Phospho-MET (Tyr1349) Antibody

What is Phospho-MET (Tyr1349) and why is it important in research?

Phospho-MET (Tyr1349) refers to the MET receptor tyrosine kinase when phosphorylated at tyrosine residue 1349. The MET receptor, also known as hepatocyte growth factor (HGF) receptor, is a proto-oncogene with tyrosine kinase activity that plays crucial roles in cellular growth, proliferation, survival, motility, and angiogenesis. Tyr1349 is located in the multi-substrate docking site of MET and is one of the key phosphorylation sites alongside Tyr1356 . When phosphorylated, these residues serve as docking sites for adaptor proteins that facilitate downstream signaling cascades including PI3K, SRC, STAT, and Ras-Raf-Mek-Erk pathways . The phosphorylation status of Tyr1349 is therefore a critical indicator of MET activation and signaling capacity, making it an important target for cancer research and therapeutic development.

How does MET become phosphorylated at Tyr1349?

MET phosphorylation at Tyr1349 occurs through a sequential process initiated by HGF binding. When HGF, the only known ligand for MET, binds to the receptor, it induces receptor dimerization and autophosphorylation . Initially, phosphorylation occurs at tyrosines 1234 and 1235 in the activation loop of the MET catalytic domain, which is required for activation of MET kinase activity . Subsequently, additional sites including Tyr1003 in the juxtamembrane domain and Tyr1349/Tyr1356 in the carboxy-terminal multi-substrate docking site become phosphorylated .

These phosphorylation events follow a hierarchical pattern, where activation loop phosphorylation (Tyr1234/1235) is prerequisite for the phosphorylation of other sites including Tyr1349. Studies with PTP1B-null animals have demonstrated that phosphorylation of activation loop tyrosines coincides with enhanced phosphorylation of non-catalytic sites, highlighting the sequential nature of this process .

What are the common applications for Phospho-MET (Tyr1349) antibodies?

Phospho-MET (Tyr1349) antibodies are versatile research tools with several key applications:

Researchers also employ these antibodies in various other methodologies including:

• Biomarker studies in cancer research, where phospho-MET levels can predict disease outcomes in cancers such as small cell lung cancer

• Pharmacological studies evaluating the effects of tyrosine kinase inhibitors on MET signaling

• Cell-based assays to monitor MET activation in response to HGF or other stimuli

• Phospho-specific ELISA assays that can quantitatively measure phosphorylated MET levels

These applications collectively provide researchers with tools to investigate MET activation in diverse experimental contexts, from basic mechanistic studies to translational cancer research.

How does phosphorylation at Tyr1349 compare with other MET phosphorylation sites?

The MET receptor contains multiple phosphorylation sites that serve distinct functions in receptor activation and downstream signaling. Comparison between these sites reveals important mechanistic insights:

Phosphorylation at Tyr1234/1235 within the activation loop is required for catalytic activity, essentially functioning as an "on/off switch" for the receptor . In contrast, phosphorylation at Tyr1349/1356 in the carboxy-terminal tail creates docking sites for adaptor proteins and does not directly affect kinase activity but is critical for signal propagation . Phosphorylation at Tyr1003 in the juxtamembrane domain primarily serves as a negative regulatory site by recruiting c-Cbl, which mediates receptor ubiquitination and degradation.

Research using phospho-specific antibodies has demonstrated that in PTP1B-null mice, HGF stimulation leads to a 4-6 fold increase in phosphorylation across multiple tyrosine residues (Tyr1234/1235, Tyr1003, and Tyr1365) compared to wild-type mice . This suggests coordinated regulation of these sites, though their individual kinetics and threshold requirements may differ.

Studies using site-directed mutagenesis have shown that substitution of Tyr1234 or Tyr1235 with phenylalanine significantly decreases the ability of phosphatases like TCPTP or PTP1B to coimmunoprecipitate with MET, highlighting the primary importance of activation loop phosphorylation in regulating MET activity .

What role does Phospho-MET (Tyr1349) play in cancer progression?

Phosphorylation of MET at Tyr1349 has significant implications for cancer biology and serves as a potential prognostic marker. Research has demonstrated that:

• Elevated phospho-MET (Tyr1349) levels correlate with poor clinical outcomes in small cell lung cancer, where statistical analysis established an optimal cutoff point using a p-MET Hscore of 5 to distinguish between positive and negative cases .

• Phosphorylation at Tyr1349 activates multiple oncogenic pathways including PI3K signaling, SRC, STAT, and Ras-Raf-Mek-Erk cascades, which collectively promote cancer cell proliferation, survival, motility, and angiogenesis .

• The multi-substrate docking site containing phosphorylated Tyr1349 serves as a platform for assembling signaling complexes that drive invasive growth programs, which are critical for metastatic progression.

• In clinical samples, phospho-MET status can be evaluated using immunohistochemistry with phospho-specific antibodies, where receiver operating curve (ROC) analysis helps establish clinically relevant thresholds .

These findings underscore the importance of phospho-MET (Tyr1349) not only as a mechanistic indicator of MET activation but also as a potential biomarker for patient stratification and therapeutic targeting in cancer treatment.

How do protein phosphatases regulate MET phosphorylation at Tyr1349?

Protein phosphatases play crucial roles in regulating MET phosphorylation through several mechanisms:

Mechanistically, PTP1B preferentially interacts with MET through the activation loop phosphotyrosines (Tyr1234/1235). Coimmunoprecipitation studies have shown that mutation of these residues significantly decreases the interaction between PTP1B and MET . Since phosphorylation of Tyr1349 is dependent on prior activation loop phosphorylation, PTP1B indirectly regulates Tyr1349 phosphorylation by controlling the catalytic activity of MET.

Another phosphatase, T-cell protein tyrosine phosphatase (TCPTP), also regulates MET phosphorylation through similar mechanisms, though with some distinct properties. While both phosphatases interact with the activation loop tyrosines, TCPTP shows some dependence on Tyr1003 for optimal interaction with MET . This suggests differential regulation of MET phosphorylation sites by different phosphatases.

These phosphatase-mediated regulatory mechanisms provide important negative feedback loops that prevent excessive MET signaling and maintain appropriate cellular responses to HGF stimulation.

What is the optimal protocol for Western Blotting with Phospho-MET (Tyr1349) antibody?

Successful Western blotting with Phospho-MET (Tyr1349) antibody requires careful attention to technical details:

Sample Preparation:

• Stimulate cells with HGF (200 ng/mL for 5 minutes) to maximize phosphorylation signal. Sodium vanadate (1 mM for 4 hours) can be added to inhibit phosphatases and preserve phosphorylation .

• Lyse cells in buffer containing phosphatase inhibitors (sodium vanadate, sodium fluoride, β-glycerophosphate) to prevent dephosphorylation during sample processing.

• For tissue samples, rapid freezing post-collection is essential to preserve phosphorylation status.

Blotting Protocol:

• Use 20 μg of protein lysate per lane for optimal detection .

• Separate proteins on 7.5% SDS-PAGE (due to MET's high molecular weight of 145 kDa).

• Transfer to PVDF membrane (nitrocellulose is acceptable but may have lower sensitivity).

• Block with 5% BSA in TBST (not milk, which contains phosphatases).

• Dilute Phospho-MET (Tyr1349) antibody 1:1000 in 5% BSA/TBST and incubate overnight at 4°C .

• Wash thoroughly with TBST (3-5 times for 5 minutes each).

• Incubate with appropriate HRP-conjugated secondary antibody.

• Develop using enhanced chemiluminescence.

Controls:

• Include both positive (HGF-stimulated) and negative (unstimulated) lysates .

• Consider including a total MET antibody blot to normalize phospho-signal to total protein expression.

• Pre-treatment with lambda phosphatase can serve as a negative control to confirm phospho-specificity.

This methodology ensures specific and sensitive detection of phosphorylated MET at Tyr1349, allowing for accurate assessment of MET activation status.

How can immunoprecipitation with Phospho-MET (Tyr1349) antibody be optimized?

Immunoprecipitation (IP) with Phospho-MET (Tyr1349) antibody requires specific optimization strategies to ensure successful enrichment of phosphorylated MET protein:

Protocol Optimization:

• Use fresh lysates (1-2 mg total protein) prepared in non-denaturing lysis buffer containing phosphatase inhibitors.

• Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Dilute Phospho-MET (Tyr1349) antibody at 1:50 ratio for IP applications .

• Incubate antibody with lysate overnight at 4°C with gentle rotation.

• Add protein A/G beads and incubate for an additional 2-4 hours.

• Wash beads 4-5 times with lysis buffer containing reduced detergent concentration.

• Elute bound proteins by boiling in SDS sample buffer.

Technical Considerations:

• For co-immunoprecipitation studies involving MET interaction partners, crosslinking may be necessary to stabilize transient interactions.

• When studying MET mutants, consider that substitution of tyrosines in the activation loop (Tyr1234/1235) significantly decreases co-immunoprecipitation efficiency, as demonstrated in studies with phosphatase trapping mutants .

• Enhanced detection can be achieved by using trapping mutants of protein tyrosine phosphatases (e.g., PTP1B-D/A) that form stable complexes with phosphorylated MET .

This optimized approach enables specific isolation of phosphorylated MET protein, facilitating downstream analyses including identification of novel interacting partners and characterization of phosphorylation-dependent protein complexes.

What quantitative methods can accurately measure Phospho-MET (Tyr1349) levels?

Several quantitative methodologies can accurately measure Phospho-MET (Tyr1349) levels with varying degrees of sensitivity and throughput:

Phospho-MET (Tyr1349) ELISA:
The MSD Phospho-Met (Tyr1349) Assay represents a highly sensitive method for quantification, with the following performance characteristics:

This assay demonstrates excellent signal-to-noise ratios across a broad range of lysate concentrations, with optimal performance observed with 5-10 μg of lysate . The assay uses a sandwich format with anti-phospho-Met capture antibody and detection with anti-total Met antibody conjugated with MSD SULFO-TAG reagent.

Immunohistochemistry (IHC) Quantification:
In clinical research, phospho-MET levels in tissue samples can be quantified using H-score methodology, which incorporates both staining intensity and percentage of positive cells . For phospho-MET (Tyr1349), a cutoff H-score of 5 has been established through receiver operating curve (ROC) analysis to discriminate between positive and negative samples in certain cancer contexts .

Western Blot Densitometry:
While less precise than ELISA methods, densitometric analysis of Western blots can provide semi-quantitative assessment of phospho-MET levels. This approach requires careful normalization to total MET expression and inclusion of standard curves when possible.

These complementary approaches provide researchers with a toolkit for phospho-MET quantification across various experimental contexts, from cell-based assays to clinical specimens.

How does Phospho-MET (Tyr1349) serve as a biomarker in cancer research?

Phospho-MET (Tyr1349) has emerged as a valuable biomarker in cancer research, with particular relevance to prognosis and treatment response prediction:

In small cell lung cancer studies, researchers have established methodologies for evaluating phospho-MET status in clinical specimens. Statistical approaches including χ2-test, Fisher's exact test, Spearman rho test, Kaplan–Meier method, and Cox proportional hazards model have been applied to correlate phospho-MET levels with clinical outcomes . These analyses have established that phospho-MET positivity, defined using an H-score cutoff of 5 as determined by receiver operating curve (ROC) analysis, is associated with poor prognosis .

The prognostic value of phospho-MET (Tyr1349) stems from its central role in activating downstream signaling pathways that drive cancer progression. When phosphorylated, Tyr1349 serves as a docking site for multiple signaling adapters that propagate oncogenic signals through PI3K, SRC, STAT, and Ras-Raf-Mek-Erk pathways . These pathways collectively promote cancer cell proliferation, survival, motility, and angiogenesis.

Methodologically, researchers typically employ immunohistochemistry with phospho-specific antibodies to evaluate phospho-MET status in tissue microarrays or whole tissue sections. Results are scored using established systems that account for both staining intensity and the percentage of positive cells, following data and statistical analysis reporting compliant with REMARK guidelines .

What considerations are important when validating Phospho-MET (Tyr1349) as a biomarker?

Validation of phospho-MET (Tyr1349) as a biomarker requires rigorous attention to both pre-analytical and analytical variables:

Pre-analytical Considerations:

• Tissue processing time significantly impacts phosphorylation preservation; samples should be fixed rapidly after collection (ideally within 30 minutes).

• Phosphatase activity during specimen handling can compromise phospho-epitopes; phosphatase inhibitors may be necessary during sample preparation.

• Fixation type and duration affect phospho-epitope detection; standardized protocols are essential for cross-study comparability.

Analytical Validation:

• Antibody specificity must be thoroughly validated using positive controls (HGF-stimulated cells) and negative controls (unstimulated cells or phosphatase-treated samples) .

• Technical reproducibility should be established through intra- and inter-laboratory testing.

• Cutoff determination requires statistical approaches such as receiver operating curve (ROC) analysis, with optimal thresholds established based on clinical outcome correlation .

Clinical Validation:

• Multivariate analysis using the Cox proportional hazards model should be performed to determine if phospho-MET status provides prognostic information independent of established clinical variables .

• Prospective studies are ultimately required to confirm the clinical utility of phospho-MET as a biomarker for patient stratification or treatment selection.

These methodological considerations are essential for the robust validation of phospho-MET (Tyr1349) as a clinically meaningful biomarker that can inform patient management decisions.

What are common problems in Phospho-MET (Tyr1349) detection and their solutions?

Researchers frequently encounter technical challenges when working with phospho-MET (Tyr1349) antibodies. The following table outlines common problems and their methodological solutions:

Quality Control Measures:

• Always include positive controls (HGF-stimulated samples) and negative controls (unstimulated or phosphatase-treated samples) .

• Perform antibody validation using phospho-Met (Tyr1349)-positive cell lysates with HGF treatment and vanadate .

• Verify signal specificity by comparing Western blot results with ELISA or other detection methods .

• For clinical samples, include internal reference standards on each experimental run to normalize inter-assay variability.

These troubleshooting approaches and quality control measures ensure reliable and reproducible detection of phospho-MET (Tyr1349) across different experimental settings.