Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Mol Cell Biol
1995 Jun 01;156:3390-7. doi: 10.1128/MCB.15.6.3390.
Show Gene links
Show Anatomy links
14-3-3 is not essential for Raf-1 function: identification of Raf-1 proteins that are biologically activated in a 14-3-3- and Ras-independent manner.
Michaud NR
,
Fabian JR
,
Mathes KD
,
Morrison DK
.
???displayArticle.abstract???
Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3. Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus oocytes and were biologically activated, as demonstrated by their ability to induced meiotic maturation of Xenopus oocytes. However, in contrast to wild-type Raf-1, activation of these mutants was independent of Ras. Our results therefore indicate that interaction with 14-3-3 is not essential for Raf-1 function.
Aitken,
14-3-3 proteins: a highly conserved, widespread family of eukaryotic proteins.
1992, Pubmed
Aitken,
14-3-3 proteins: a highly conserved, widespread family of eukaryotic proteins.
1992,
Pubmed
Cleghon,
Raf-1 interacts with Fyn and Src in a non-phosphotyrosine-dependent manner.
1994,
Pubmed
Dent,
Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.
1994,
Pubmed
Dent,
Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts.
1994,
Pubmed
Dickson,
Raf functions downstream of Ras1 in the Sevenless signal transduction pathway.
1992,
Pubmed
Fabian,
Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes.
1993,
Pubmed
,
Xenbase
Fabian,
Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase.
1993,
Pubmed
,
Xenbase
Fabian,
A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function.
1994,
Pubmed
,
Xenbase
Fantl,
Activation of Raf-1 by 14-3-3 proteins.
1994,
Pubmed
,
Xenbase
Finney,
Association of pRas and pRaf-1 in a complex correlates with activation of a signal transduction pathway.
1993,
Pubmed
Ford,
14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast.
1994,
Pubmed
Freed,
Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation.
1994,
Pubmed
Fu,
The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family.
1993,
Pubmed
Fu,
Interaction of the protein kinase Raf-1 with 14-3-3 proteins.
1994,
Pubmed
Furukawa,
Demonstration of the phosphorylation-dependent interaction of tryptophan hydroxylase with the 14-3-3 protein.
1993,
Pubmed
Hallberg,
Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.
1994,
Pubmed
Han,
C. elegans lin-45 raf gene participates in let-60 ras-stimulated vulval differentiation.
1993,
Pubmed
Heidecker,
The role of Raf-1 phosphorylation in signal transduction.
1992,
Pubmed
Heidecker,
Mutational activation of c-raf-1 and definition of the minimal transforming sequence.
1990,
Pubmed
Irie,
Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein kinase.
1994,
Pubmed
Koide,
GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells.
1993,
Pubmed
Kolch,
Protein kinase C alpha activates RAF-1 by direct phosphorylation.
1993,
Pubmed
Krieg,
Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs.
1984,
Pubmed
,
Xenbase
Kyriakis,
Mitogen regulation of c-Raf-1 protein kinase activity toward mitogen-activated protein kinase-kinase.
1993,
Pubmed
Leevers,
Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane.
1994,
Pubmed
Moodie,
The 3Rs of life: Ras, Raf and growth regulation.
1994,
Pubmed
Moodie,
Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase.
1993,
Pubmed
Morrison,
The Raf-1 kinase as a transducer of mitogenic signals.
1990,
Pubmed
Morrison,
14-3-3: modulators of signaling proteins?
1994,
Pubmed
Morrison,
Identification of the major phosphorylation sites of the Raf-1 kinase.
1993,
Pubmed
Nishida,
The MAP kinase cascade is essential for diverse signal transduction pathways.
1993,
Pubmed
,
Xenbase
Pallas,
Association of polyomavirus middle tumor antigen with 14-3-3 proteins.
1994,
Pubmed
Pumiglia,
Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction.
1995,
Pubmed
,
Xenbase
Rapp,
Role of Raf-1 serine/threonine protein kinase in growth factor signal transduction.
1991,
Pubmed
Rapp,
raf family serine/threonine protein kinases in mitogen signal transduction.
1988,
Pubmed
Reuther,
Association of the protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3 family.
1994,
Pubmed
Roberts,
Cell biology. A signal chain of events.
1992,
Pubmed
Smith,
Requirement for c-ras proteins during viral oncogene transformation.
,
Pubmed
Stanton,
Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.
1989,
Pubmed
Stokoe,
Activation of Raf as a result of recruitment to the plasma membrane.
1994,
Pubmed
Troppmair,
Ras controls coupling of growth factor receptors and protein kinase C in the membrane to Raf-1 and B-Raf protein serine kinases in the cytosol.
1992,
Pubmed
Tsuda,
A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila.
1993,
Pubmed
Van Aelst,
Complex formation between RAS and RAF and other protein kinases.
1993,
Pubmed
Vojtek,
Mammalian Ras interacts directly with the serine/threonine kinase Raf.
1993,
Pubmed
Warne,
Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro.
1993,
Pubmed
Wigler,
Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor.
1978,
Pubmed
Williams,
Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase.
1992,
Pubmed
Wood,
ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK.
1992,
Pubmed
Zhang,
Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1.
1993,
Pubmed
