Q99N57 (RAF1_MOUSE) Reviewed, UniProtKB/Swiss-Prot
Last modified February 19, 2014. Version 125. History...
Names and origin
|Protein names||Recommended name:|
RAF proto-oncogene serine/threonine-protein kinase
|Organism||Mus musculus (Mouse) [Reference proteome]|
|Taxonomic identifier||10090 [NCBI]|
|Taxonomic lineage||Eukaryota › Metazoa › Chordata › Craniata › Vertebrata › Euteleostomi › Mammalia › Eutheria › Euarchontoglires › Glires › Rodentia › Sciurognathi › Muroidea › Muridae › Murinae › Mus › Mus|
|Sequence length||648 AA.|
|Protein existence||Evidence at protein level|
General annotation (Comments)
Serine/threonine-protein kinase that acts as a regulatory link between the membrane-associated Ras GTPases and the MAPK/ERK cascade, and this critical regulatory link functions as a switch determining cell fate decisions including proliferation, differentiation, apoptosis, survival and oncogenic transformation. RAF1 activation initiates a mitogen-activated protein kinase (MAPK) cascade that comprises a sequential phosphorylation of the dual-specific MAPK kinases (MAP2K1/MEK1 and MAP2K2/MEK2) and the extracellular signal-regulated kinases (MAPK3/ERK1 and MAPK1/ERK2). The phosphorylated form of RAF1 (on residues Ser-338 and Ser-339, by PAK1) phosphorylates BAD/Bcl2-antagonist of cell death at 'Ser-75'. Phosphorylates adenylyl cyclases: ADCY2, ADCY5 and ADCY6, resulting in their activation. Phosphorylates PPP1R12A resulting in inhibition of the phosphatase activity. Phosphorylates TNNT2/cardiac muscle troponin T. Can promote NF-kB activation and inhibit signal transducers involved in motility (ROCK2), apoptosis (MAP3K5/ASK1 and STK3/MST2), proliferation and angiogenesis (RB1). Can protect cells from apoptosis also by translocating to the mitochondria where it binds BCL2 and displaces BAD/Bcl2-antagonist of cell death. Plays a role in the oncogenic transformation of epithelial cells via repression of the TJ protein, occludin (OCLN) by inducing the up-regulation of a transcriptional repressor SNAI2/SLUG, which induces down-regulation of OCLN. Restricts caspase activation in response to selected stimuli, notably Fas stimulation, pathogen-mediated macrophage apoptosis, and erythroid differentiation By similarity. Regulates Rho signaling and migration, and is required for normal wound healing. Ref.5
ATP + a protein = ADP + a phosphoprotein.
Binds 2 zinc ions per subunit By similarity.
Regulation is a highly complex process involving membrane recruitment, protein-protein interactions, dimerization, and phosphorylation/dephosphorylation events. Ras-GTP recruits RAF1 to the membrane, thereby promoting its activation. The inactive conformation of RAF1 is maintained by autoinhibitory interactions occurring between the N-terminal regulatory and the C-terminal catalytic domains and by the binding of a 14-3-3 protein that contacts two phosphorylation sites, Ser-259 and Ser-621. Upon mitogenic stimulation, Ras and PPP2R1A cooperate to release autoinhibition and the subsequent phosphorylation of activating sites: Ser-338, Tyr-341, Thr-491, and Ser-494, yields a fully active kinase. Through a negative feedback mechanism involving MAPK1/ERK2, RAF1 is phosphorylated on Ser-29, Ser-43, Ser-289, Ser-296, Ser-301 and Ser-642 by MAPK1/ERK2, which yields an inactive, desensitized kinase. The signaling-competent conformation of RAF1 is finally re-established by the coordinated action of PIN1, a prolyl isomerase that converts pSer and pThr residues from the cis to the trans conformation, which is preferentially recognized and dephosphorylated by PPP2R1A. Activated by homodimerization and heterodimerization (with BRAF). Also regulated through association with other proteins such as KSR2, CNKSR1/CNK1, PEBP1/RKIP, PHB/prohibitin and SPRY4. PEBP1/RKIP acts by dissociating RAF1 from its substrates MAP2K1/MEK1 and MAP2K2/MEK2. PHB/prohibitin facilitates the displacement of 14-3-3 from RAF1 by activated Ras, thereby promoting cell membrane localization and phosphorylation of RAF1 at the activating Ser-338. SPRY4 inhibits Ras-independent, but not Ras-dependent, activation of RAF1. CNKSR1/CNK1 regulates Src-mediated RAF1 activation By similarity. Ref.6
Monomer. Homodimer. Heterodimerizes with BRAF and this heterodimer possesses a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimerization is mitogen-regulated and enhanced by 14-3-3 proteins. MAPK1/ERK2 activation can induce a negative feedback that promotes the dissociation of the heterodimer. Forms a multiprotein complex with Ras (M-Ras/MRAS), SHOC2 and protein phosphatase 1 (PPP1CA, PPP1CB and PPP1CC). Interacts with Ras proteins; the interaction is antagonized by RIN1. Weakly interacts with RIT1. Interacts (via N-terminus) with RGS14 (via RBD domains); the interaction mediates the formation of a ternary complex with BRAF, a ternary complex inhibited by GNAI1 By similarity. Interacts with STK3/MST2; the interaction inhibits its pro-apoptotic activity. Interacts (when phosphorylated at Ser-259) with YWHAZ (unphosphorylated at 'Thr-232'). Interacts with MAP2K1/MEK1 and MAP2K2/MEK2 By similarity. Interacts with MAP3K5/ASF1 (via N-terminus) and this interaction inhibits the proapoptotic function of MAP3K5/ASK1. Interacts with PAK1 (via kinase domain). The phosphorylated form interacts with PIN1. The Ser-338 and Ser-339 phosphorylated form (by PAK1) interacts with BCL2. Interacts with PEBP1/RKIP and this interaction is enhanced if RAF1 is phosphorylated on residues Ser-338, Ser-339, Tyr-340 and Tyr-341. Interacts with ADCY2, ADCY5, ADCY6, DGKH, RCAN1/DSCR1, PPP1R12A, PKB/AKT1, SPRY2, SPRY4, CNKSR1/CNK1, KSR2 and PHB/prohibitin By similarity. The phosphorylated form interacts with PIN1. Interacts with PPP2CA, PPP2R1B and ROCK2. In its active form, interacts with PRMT5 By similarity. Ref.5 Ref.6
Cytoplasm By similarity. Cell membrane By similarity. Mitochondrion By similarity. Nucleus. Note: Colocalizes with RGS14 and BRAF in both the cytoplasm and membranes. Phosphorylation at Ser-259 impairs its membrane accumulation. Recruited to the cell membrane by the active Ras protein. Phosphorylation at Ser-338 and Ser-339 by PAK1 is required for its mitochondrial localization By similarity. Retinoic acid-induced Ser-621 phosphorylated form of RAF1 is predominantly localized at the nucleus. Ref.8
Present in all tissues tested: testis, ovary, small intestine, colon, peripheral blood leukocytes, fetal liver, bone marrow, thymus, lymph node and spleen, and the cell lines melanoma G-361, lung carcinoma A-549, colorectal adenocarcinoma SW480, Burkitt's lymphoma Raji and lymphoblastic leukemia MOLT-4. In skeletal muscle, isoform 1 is more abundant than isoform 2. Ref.1 Ref.4
Phosphorylation at Thr-269, Ser-338, Tyr-341, Thr-491 and Ser-494 results in its activation. Phosphorylation at Ser-29, Ser-43, Ser-289, Ser-296, Ser-301 and Ser-642 by MAPK1/ERK2 results in its inactivation. Phosphorylation at Ser-259 induces the interaction with YWHAZ and inactivates kinase activity. Dephosphorylation of Ser-259 by the complex containing protein phosphatase 1, SHOC2 and M-Ras/MRAS relieves inactivation, leading to stimulate RAF1 activity. Phosphorylation at Ser-338 by PAK1 and PAK7/PAK5 and Ser-339 by PAK1 is required for its mitochondrial localization By similarity. Phosphorylation at Ser-621 in response to growth factor treatment stabilizes the protein, possibly by preventing proteasomal degradation. Phosphorylation at Ser-289, Ser-296, Ser-301, Ser-338 and Ser-621 are somehow linked to the methylation potential of cells. Treatment of cells with HGF in the presence of the methylation inhibitor 5'-methylthioadenosine (MTA) results in increased phosphorylation at Ser-338 and Ser-621 and decreased phosphorylation at Ser-296, Ser-301 and Ser-338. Dephosphorylation at SER-338 by PPP5C results in a decreased of activity By similarity.
Methylated at Arg-563 in response to EGF treatment By similarity. This modification leads to destabilization of the protein, possibly through proteasomal degradation By similarity.
Contains 1 phorbol-ester/DAG-type zinc finger.
Contains 1 protein kinase domain.
Contains 1 RBD (Ras-binding) domain.
|This entry describes 2 isoforms produced by alternative splicing. [Align] [Select]|
|Isoform 1 (identifier: Q99N57-1) |
Also known as: 6C;
This isoform has been chosen as the 'canonical' sequence. All positional information in this entry refers to it. This is also the sequence that appears in the downloadable versions of the entry.
|Isoform 2 (identifier: Q99N57-2) |
Also known as: 1A;
The sequence of this isoform differs from the canonical sequence as follows:
278-278: E → ESNSLNASPRACSRRFCLRGR
Sequence annotation (Features)
|Feature key||Position(s)||Length||Description||Graphical view||Feature identifier|
|Chain||1 – 648||648||RAF proto-oncogene serine/threonine-protein kinase||PRO_0000086597|
|Domain||56 – 131||76||RBD|
|Domain||349 – 609||261||Protein kinase|
|Zinc finger||138 – 184||47||Phorbol-ester/DAG-type|
|Nucleotide binding||355 – 363||9||ATP By similarity|
|Region||331 – 349||19||Interaction with PEBP1/RKIP By similarity|
|Active site||468||1||Proton acceptor By similarity|
|Metal binding||139||1||Zinc 1 By similarity|
|Metal binding||152||1||Zinc 2 By similarity|
|Metal binding||155||1||Zinc 2 By similarity|
|Metal binding||165||1||Zinc 1 By similarity|
|Metal binding||168||1||Zinc 1 By similarity|
|Metal binding||173||1||Zinc 2 By similarity|
|Metal binding||176||1||Zinc 2 By similarity|
|Metal binding||184||1||Zinc 1 By similarity|
|Binding site||375||1||ATP By similarity|
Amino acid modifications
|Modified residue||29||1||Phosphoserine; by MAPK1 Ref.6|
|Modified residue||43||1||Phosphoserine; by PKA and MAPK1 Ref.6|
|Modified residue||233||1||Phosphoserine; by PKA By similarity|
|Modified residue||252||1||Phosphoserine By similarity|
|Modified residue||259||1||Phosphoserine; by PKA, PKC and PKB/AKT1 By similarity|
|Modified residue||268||1||Phosphothreonine; by autocatalysis By similarity|
|Modified residue||269||1||Phosphothreonine; by PKA By similarity|
|Modified residue||289||1||Phosphoserine; by MAPK1 Ref.6|
|Modified residue||296||1||Phosphoserine; by MAPK1 Ref.6|
|Modified residue||301||1||Phosphoserine; by MAPK1 Ref.6|
|Modified residue||338||1||Phosphoserine; by PAK1, PAK2, PAK3 and PAK7/PAK5 By similarity|
|Modified residue||339||1||Phosphoserine; by PAK1, PAK2 and PAK3 By similarity|
|Modified residue||340||1||Phosphotyrosine; by SRC By similarity|
|Modified residue||341||1||Phosphotyrosine; by SRC By similarity|
|Modified residue||471||1||Phosphoserine By similarity|
|Modified residue||491||1||Phosphothreonine By similarity|
|Modified residue||494||1||Phosphoserine By similarity|
|Modified residue||497||1||Phosphoserine; by PKC By similarity|
|Modified residue||499||1||Phosphoserine; by PKC By similarity|
|Modified residue||563||1||Symmetric dimethylarginine; by PRMT5 By similarity|
|Modified residue||621||1||Phosphoserine Ref.6|
|Modified residue||642||1||Phosphoserine; by MAPK1 Ref.6|
|Alternative sequence||278||1||E → ESNSLNASPRACSRRFCLRG R in isoform 2.||VSP_034629|
|Sequence conflict||522||1||D → N in AAH92040. Ref.3|
|Sequence conflict||543||1||A → T in AAH92040. Ref.3|
|||"Phylogenetic conservation of the makorin-2 gene, encoding a multiple zinc-finger protein, antisense to the raf1 proto-oncogene."|
Gray T.A., Azama K., Whitmore K., Min A., Abe S., Nicholls R.D.
Genomics 77:119-126(2001) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [MRNA] (ISOFORM 1), TISSUE SPECIFICITY.
|||"The transcriptional landscape of the mammalian genome."|
Carninci P., Kasukawa T., Katayama S., Gough J., Frith M.C., Maeda N., Oyama R., Ravasi T., Lenhard B., Wells C., Kodzius R., Shimokawa K., Bajic V.B., Brenner S.E., Batalov S., Forrest A.R., Zavolan M., Davis M.J. Hayashizaki Y.
Science 309:1559-1563(2005) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA] (ISOFORM 1).
|||"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC)."|
The MGC Project Team
Genome Res. 14:2121-2127(2004) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA] (ISOFORM 1).
Strain: Czech II and FVB/N.
Tissue: Kidney and Mammary tumor.
|||"An alternatively spliced c-mil/raf mRNA is predominantly expressed in chicken muscular tissues and conserved among vertebrate species."|
Dozier C., Ansieau S., Ferreira E., Coll J., Stehelin D.
Oncogene 6:1307-1311(1991) [PubMed] [Europe PMC] [Abstract]
Cited for: PARTIAL NUCLEOTIDE SEQUENCE [GENOMIC DNA], ALTERNATIVE SPLICING, TISSUE SPECIFICITY.
|||"Raf-1 regulates Rho signaling and cell migration."|
Ehrenreiter K., Piazzolla D., Velamoor V., Sobczak I., Small J.V., Takeda J., Leung T., Baccarini M.
J. Cell Biol. 168:955-964(2005) [PubMed] [Europe PMC] [Abstract]
Cited for: FUNCTION, INTERACTION WITH ROCK2.
|||"Regulation of Raf-1 by direct feedback phosphorylation."|
Dougherty M.K., Muller J., Ritt D.A., Zhou M., Zhou X.Z., Copeland T.D., Conrads T.P., Veenstra T.D., Lu K.P., Morrison D.K.
Mol. Cell 17:215-224(2005) [PubMed] [Europe PMC] [Abstract]
Cited for: PHOSPHORYLATION AT SER-29; SER-43; SER-259; SER-289; SER-296; SER-301; SER-338; SER-621 AND SER-642, ENZYME REGULATION, INTERACTION WITH PIN1; PPP2CA AND PPP2R1B.
|||"Large-scale phosphorylation analysis of mouse liver."|
Villen J., Beausoleil S.A., Gerber S.A., Gygi S.P.
Proc. Natl. Acad. Sci. U.S.A. 104:1488-1493(2007) [PubMed] [Europe PMC] [Abstract]
Cited for: IDENTIFICATION BY MASS SPECTROMETRY [LARGE SCALE ANALYSIS].
|||"Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells."|
Smith J., Bunaciu R.P., Reiterer G., Coder D., George T., Asaly M., Yen A.
Exp. Cell Res. 315:2241-2248(2009) [PubMed] [Europe PMC] [Abstract]
Cited for: SUBCELLULAR LOCATION.
|+||Additional computationally mapped references.|
|AB057655 mRNA. Translation: BAB39748.1.|
AB057663 mRNA. Translation: BAB39743.2.
AK141745 mRNA. Translation: BAE24820.1.
BC015273 mRNA. Translation: AAH15273.1.
BC092040 mRNA. Translation: AAH92040.1.
X55432 Genomic DNA. No translation available.
|RefSeq||NP_084056.1. NM_029780.3. |
3D structure databases
|SMR||Q99N57. Positions 56-131, 136-187, 307-624. |
Protein-protein interaction databases
|BioGrid||225343. 15 interactions.|
|IntAct||Q99N57. 8 interactions.|
Protocols and materials databases
Genome annotation databases
|Ensembl||ENSMUST00000000451; ENSMUSP00000000451; ENSMUSG00000000441. |
ENSMUST00000112949; ENSMUSP00000108571; ENSMUSG00000000441.
|UCSC||uc009dix.1. mouse. |
|MGI||MGI:97847. Raf1. |
Gene expression databases
Family and domain databases
|InterPro||IPR020454. DAG/PE-bd. |
|Pfam||PF00130. C1_1. 1 hit. |
PF07714. Pkinase_Tyr. 1 hit.
PF02196. RBD. 1 hit.
|PRINTS||PR00008. DAGPEDOMAIN. |
|SMART||SM00109. C1. 1 hit. |
SM00455. RBD. 1 hit.
|SUPFAM||SSF56112. SSF56112. 1 hit. |
|PROSITE||PS00107. PROTEIN_KINASE_ATP. 1 hit. |
PS50011. PROTEIN_KINASE_DOM. 1 hit.
PS00108. PROTEIN_KINASE_ST. 1 hit.
PS50898. RBD. 1 hit.
PS00479. ZF_DAG_PE_1. 1 hit.
PS50081. ZF_DAG_PE_2. 1 hit.
|ChiTaRS||RAF1. mouse. |
|Accession||Primary (citable) accession number: Q99N57|
Secondary accession number(s): Q3UR68 Q9QUU8
|Entry status||Reviewed (UniProtKB/Swiss-Prot)|
|Annotation program||Chordata Protein Annotation Program|