Activation of mitogen-activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C-dependent mechanism.
Arachidonic acid (20:4(n-6)), which is released by cells responding to a wide range of stimuli, may play an important role in intracellular signaling. We now report that incubation of WB cells with 20:4(n-6) resulted in the appearance of several tyrosine-phosphorylated cytosolic proteins. Two of the phosphotyrosine-containing proteins, migrating in SDS-polyacrylamide gels of approximately 43 and 45 kDa, corresponded in mobility to phosphorylated species of the 42- and 44-kDa mitogen-activated protein kinase (MAPK) isoforms. Immunoblots of soluble fractions from unstimulated WB cells with anti-MAPK antibodies revealed the presence of the 42- and 44-kDa isoforms of MAPK. Upon incubation with 20:4(n-6), the mobility of both isoforms was retarded, consistent with their activation by phosphorylation. Chromatography of soluble fractions from these cells on Mono Q columns revealed early and late eluting peaks of myelin basic protein kinase activity, which contained the 42- and 44-kDa MAPK isoforms, respectively. Activation of MAPK was transient, peaking at 5 min, and was detectable at 5 microM 20:4(n-6). Further studies into the mechanisms by which MAPK was activated by 20:4(n-6) strongly suggested the involvement of protein kinase C (PKC). Not only did incubation of WB cells with 20:4(n-6) result in the translocation of PKC alpha, delta, and epsilon to a particulate fraction, it was found that the fatty acid failed to activate MAPK in cells pretreated for 26 h with phorbol 12-myristate 13-acetate, which depleted WB cells of PKC alpha, delta and epsilon. In addition, fatty acids of the n-3 series were effective activators of MAPK. The present study, to our knowledge, is the first to report that polyunsaturated fatty acids can cause the activation of MAPK.
Hii
C S
CS
Department of Immunology, Women's and Children's Hospital, North Adelaide, Australia.
Ferrante
A
A
Edwards
Y S
YS
Huang
Z H
ZH
Hartfield
P J
PJ
Rathjen
D A
DA
Poulos
A
A
Murray
A W
AW
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D011493:Protein Kinase C / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005231:Fatty Acids, Unsaturated; D016718:Arachidonic Acid; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate