Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins.
We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins. Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain. Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells. 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system. However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain. The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells. We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.
Li
S
S
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520.
Janosch
P
P
Tanji
M
M
Rosenfeld
G C
GC
Waymire
J C
JC
Mischak
H
H
Kolch
W
W
Sedivy
J M
JM
D048948:14-3-3 Proteins; D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006133:Growth Substances / Q000097:blood; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D006133:Growth Substances; D011506:Proteins; D011518:Proto-Oncogene Proteins; D014446:Tyrosine 3-Monooxygenase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf