Negative modulation of membrane localization of the Raf-1 protein kinase by hyperphosphorylation.
The serine/threonine-specific protein kinase Raf-1 plays a key role in mitogenic signal transduction by coupling Ras to the mitogen-activated protein (MAP) kinase cascade. Ras-mediated translocation to the plasma membrane represents a crucial step in the process of serum-stimulated Raf-1 kinase activation. The exact role of the multisite phosphorylation in Raf regulation, however, is not clear. We have previously reported that the mobility shift-associated hyperphosphorylation of Raf correlates with a reduction of serum-stimulated Raf kinase activity (Wartmann, M., and Davis, R. J. (1994) J. Biol. Chem. 269, 6695-6701). Here we show that incubation of serum-starved CHO cells with D609, a purported inhibitor of phosphatidylcholine-specific phospholipase C, also results in a mobility shift of Raf-1 that is due to hyperphosphorylation on sites identical to those observed following mitogen stimulation. Subcellular fractionation analyses revealed that D609-induced mobility shift-associated hyperphosphorylation was paralleled by a decreased membrane association of Raf-1. Similar results were obtained in an in vitro reconstitution system. Furthermore, PD98059, a specific inhibitor of activation of the MAP kinase kinase MEK, prevented D609-induced Raf hyperphosphorylation and restored the amount of membrane-bound Raf to control levels. Taken together, these data suggest that mobility shift-associated hyperphosphorylation of Raf-1, by virtue of reducing the amount of plasma membrane-bound Raf-1, represents a negative feedback mechanism contributing to the desensitization of the MAP kinase signaling cascade.
Wartmann
M
M
Friedrich Miescher Institute, P. O. Box 2543, CH-4002 Basel, Switzerland.
Hofer
P
P
Turowski
P
P
Saltiel
A R
AR
Hynes
N E
NE
D000818:Animals; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009636:Norbornanes; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D001952:Bridged-Ring Compounds; D005419:Flavonoids; D008565:Membrane Proteins; D009636:Norbornanes; D011518:Proto-Oncogene Proteins; D013859:Thiocarbamates; D013871:Thiones; C046498:tricyclodecane-9-yl-xanthogenate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D010738:Type C Phospholipases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one