Phospho-RAF1 (Ser338) Antibody
Description
Definition and Target Specificity
Phospho-RAF1 (Ser338) Antibody is a polyclonal rabbit antibody generated against a synthetic phosphopeptide corresponding to residues surrounding Ser338 of human RAF1. It selectively recognizes RAF1 phosphorylated at Ser338 (pS338-RAF1) and does not cross-react with non-phosphorylated forms or other RAF isoforms (e.g., BRAF) .
| Parameter | Specification |
|---|---|
| Target Protein | RAF1 (UniProt: P04049) |
| Phosphorylation Site | Serine 338 |
| Host Species | Rabbit |
| Clonality | Polyclonal |
| Applications | Western Blot (WB, 1:1000 dilution), Dot Blot (DB, 1:500), ELISA (E) |
| Reactivity | Human, predicted in bovine, mouse, rat, and chicken |
| Storage | -20°C in aliquots; avoid freeze-thaw cycles |
Functional Significance of Ser338 Phosphorylation
Phosphorylation at Ser338 is essential for RAF1 activation. Key findings include:
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Regulatory Role: pS338-RAF1 initiates the MAPK/ERK cascade by phosphorylating MEK1/2, which subsequently activates ERK1/2 .
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Upstream Activators: PAK1 (p21-activated kinase 1) phosphorylates Ser338 in response to growth factors (e.g., EGF) or phorbol esters (e.g., TPA) .
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Autophosphorylation: Mutational studies (e.g., K375M, S471A) reveal that RAF1 autophosphorylates Ser338 during activation, independent of PAK1 .
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Dimerization Dependency: Drug-induced RAF1 dimerization (e.g., via AP1510) enhances Ser338 phosphorylation, suggesting a role in RAF oligomerization .
Key Uses:
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Mechanistic Studies: Investigate RAF1 activation in cancer, apoptosis, and mitochondrial signaling .
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Pathway Analysis: Monitor MAPK/ERK cascade dynamics under stimuli like EGF or oxidative stress .
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Disease Models: Used in studies of Huntington’s disease, melanoma, and colorectal cancer .
Example Protocol (Western Blot):
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Lysate Preparation: Use RIPA buffer with phosphatase inhibitors.
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Electrophoresis: Load 20–30 µg of protein per lane on 10% SDS-PAGE.
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Transfer: PVDF membrane, 100 V for 1 hr.
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Blocking: 5% non-fat milk in TBST, 1 hr.
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Primary Antibody: Dilute 1:1000 in TBST, incubate overnight at 4°C .
Table: Select Studies Using Phospho-RAF1 (Ser338) Antibody
| Study Focus | Major Finding | Citation Source |
|---|---|---|
| Huntington’s Disease | Identified RRAS signaling dysregulation linked to pS338-RAF1 activity | Miller et al., 2012 |
| EGFR Fate Determination | Demonstrated RIN1-dependent RAF1 activation via Ser338 phosphorylation | Balaji et al., 2012 |
| Melanoma Metastasis | Filamin A modulates RAF1 activity via Ser338 to regulate MMP-9 expression | Zhu et al., 2007 |
| pH-Dependent mTORC1 Regulation | Linked extracellular pH changes to RAF1/ERK signaling | Balgi et al., 2011 |
Technical Considerations
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Specificity Controls: Always include non-phosphorylated RAF1 and PAK1-knockout samples to confirm signal specificity .
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Inhibitor Interference: Raf inhibitors (e.g., Sarofenib) reduce Ser338 phosphorylation, necessitating careful experimental timing .
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Cross-Reactivity: No observed cross-reactivity with B-Raf or A-Raf isoforms .
Limitations and Alternatives
Product Specs
Target Background
- Functional assessments supported the pathogenicity of RAF1 and RIT1 variants of unknown significance (VUSs), while the significance of two VUSs in A2ML1 remained unclear. PMID: 29402968
- This report presents the second familial case of Noonan syndrome due to a germline p.S427G substitution in RAF1, without any occurrence of a malignant tumor. This finding may suggest that carrying a germline mutation in the RAF1 oncogene is not associated with an increased risk of tumor development, despite the observation of RAF1 mutations as somatic events in many cancer types. PMID: 30204961
- Data indicate that Raf-1 proto-oncogene, serine-threonine kinase (RAF1) acts as a negative regulator of hepatocarcinogenesis. PMID: 28000790
- We report a patient with an inherited RAF1-associated Noonan syndrome presenting with an antenatally diagnosed abnormality of skull shape, bilateral subdural haematomas (of unknown cause), delayed myelination, and polymicrogyria. PMID: 27753652
- Raf1 may serve as a novel prognostic factor and potential target for improving the long-term outcome of nonsmall cell lung cancer (NSCLC). PMID: 29484414
- Results provide evidence that RAF1 binding to SPRY4 is regulated by miR-1908 in glioma tumors. PMID: 29048686
- High RAF1 expression is associated with malignant melanoma. PMID: 28677804
- Two premature neonates with progressive biventricular hypertrophy found to have RAF1 variants in the CR2 domain are reported. PMID: 28777121
- Data indicate connector enhancer of kinase suppressor of Ras 1 protein (CNK1) as a molecular platform that controls c-raf protein (RAF) and c-akt protein (AKT) signaling and determines cell fate decisions in a cell type- and cell stage-dependent manner. PMID: 27901111
- CRAF is a bona fide alternative oncogene for BRAF/NRAS/GNAQ/GNA11 wild type melanomas. PMID: 27273450
- Authors evaluated the expression of known targets of miR-125a and found that sirtuin-7, matrix metalloproteinase-11, and c-Raf were up-regulated in tumor tissue by 2.2-, 3-, and 1.7-fold, respectively. Overall, these data support a tumor suppressor role for miR-125a. PMID: 28445974
- Overexpression of ciRS-7 in HCT116 and HT29 cells led to the blocking of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted the inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes. PMID: 28174233
- miR-497 could serve as a tumor suppressor and a potential early diagnostic marker of gastric cancer by targeting Raf-1 proto-oncogene. PMID: 28586056
- RAF1 may have a role in survival in hepatocellular carcinoma, and indicate whether sorafenib should be used as a postoperative adjuvant. PMID: 26981887
- Mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy. PMID: 27391150
- Results indicate that des-gamma-carboxy prothrombin (DCP) antagonizes the inhibitory effects of Sorafenib on hepatocellular carcinoma (HCC) through activation of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways. PMID: 27167344
- DiRas3 binds to KSR1 independently of its interaction with activated Ras and RAF. PMID: 27368419
- RhoA/ROCK and Raf-1/CK2 pathway are responsible for TNF-alpha-mediated endothelial cytotoxicity via regulation of the vimentin cytoskeleton. PMID: 28743511
- Although Raf-1 gene is not mutated, an abnormality of Raf-1 kinase feedback regulation enhances its antiapoptotic function, and Raf-1 can still be a pharmaceutical target to increase chemotherapy or radiotherapy sensitivity in these cancer cells. PMID: 27841865
- RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS. PMID: 27670374
- This finding suggests that stringent assemblage of Hsp90 keeps CRAF kinase equipped for participating in the MAPK pathway. Thus, the role of Hsp90 in CRAF maturation and activation acts as a limiting factor to maintain the function of a strong client like CRAF kinase. PMID: 27703006
- Oncogenic NFIA:RAF1 fusion activation of the MAPK pathway is associated with pilocytic astrocytoma. PMID: 27810072
- IGF2BP2 acts as a post-transcriptional regulatory mRNA-binding factor, interfering with Raf-1 degradation by miR-195, that contributes to Colorectal carcinogenesis. PMID: 27153315
- Data show that when microRNA miR-125b was over-expressed in THP-1 macrophages, the expression of Raf1 proto-oncogene serine/threonine protein kinase (RAF1) was reduced to promote the apoptosis of macrophages. PMID: 27363278
- Data show that Griffipavixanthone (GPX), a dimeric xanthone isolated from Garcinia esculenta, is a B-RAF and C-RAF inhibitor against esophageal cancer cells. PMID: 26646323
- Up-regulation of Raf-1 is associated with triple-negative breast cancer. PMID: 26513016
- This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3zeta protein and gives structural insights responsible for phosphorylation-mediated protein binding. PMID: 26295714
- A model suggests that CD166 regulates MCAM through a signaling flow from activation of PI3K/AKT and c-Raf/MEK/ERK signaling to the inhibition of potential MCAM ubiquitin E3 ligases, betaTrCP and Smurf1. PMID: 26004137
- Suggest an interrelated kinase module involving c-Raf/PI3K/Lyn and perhaps Fgr functions in a nontraditional way during retinoic acid-induced maturation or during rescue of RA induction therapy using inhibitor co-treatment in RA-resistant leukemia cells. PMID: 25817574
- Abnormal activation of the Ras/MAPK pathway may play a significant role in the development of pulmonary vascular disease in the subset of patients with Noonan syndrome and a specific RAF1 mutation. PMID: 25706034
- Raf-1 may be an important biomarker in predicting the prognosis of chordoma patients. PMID: 25755752
- In the presence of Raf1, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-Ras binding domain. PMID: 25684575
- Besides mediating the anticancer effect, pDAPK(S308) may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation. PMID: 26100670
- DJ-1 directly binds to the kinase domain of c-Raf to stimulate its self-phosphorylation, followed by phosphorylation of MEK and ERK1/2 in EGF-treated cells. PMID: 26048984
- Truncated RAF1 and BRAF proteins, recently described as products of genomic rearrangements in gastric cancer and other malignancies, have the ability to render neoplastic cells resistant to RTK-targeted therapy. PMID: 25473895
- Our study demonstrated that miR-455-RAF1 may represent a new potential therapeutic target for colorectal carcinoma treatment. PMID: 25355599
- This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. PMID: 25512530
- Aberrant expression of A-, B-, and C-RAF, and COT is frequent in PTC; increased expression of COT is correlated with recurrence of PTC. PMID: 25674762
- Authors demonstrate that the N-terminus of human Raf1 kinase (hRaf11-147aa) binds with human RKIP (hRKIP) at its ligand-binding pocket, loop "127-149", and the C-terminal helix by nuclear magnetic resonance experiments. PMID: 24863296
- Including several anti-apoptotic Bcl-2 family members and c-Raf. PMID: 24969872
- These data suggest that miR-7-5p functions as a tumor suppressor gene to regulate glioblastoma microvascular endothelial cell proliferation potentially by targeting the RAF1 oncogene. PMID: 25027403
- A novel mechanism for response was discovered whereby high expression level of CAV-1 at the plasma membrane disrupts the BRaf/CRaf heterodimer and thus inhibits the activation of MAPK pathway during dasatinib treatment. PMID: 24486585
- Results show that ubiquitination and levels of RAF-1 is controlled by both Shoc2 and HUWE1. PMID: 25022756
- Raf-1/JNK /p53/p21 pathway may be involved in apoptosis, and NFkappaB1 may play a possible role in inhibiting apoptosis. PMID: 22282237
- The higher expression of RAF1 mRNA and the activation of AKT/ERK proteins in vinorelbine-resistant non-small cell lung cancer cell lines may confer resistance to vinorelbine. PMID: 24427333
- Analysis of RAF1 mutations in cohorts of South Indian, North Indian, and Japanese patients with childhood-onset dilated cardiomyopathy. PMID: 24777450
- Expression of miR-195 or knockdown of Raf-1 can similarly reduce tumor cell survival. PMID: 23760062
- We hypothesize a potential direct or indirect role for SRC, RAF1, PTK2B genes in neurotransmission and in central nervous system signaling processes. PMID: 24108181
- We identified multiple C-RAF mutations that produced biochemical and pharmacologic resistance in melanoma cell lines. PMID: 23737487
- ARAF seems to stabilize BRAF:CRAF complexes in cells treated with RAF inhibitors and thereby regulate cell signaling in a subtle manner to ensure signaling efficiency. PMID: 22926515
Q&A
What is the significance of RAF1 Ser338 phosphorylation in cellular signaling?
RAF1 (also known as C-RAF) is a serine/threonine protein kinase that functions as a regulatory link between membrane-associated Ras GTPases and the MAPK/ERK cascade. Phosphorylation at Ser338 is a crucial event for RAF1 activation and serves as a molecular switch determining cell fate decisions including proliferation, differentiation, apoptosis, and survival . The phosphorylated form of RAF1 (at residues Ser-338 and Ser-339) initiates a mitogen-activated protein kinase cascade comprising sequential phosphorylation of MAP2K1/MEK1, MAP2K2/MEK2, and the extracellular signal-regulated kinases (MAPK3/ERK1 and MAPK1/ERK2) . This phosphorylation event is critical for transducing signals from the cell membrane to the nucleus, making it a central point of interest in cancer research and cellular biology.
How does Phospho-RAF1 (Ser338) detection contribute to understanding signaling pathways?
Detection of phosphorylated RAF1 at Ser338 provides crucial insights into the activation status of the RAF-MEK-ERK pathway. Low levels of basal Ser338 phosphorylation are typically observed in resting cells, but upon stimulation with growth factors like EGF, phosphorylation is rapidly elevated within 2 minutes and continues to rise over extended periods (up to 60 minutes) . By monitoring the phosphorylation status of Ser338, researchers can assess pathway activation in response to various stimuli, inhibitors, or genetic modifications. This detection is particularly valuable when studying oncogenic signaling, as aberrant activation of the RAF-MEK-ERK pathway is common in many cancers.
What are the competing models for RAF1 Ser338 phosphorylation?
Two major models exist regarding RAF1 Ser338 phosphorylation:
Model 1: Autophosphorylation Model
Research has demonstrated that mutations of Lys375 to Met and Ser471 to Ala in the activation segment both abolish Ser338 phosphorylation of RAF1 in response to EGF or TPA, while these mutants are still properly phosphorylated by PAK1 . The phosphorylation is also suppressed by treating cells with the RAF inhibitor Sorafenib but not by the MEK inhibitor U0126 . These findings suggest that Ser338 undergoes autophosphorylation during RAF1 activation induced by mitogens.
Model 2: PAK-dependent Phosphorylation Model
Earlier studies suggested that p21-activated kinases (PAKs), particularly PAK1 and PAK5, can directly phosphorylate RAF1 at Ser338 . This model proposes that RAF1 is recruited to the plasma membrane by Ras, where it becomes accessible to PAK-mediated phosphorylation.
The current consensus indicates both mechanisms may operate in different cellular contexts, with autophosphorylation predominating in response to certain growth factors.
How does RAF1 dimerization influence Ser338 phosphorylation?
Experimental evidence shows that RAF1 dimerization plays a significant role in Ser338 phosphorylation. When inactive RAF1 is dimerized with an active mutant of RAF1 in cells, Ser338 becomes phosphorylated on the inactive RAF1 molecule . Additionally, artificial dimerization of RAF1 using dimerization drugs like AP1510 causes progressive enhancement of Ser338 phosphorylation as drug doses increase, accompanied by increased ERK phosphorylation . These findings suggest that:
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RAF1 dimers can trans-phosphorylate each other at Ser338
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Dimerization is a key mechanism for RAF1 activation
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Ser338 phosphorylation likely occurs through an interdimer mechanism
This discovery has significant implications for understanding RAF inhibitor resistance in cancer therapy, as paradoxical pathway activation through RAF dimerization is a documented resistance mechanism.
What are the optimal conditions for Western blot detection of phospho-RAF1 (Ser338)?
Based on manufacturer recommendations and research protocols, the following conditions are optimal for Western blot detection:
For optimal results, researchers should:
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Use fresh samples or properly stored frozen samples (-80°C)
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Include phosphatase inhibitors in all buffers
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Confirm specificity using dephosphorylation controls
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Consider using gradient gels (4-12%) for better resolution of high molecular weight proteins
How can you validate the specificity of phospho-RAF1 (Ser338) antibodies?
Validating antibody specificity is crucial for obtaining reliable results. Multiple approaches should be employed:
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Phosphatase treatment: Treat one sample with lambda phosphatase before immunoblotting. Specific phospho-antibodies will show diminished or absent signal in the treated sample .
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Peptide competition: Pre-incubate the antibody with a phospho-Ser338 peptide before Western blotting. A specific antibody will show reduced signal when pre-blocked with the corresponding phosphopeptide.
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Site-directed mutagenesis: Compare wild-type RAF1 with S338A mutant expressed in cells. The antibody should not detect the S338A mutant.
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Knockout/knockdown controls: Use RAF1 knockout or knockdown cells to confirm absence of signal.
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Stimulus-response validation: Verify that known activators of RAF1 (e.g., EGF, TPA) increase phospho-Ser338 signal in a time-dependent manner consistent with published data .
What factors can lead to inconsistent detection of phospho-RAF1 (Ser338)?
Several factors can affect the consistency and reliability of phospho-RAF1 (Ser338) detection:
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Rapid dephosphorylation: Ser338 can be rapidly dephosphorylated by cellular phosphatases. Ensure samples are collected and processed quickly with phosphatase inhibitors.
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Cell confluency effects: Cells at different confluency levels may show variable baseline Ser338 phosphorylation. Standardize cell density across experiments.
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Serum factors: Serum contains growth factors that can activate RAF1. Proper serum starvation (typically 16-24h) before stimulation experiments is essential.
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Antibody cross-reactivity: Some phospho-Ser338 antibodies may cross-react with phosphorylated B-RAF at the equivalent site (Ser445) . Validate using appropriate controls.
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Inhibitor specificity: When using kinase inhibitors like Sorafenib, consider that they may affect multiple pathways, potentially leading to misinterpretation of results.
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Cell type variation: Different cell types show varying levels of basal and stimulated Ser338 phosphorylation. Establish baseline readings for each cell type used.
How can researchers distinguish between direct and indirect effects on RAF1 Ser338 phosphorylation?
Distinguishing between direct and indirect effects requires carefully designed experiments:
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Time-course analysis: Direct effects typically occur rapidly (seconds to minutes) while indirect effects take longer (tens of minutes to hours).
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In vitro kinase assays: Purified kinases can be used to test direct phosphorylation of RAF1 at Ser338 in cell-free systems.
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Selective inhibitors: Use specific inhibitors of suspected upstream regulators. For example, research has shown that PI3K inhibitors (LY294002 and wortmannin) at appropriate concentrations do not block EGF-induced Ser338 phosphorylation, suggesting PI3K is not directly involved .
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Genetic approaches: Use dominant-negative or constitutively active mutants of potential upstream regulators to establish causality.
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Structural studies: Co-crystallization or biophysical interaction studies can establish direct binding relationships.
How does phospho-RAF1 (Ser338) antibody contribute to cancer research and therapeutics?
Phospho-RAF1 (Ser338) antibodies have become valuable tools in cancer research and therapeutic development:
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Biomarker development: Phospho-Ser338 levels can serve as biomarkers for RAF-MEK-ERK pathway activation in tumors and predictors of response to RAF or MEK inhibitors.
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Resistance mechanism studies: Increased RAF1 Ser338 phosphorylation is often observed in tumors resistant to BRAF inhibitors. The antibody helps monitor this adaptive response.
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Drug discovery applications: Phospho-Ser338 antibodies are used in high-throughput screens to identify compounds that modulate this critical regulatory site.
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Combination therapy assessment: These antibodies help evaluate the effects of combining different targeted therapies (e.g., RAF inhibitors with MEK inhibitors).
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Feedback loop investigation: The antibody enables detailed study of feedback mechanisms in the RAF-MEK-ERK pathway, which is crucial for understanding therapeutic resistance.
Notable research has shown that Sorafenib, a clinically used multikinase inhibitor, suppresses RAF1 Ser338 phosphorylation, while MEK inhibitors like U0126 do not . This finding has implications for developing more effective targeted therapy combinations.
What new insights have emerged regarding RAF1 Ser338 phosphorylation in cell biology?
Recent research has revealed several important insights:
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Autophosphorylation mechanism: Contrary to earlier beliefs that Ser338 is exclusively phosphorylated by upstream kinases like PAK, evidence now supports that RAF1 can autophosphorylate this site during activation .
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Dimerization-dependent regulation: RAF1 dimerization induces Ser338 phosphorylation, which correlates with ERK activation . This provides a mechanistic understanding of how dimerization contributes to RAF activation.
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Differential regulation from B-RAF: While B-RAF has constitutive phosphorylation at the equivalent site (Ser445), RAF1 Ser338 phosphorylation is stimulus-dependent . This difference may explain the distinct roles of these isoforms in signaling.
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Independence from PI3K activity: Though earlier studies suggested PI3K regulates RAF1 Ser338 phosphorylation, newer evidence indicates that EGF-mediated Ser338 phosphorylation occurs even when PI3K activity is completely blocked .
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Role in subcellular localization: Phosphorylation at Ser338 by PAK1/PAK5 and Ser339 by PAK1 is required for RAF1's mitochondrial localization, suggesting a role beyond canonical MAPK pathway activation .
These discoveries continue to reshape our understanding of RAF1 regulation and function in both normal and pathological contexts.
