Interleukin-1 activates p54 mitogen-activated protein (MAP) kinase/stress-activated protein kinase by a pathway that is independent of p21ras, Raf-1, and MAP kinase kinase.
In KB epidermoid cells, we previously showed that interleukin-1 alpha (IL-1) and various mitogens activate the mitogen-activated protein (MAP) kinases ERK1 and ERK2, which phosphorylate both myelin basic protein (MBP) and a peptide containing Thr669 of the epidermal growth factor receptor. In cell-free extracts made from gingival fibroblasts treated with platelet-derived growth factor or HepG2 hepatoma cells stimulated with phorbol myristate acetate, MBP and Thr669 kinase were both elevated 4-fold, and ERK1 and ERK2 were tyrosine-phosphorylated. In these cells IL-1 activated a kinase(s) that phosphorylated Thr669 peptide but not MBP and failed to cause tyrosine phosphorylation of ERK1/ERK2. Ceramide has been proposed as an intracellular mediator of IL-1 action, but C2-ceramide or sphingosine stimulated predominantly MBP-specific kinase activity in fibroblasts and had no effect in HepG2 cells. p54 MAP kinase (also called stress-activated protein kinase) is a c-Jun kinase first isolated from livers of cycloheximide-treated rats. After IL-1 stimulation, immunoprecipitates of lysates made from all three cell types with specific anti-p54 MAP kinase serum contained Thr669 and c-Jun phosphorylating activity, whereas precipitates from unstimulated cells contained no detectable p54 kinase activity. The major peak of IL-1-stimulated HepG2 Thr669 kinase activity co-chromatographed on Mono Q and phenyl-Superose with immunodetectable p54 MAP kinase. IL-1 did not cause p21ras activation in any cell type. Induction of Thr 669 kinase activity was not abrogated by elevation of cAMP levels, which has been shown to interfere with the activation of Raf-1. We could not detect MAP kinase kinase phosphorylating activity in unfractionated lysates made from IL-1-stimulated fibroblasts or HepG2 cells. KB cells contained a small amount of this activity, but it was not precipitated with an anti-Raf-1 antibody. We conclude that most of the IL-1-activated Thr669 kinase activity in fibroblasts and HepG2 cells, and a portion in KB cells, is due to p54 MAP kinase and that its activation is Ras-, Raf-, and MAP kinase kinase-independent.
Bird
T A
TA
Department of Biochemistry, Immunex Corporation, Seattle, Washington 98101.
Kyriakis
J M
JM
Tyshler
L
L
Gayle
M
M
Milne
A
A
Virca
G D
GD
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002518:Ceramides / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005881:Gingiva / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000494:pharmacology; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D002518:Ceramides; D007375:Interleukin-1; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D010769:Phosphothreonine; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D000242:Cyclic AMP; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate