Retinoic acid causes MEK-dependent RAF phosphorylation through RARalpha plus RXR activation in HL-60 cells.
Retinoic acid (RA) is known to cause the myeloid differentiation of HL-60 human myeloblastic leukemia cells in a process requiring MEK-dependent ERK2 activation. This RA-induced ERK2 activation appears after approximately 4 h and persists until the cells are differentiated and G0 arrested (Yen et al, 1998). This motivates the question of whether RA also activated RAF as part of a typical RAF/MEK/MAPK cascade. Retinoic acid is shown here to also increase the phosphorylation of RAF, but in an unusual way. Surprisingly, increased RAF phosphorylation is first detectable after 12 to 24 hours by phosphorylation-induced retardation of polyacrylamide gel electrophoretic mobility. The RA-induced increased RAF phosphorylation is still apparent after 72 hours of treatment when most cells are differentiated and G0 arrested. There is a progressive dose-response relationship with 10(-8), 10(-7), and 10(-6) M RA. The RA-induced RAF phosphorylation corresponds to increased in vitro kinase activity. Inhibition of MEK with a PD98059 dose which inhibits ERK2 phosphorylation and subsequent cell differentiation also inhibits RAF phosphorylation. RA-induced MEK-dependent RAF phosphorylation is not due to changes in the amount of cellular MEK. The induced RAF phosphorylation, as well as anteceding ERK2 activation, depends on ligand-induced activation of both an RARalpha receptor and an RXR receptor. This and the slow kinetics of activation suggest a need for prior RA-induced gene expression. In summary, RA induces a MEK-dependent prolonged RAF activation, whose slow onset occurs after ERK2 activation but still well before cell cycle arrest and cell differentiation. The RA-induced increased RAF phosphorylation thus differs from typical mitogenic growth factor signaling, features that may contribute to cell cycle arrest and differentiation instead of division as the cellular outcome.
Hong
H Y
HY
Department of Biomedical Science, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Varvayanis
S
S
Yen
A
A
D002454:Cell Differentiation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007399:Interphase / Q000187:drug effects; D048728:MAP Kinase Kinase Kinase 1; D008301:Maleimides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000187:drug effects / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D019311:Staurosporine / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014212:Tretinoin / Q000378:metabolism / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D008301:Maleimides; D009281:Naphthalenes; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014212:Tretinoin; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D019311:Staurosporine; C058819:calphostin C; C070515:bisindolylmaleimide I; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one