Rabbit anti-Rat Raf-1 (Ser642) Polyclonal Antibody | anti-RAF1 antibody

Anti-Phospho-Ser642 Raf-1

Cat. Num. AAA502111

• Reactivity: Rat
• Applications: Western Blot
• Purity: Affinity Purified (Prepared from rabbit serum by affinity purification via sequential chromatography on phospho- and non-phosphopeptide affinity columns.)
• Synonyms: Raf-1 (Ser642); Polyclonal Antibody; Anti-Phospho-Ser642 Raf-1; anti-RAF1 antibody

• Host: Rabbit
• Reactivity: Rat
• Clonality: Polyclonal
• Specificity: Specific for the ~74k Raf-1 protein phosphorylated at Ser642.
• Purity/Purification: Affinity Purified (Prepared from rabbit serum by affinity purification via sequential chromatography on phospho- and non-phosphopeptide affinity columns.)
• Form/Format: 100 ul in 10 mM HEPES (pH 7.5), 150 mM NaCl, 100 ug per ml BSA and 50% glycerol. Adequate amount of material to conduct 10-mini Western Blots.
• Sequence Length: 648

• Applicable Applications for anti-RAF1 antibody: Western Blot (WB)
• Application Notes: Quality Control: Western blots performed on each lot.; WB: 1:1000
• Antigen: Phosphopeptide corresponding to amino acid residues surrounding the phospho-Ser642 of rat Raf-1.
• Immunogen Information: Synthetic phospho-peptide corresponding to amino acid residues surrounding Ser642 conjugated to KLH
• Immunogen Species: Rat
• Reactivity Assumed Based on 100% Sequence Homology: Bovine, canine, chicken, human, mouse, and non human primates
• Species Reactivity Note: The antibody has been directly tested for reactivity in Western blots with rat tissue. It is anticipated that the antibody will react with bovine, canine, chicken, human, mouse, and non human primates based on the fact that these species have 100% homology with the amino acid sequence used as antigen.
• Biological Significance: The Ras pathway is a critical signal transduction cascade involved in regulating cellular proliferation, differentiation, survival, and oncogenic transformation. Members of the Raf serine/threonine kinase family are key intermediates in this cascade, functioning to relay signals from activated Ras to the downstream protein kinases MEK and ERK (Marshall, 1996). Previous studies have shown that phosphorylation is required for Raf-1 activation (Dhillon and Kolch, 2002; Chong et al., 2003). Recent work has demonstrated that phosphorylation also regulates the downregulation of Raf (Dougherty et al., 2005) with two sites participating: Ser301 and Ser642.
• Preparation and Storage: For long term storage -20 degree C is recommended. Stable at -20 degree C for at least 1 year.

Related Product Information for anti-RAF1 antibody

Affinity purified rabbit polyclonal antibody

References

• Chong H, Vikis HG, Guan KL (2003) Mechanisms of regulating the Raf kinase family. Cellular Signalling 15:463-469.
• Dhillon AS, Kolch W (2002) Untying the regulation of the Raf-1 kinase. Arch Biochem Biophys 404:3-9.
• Dougherty MK, Muller J, Ritt DA, Zhou M, Zhou XZ, Copeland TD, Conrads TP, Veenstra TD, Lu KP, Morrison DK (2005) Regulation of Raf-1 by Direct Feedback Phosphorylation. Mol Cell 17:215-224.
• Marshall CJ (1996) Ras effectors. current opinion in cell biology 8:197-204.

NCBI and Uniprot Product Information

• NCBI GI #: 6981458
• NCBI GeneID: 24703
• NCBI Accession #: NP_036771.1
• NCBI GenBank Nucleotide #: NM_012639.2
• UniProt Accession #: P11345
• Molecular Weight: 74
• NCBI Official Full Name: RAF proto-oncogene serine/threonine-protein kinase
• NCBI Official Synonym Full Names: v-raf-leukemia viral oncogene 1
• NCBI Official Symbol: Raf1
• NCBI Protein Information: RAF proto-oncogene serine/threonine-protein kinase; cRaf; C-RAF; raf-1; proto-oncogene c-RAF; v-raf-1 murine leukemia viral oncogene homolog 1; murine leukemia viral (v-raf-1) oncogene homolog 1 (3611-MSV)
• UniProt Protein Name: RAF proto-oncogene serine/threonine-protein kinase
• Protein Family: Rik1-associated factor
• UniProt Gene Name: Raf1
• UniProt Synonym Gene Names: Raf; cRaf
• UniProt Entry Name: RAF1_RAT

NCBI Description

acts as a mitogenic protein kinase; mutant forms may play a role in transformation [RGD, Feb 2006]

Uniprot Description

Function: 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. 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. Regulates Rho signaling and migration, and is required for normal wound healing. 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. Phosphorylates TNNT2/cardiac muscle troponin T. Ref.3 Ref.6

Catalytic activity: ATP + a protein = ADP + a phosphoprotein.

Cofactor: Binds 2 zinc ions per subunit

By similarity.

Enzyme regulation: 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.

Subunit structure: 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

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')

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, ROCK2, PPP1R12A, PKB/AKT1, PPP2CA, PPP2R1B, SPRY2, SPRY4, CNKSR1/CNK1, KSR2 and PHB/prohibitin

By similarity. 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. Interacts with MAP2K1/MEK1 and MAP2K2/MEK2. In its active form, interacts with PRMT5

By similarity. Ref.4 Ref.5 Ref.6

Subcellular location: Cytoplasm

By similarity. Cell membrane

By similarity. Mitochondrion

By similarity. Nucleus

By similarity. 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. Retinoic acid-induced Ser-621 phosphorylated form of RAF1 is predominantly localized at the nucleus

By similarity. Ref.5

Post-translational modification: 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 in response to EGF treatment. This modification leads to destabilization of the protein, possibly through proteasomal degradation

By similarity. Ref.7

Sequence similarities: Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. RAF subfamily.Contains 1 phorbol-ester/DAG-type zinc finger.Contains 1 protein kinase domain.Contains 1 RBD (Ras-binding) domain.