Differential Raf requirement for activation of mitogen-activated protein kinase by growth factors, phorbol esters, and calcium.
Although a pathway that requires sequential activation of Ras, Raf, and MAP kinase kinase has been proposed as the major mechanism for stimulation of mitogen-activated protein kinase (MAP kinase), alternative pathways also exist. A wide variety of extracellular stimuli have been shown to activate MAP kinase; however, the precise mechanisms by which these stimuli mediate the signaling events have not been elucidated. Using a Balb/c-derived cell line expressing a dominant-negative mutant of Raf, we determined whether Raf is required for the activation of MAP kinase by growth factors, phorbol esters, and calcium. Insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and phorbol 12,13-dibutyrate activated Ras in both mutant and control cells. However, stimulation of MAP kinase by IGF-I was nearly abolished in the dominant-negative Raf mutant. Stimulation of MAP kinase by the Ca2+ mobilizer thapsigargin was also inhibited in the presence of the Raf mutant. In contrast, EGF and phorbol 12,13-dibutyrate remained potent stimulators of MAP kinase in the dominant-negative Raf cells. The activation of MAP kinase by these stimuli can be further distinguished by differential requirements for Ca2+ and protein kinase C. These results suggest that Raf is required for the activation of MAP kinase by IGF-I and calcium, whereas EGF and possibly phorbol esters may employ alternative Raf-independent pathways for MAP kinase activation.
Chao
T S
TS
Ben May Institute, University of Chicago, Illinois 60637.
Foster
D A
DA
Rapp
U R
UR
Rosner
M R
MR
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D015536:Down-Regulation; D004789:Enzyme Activation; D006133:Growth Substances / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D019950:Mitogen-Activated Protein Kinase 1; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D006133:Growth Substances; D011518:Proto-Oncogene Proteins; D013729:Terpenes; D015240:Phorbol 12,13-Dibutyrate; D019284:Thapsigargin; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D002118:Calcium