Interactions of c-Raf-1 with phosphatidylserine and 14-3-3.
Activation of Raf-1 occurs at the plasma membrane. We recently showed that 14-3-3 must be complexed with Raf-1 for efficient recruitment to the plasma membrane and activation by Ras, but that 14-3-3 is completely displaced from Raf-1 following plasma membrane binding. We show here that the Raf-1 zinc finger is not absolutely required for 14-3-3 binding but is required to stabilize the interaction between Raf-1 and 14-3-3. Incubation of Raf-1 with phosphatidylserine, an inner plasma membrane phospholipid, results in removal of 14-3-3 and an increase in Raf-1 kinase activity, whereas removal of 14-3-3 from Raf-1 using specific phosphopeptides substantially reduces Raf-1 basal kinase activity. Displacement of 14-3-3 from activated Raf-1 by phosphopeptides has no effect on kinase activity if Raf-1 is first removed from solution, but completely eradicates kinase activity of soluble activated Raf-1. These results suggest a mechanism for the removal of 14-3-3 from Raf-1 at the plasma membrane and show that removal of 14-3-3 from Raf-1 has markedly different effects depending on experimental conditions.
McPherson
R A
RA
Queensland Cancer Fund Laboratory for Experimental Oncology, Department of Pathology, University of Queensland Medical School, Herston, Australia.
Harding
A
A
Roy
S
S
Lane
A
A
Hancock
J F
JF
D048948:14-3-3 Proteins; D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase; D016335:Zinc Fingers / Q000502:physiology
D048948:14-3-3 Proteins; D010718:Phosphatidylserines; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D003545:Cysteine