61644-8