Phospho-CSF1R (Tyr809) Antibody

What is CSF1R and what is the significance of Tyr809 phosphorylation?

CSF1R (Colony-Stimulating Factor 1 Receptor), encoded by the c-fms proto-oncogene, is a receptor tyrosine kinase primarily expressed in monocytes, macrophages, and their progenitors. This receptor plays a crucial role in driving the growth and development of the monocyte/macrophage lineage .

Tyrosine 809 phosphorylation represents one of at least five major autophosphorylation sites on the receptor. When phosphorylated, Tyr809 serves as a docking site for the adapter protein Shc, which facilitates downstream signal transduction . This phosphorylation event is critical for full receptor activation and subsequent cellular responses, including proliferation and differentiation of macrophages .

Research has demonstrated that phosphorylation at Tyr809 is a key mediator in the CSF1R signaling cascade and can significantly influence cellular outcomes in both normal physiological processes and pathological conditions .

How does CSF1R activation occur and what role does Tyr809 play in this process?

CSF1R activation follows a well-characterized sequence of events. The binding of CSF-1 (M-CSF) to the receptor induces receptor dimerization, which triggers activation of the intrinsic tyrosine kinase domain. This activation leads to autophosphorylation of multiple cytoplasmic tyrosine residues that serve as docking sites for SH2-containing signaling proteins .

The phosphorylation of Tyr809 occurs as part of this activation cascade and provides a specific docking site for the adapter protein Shc . Experimental evidence indicates that Tyr809 phosphorylation is crucial for full receptor activation and downstream signaling. In studies using mutant receptors, it was shown that Tyr-807 (equivalent to Tyr809 in some numbering systems) drives proliferation, while other tyrosine residues like Tyr-559 confer ligand dependence to the receptor's activity .

The activation mechanism can be visualized as follows:

• CSF-1 binds to CSF1R

• Receptor dimerization occurs

• Tyrosine kinase domains become activated

• Autophosphorylation of multiple tyrosines, including Tyr809

• Recruitment of signaling proteins (such as Shc to pTyr809)

• Activation of downstream signaling pathways

What are the recommended applications for Phospho-CSF1R (Tyr809) Antibody?

The Phospho-CSF1R (Tyr809) Antibody is primarily recommended for Western Blotting applications at a dilution of 1:1000 . This application enables researchers to detect endogenous levels of CSF1R only when phosphorylated at Tyr809, making it valuable for studying receptor activation status.

The antibody has demonstrated reactivity with human and mouse samples, with a detected molecular weight of approximately 175 kDa, corresponding to the full-length phosphorylated receptor . While Western Blotting is the primary validated application, researchers should note that optimal conditions may vary depending on specific experimental systems and should be determined empirically.

It's important to note that the antibody detects endogenous levels of the target protein, providing physiologically relevant data when examining CSF1R signaling in various experimental contexts .

How does the phosphorylation status of Tyr809 correlate with macrophage proliferation and differentiation?

Research has established a critical relationship between Tyr809 phosphorylation and macrophage proliferation. Studies using mutational analysis have revealed that Tyr-807 (equivalent to Tyr809) confers sufficient kinase activity for strong CSF-1-independent proliferation . This finding suggests that phosphorylation at this site serves as a key regulator of proliferative responses.

In experimental systems where all eight tyrosine phosphorylation sites were mutated to phenylalanine (YEF mutant), the addition of Tyr-807 alone (Y807AB mutant) led to CSF-1-independent but receptor kinase-dependent proliferation . This occurred without detectable activation loop Tyr-807 phosphorylation, suggesting that the presence of this tyrosine residue, rather than its phosphorylation status, may be sufficient to permit a baseline level of proliferative signaling.

The relationship between Tyr809 phosphorylation and macrophage differentiation appears to be complex and interconnected with other phosphorylation sites. For instance, while Tyr-807 drives proliferation, Tyr-559 in the juxtamembrane domain maintains the receptor in an inactive state until ligand binding occurs, at which point Tyr-559 phosphorylation releases this inhibition and contributes to the CSF-1-regulated proliferative response .

What is the relationship between TNFAIP2 and CSF1R Tyr809 phosphorylation?

Recent research has identified TNFAIP2 (Tumor Necrosis Factor Alpha-Induced Protein 2) as a unique cellular regulator of CSF1R signaling. TNFAIP2 expression has been shown to augment CSF-1–mediated activation of MAP kinases and increase phosphorylation of CSF1R at Tyr809, which is described as "the major autophosphorylation site important for full activation of CSF1R" .

The mechanistic relationship appears to involve TNFAIP2-mediated enhancement of CSF1R aggregate formation. In CSF1R-transfected 293 cells, TNFAIP2 expression augmented the formation of large CSF1R aggregates and increased their size . This aggregation phenomenon appears to facilitate more efficient CSF-1–induced activation of CSF1R, as evidenced by enhanced Tyr809 phosphorylation.

Interestingly, phosphatidylinositol 4,5-bisphosphate (PIP2) binding seems to play a role in this process, as CSF1R mutants lacking the motif that mediates binding to PIP2 fail to form large aggregates even in the presence of TNFAIP2 . This suggests a complex interplay between membrane phospholipids, TNFAIP2, and CSF1R in regulating receptor aggregation and subsequent phosphorylation events, including at Tyr809.

How do mutations affecting Tyr809 impact signaling pathway activation?

Mutational studies have provided significant insights into the role of Tyr809 in CSF1R signaling. The Y807F mutation (equivalent to Y809F in some numbering systems) severely compromises CSF-1-regulated proliferation and differentiation . This indicates that this tyrosine residue plays a non-redundant role in receptor function that cannot be compensated by other phosphorylation sites.

Experimental evidence suggests that constitutive proliferation mediated by Tyr807 (Tyr809) is primarily dependent on the phosphatidylinositol 3-kinase (PI3K) and ERK1/2 pathways . This was demonstrated through inhibitor experiments where proliferation of Y807AB macrophages (with Tyr807 as the only tyrosine add-back in the receptor) was blocked by PI3K and ERK pathway inhibitors.

In contrast, proliferation of wild-type and Y559,807AB macrophages also involved Src family kinase (SFK)-dependent pathways . This indicates that the presence and phosphorylation status of Tyr809 not only affects receptor activation but also determines which downstream signaling pathways become engaged.

The differential activation of signaling pathways based on Tyr809 status has important implications for therapeutic interventions targeting CSF1R, particularly in contexts such as cancer where aberrant receptor activation may contribute to disease progression.

What are the optimal protocols for detecting Phospho-CSF1R (Tyr809) in experimental systems?

For optimal detection of phosphorylated CSF1R at Tyr809, Western blotting is the recommended method using the phospho-specific antibody at a 1:1000 dilution . The following protocol recommendations will help ensure reliable and reproducible results:

Sample preparation:

• Treat cells with CSF-1 (360 ng/ml) to stimulate receptor phosphorylation

• Lyse cells in an appropriate buffer containing phosphatase inhibitors to preserve phosphorylation status

• Clear lysates by centrifugation and determine protein concentration

Western blotting procedure:

• Separate proteins by SDS-PAGE (typically 7.5% gels work well for the 175 kDa CSF1R)

• Transfer proteins to PVDF or nitrocellulose membrane

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

• Incubate with Phospho-CSF1R (Tyr809) antibody (1:1000) overnight at 4°C

• Wash and incubate with HRP-conjugated secondary antibody

• Develop using enhanced chemiluminescence

For pulse-chase experiments to study receptor dynamics, cells can be labeled with 35S-LABEL™ No-Thaw (1049 Ci/mmol) and chased in 100-fold excess of unlabeled Met and Cys . Alternatively, cell surface biotinylation with EZ-Link Sulfo-NHS-Biotin followed by receptor immunoprecipitation can provide insights into receptor trafficking and phosphorylation kinetics .

How can researchers effectively use inhibitor studies to elucidate Tyr809-dependent signaling pathways?

Inhibitor studies represent a powerful approach to dissect the specific signaling pathways dependent on Tyr809 phosphorylation. Based on the literature, the following methodology has proven effective:

Experimental design for inhibitor studies:

• Culture cells with or without CSF-1 (360 ng/ml) in appropriate medium

• Preincubate cells with vehicle control (DMSO) or specific inhibitors:

  • SU6656 (2 μM) for Src family kinases

  • PD98058 (50 μM) for MEK/ERK pathway

  • LY294002 (10 μM) for PI3K pathway

  • JNK inhibitor (10 μM)

  • p38 MAPK inhibitor (5 μM)

  • CSF1R inhibitors such as PLX3397 (15 μM) or PLX5622 (15 μM)

• Analyze effects on:

  • Proliferation using EdU incorporation

  • CSF1R phosphorylation status by Western blotting

  • Downstream pathway activation

This approach has revealed that the constitutive proliferation observed in Y807AB macrophages is mediated primarily by PI3K and ERK1/2 pathways, whereas proliferation of wild-type and Y559,807AB macrophages additionally involves Src family kinase-dependent pathways .

To maximize experimental rigor, researchers should include appropriate controls, such as total CSF1R blotting alongside phospho-specific detection, and consider dose-response studies with inhibitors to establish specificity.

What approaches can be used to study the interaction between TNFAIP2 and CSF1R phosphorylation at Tyr809?

To investigate the relationship between TNFAIP2 and CSF1R phosphorylation at Tyr809, researchers can employ several complementary approaches:

Co-expression studies:

• Transfect cells (e.g., 293 cells) with CSF1R with or without TNFAIP2

• Stimulate with CSF-1 and analyze:

  • CSF1R aggregation through microscopy

  • Phosphorylation at Tyr809 via Western blotting

  • Activation of downstream pathways such as MAP kinases

Aggregate analysis:

• Quantify the size and number of CSF1R aggregates in the presence/absence of TNFAIP2

• Correlate aggregate formation with Tyr809 phosphorylation status

• Investigate the role of membrane components like PIP2 using CSF1R mutants lacking the PIP2-binding motif

Signaling pathway analysis:

• Monitor MAP kinase activation in parallel with Tyr809 phosphorylation

• Use phospho-specific antibodies to detect activated ERK, p38, and JNK

• Compare signaling kinetics between TNFAIP2-positive and TNFAIP2-negative cells

These approaches have revealed that TNFAIP2 expression augments CSF-1–mediated activation of MAP kinases and increases phosphorylation of CSF1R at Tyr809, likely through enhancing receptor aggregation .

How should researchers address discrepancies in detected phospho-CSF1R (Tyr809) levels between different experimental systems?

When confronting discrepancies in phospho-CSF1R (Tyr809) detection across different experimental systems, researchers should systematically evaluate several factors:

Antibody validation and specificity:

• Confirm antibody specificity using positive and negative controls

• Consider using multiple antibodies targeting the same phospho-site

• Include phosphatase-treated samples as controls for phospho-specificity

Cell type and receptor expression levels:

• Quantify total CSF1R expression in different cell systems

• Consider the ratio of phosphorylated to total receptor rather than absolute levels

• Note that endogenous detection works best in cells with high CSF1R expression like monocytes and macrophages

Experimental conditions affecting phosphorylation:

• Standardize CSF-1 stimulation protocols (concentration, duration, temperature)

• Ensure phosphatase inhibitors are fresh and used at appropriate concentrations

• Minimize sample processing time to prevent dephosphorylation

Technical considerations:

• For Western blotting, try different blockers (BSA vs. milk products)

• Adjust exposure times to capture potentially weak signals

• Consider using more sensitive detection methods for low-expression systems

By systematically addressing these factors, researchers can better understand whether discrepancies represent biological differences or technical artifacts, leading to more reliable interpretation of phospho-CSF1R (Tyr809) data across experimental systems.

What are the critical controls needed when studying CSF1R Tyr809 phosphorylation in relation to cellular functions?

When investigating the relationship between CSF1R Tyr809 phosphorylation and cellular functions, several critical controls should be incorporated:

Phosphorylation-specific controls:

• Total CSF1R expression controls alongside phospho-specific detection

• Phosphatase treatment controls to confirm phospho-specificity

• Time-course analysis of phosphorylation to determine optimal sampling points

Genetic controls:

• CSF1R knockout or knockdown cells as negative controls

• Y809F mutant receptor expression to confirm specificity

• Add-back experiments with mutant receptors (as described in the Y807AB studies)

Pharmacological controls:

• CSF1R kinase inhibitors (e.g., PLX3397, PLX5622) to block all receptor activity

• Pathway-specific inhibitors to dissect downstream contributions

• Appropriate vehicle controls for all inhibitors

Functional assay controls:

• For proliferation assays, include both positive stimulators (e.g., serum) and growth arrest controls

• For differentiation assessments, include multiple markers beyond morphology

• For signaling studies, validate pathway activation using multiple methods

A particularly informative approach is the comparison between wild-type CSF1R, the Y807F (Y809F) mutant, and the YEF mutant with Y807 add-back, which has revealed distinct roles for Tyr809 in proliferation and pathway activation .

How can researchers differentiate between direct effects of Tyr809 phosphorylation and indirect consequences on CSF1R signaling?

Distinguishing direct effects of Tyr809 phosphorylation from indirect consequences on CSF1R signaling requires sophisticated experimental approaches:

Site-specific mutational analysis:

• Compare Y809F single mutants with wild-type CSF1R

• Use the YEF background with single tyrosine add-back at position 809 (Y809AB)

• Create phosphomimetic mutants (Y809E or Y809D) to simulate constitutive phosphorylation

Temporal signaling analysis:

• Perform detailed time-course studies of Tyr809 phosphorylation

• Compare kinetics of Tyr809 phosphorylation with activation of downstream pathways

• Use rapid inhibition approaches to determine pathway dependencies

Protein-protein interaction studies:

• Identify direct binding partners of phospho-Tyr809 using pull-down assays with phosphopeptides

• Confirm that Shc binding occurs directly to phospho-Tyr809 using co-immunoprecipitation

• Employ proximity ligation assays to visualize interactions in intact cells

Pathway-specific interventions:

• Use RNA interference to deplete specific signaling components

• Apply pharmacological inhibitors in combination with site-specific mutants

• Reconstitute signaling in minimal systems to identify essential components

What novel roles for CSF1R Tyr809 phosphorylation have been discovered in disease contexts?

Recent research has revealed several important roles for CSF1R Tyr809 phosphorylation in disease contexts:

Cancer progression and prognosis:
The activated CSF-1 receptor, including phosphorylation at Tyr809, has been identified as a predictor of poor outcome in advanced epithelial ovarian carcinoma and breast cancer . This suggests that phospho-Tyr809 detection might serve as a biomarker for disease aggressiveness and potential therapeutic response.

Overactivation of the receptor, which includes aberrant Tyr809 phosphorylation, can lead to malignant phenotypes in various cell systems . This has implications for cancers where CSF1R signaling contributes to disease progression, particularly those with a significant tumor-associated macrophage component.

Inflammatory conditions:
The discovery that TNFAIP2 enhances CSF1R phosphorylation at Tyr809 and subsequent MAP kinase activation connects inflammatory signaling networks with macrophage proliferation and activation. This may have relevance for inflammatory diseases where macrophage dysregulation plays a central role.

Future clinical applications:
The specific detection of phospho-Tyr809 could potentially be developed into diagnostic or prognostic tools for cancers where CSF1R activation correlates with disease outcomes. Therapeutic approaches targeting this specific phosphorylation event or its downstream consequences might offer more selective intervention strategies compared to general CSF1R inhibition.

How does the regulatory relationship between Tyr559 and Tyr809 influence development of CSF1R-targeted therapeutics?

The complex regulatory relationship between Tyr559 and Tyr809 has significant implications for CSF1R-targeted therapeutic development:

Dual targeting strategies:
Research has revealed that Tyr-807 (Tyr809) drives proliferation, while Tyr-559 maintains the receptor in an inactive state . This suggests that therapeutics might benefit from addressing both sites—either stabilizing the autoinhibitory function of Tyr559 or interfering with the proliferative capacity of Tyr809.

Pathway-selective inhibition:
Studies indicate that the constitutive proliferation mediated by Tyr807 (Tyr809) involves primarily PI3K and ERK1/2 pathways, whereas proliferation dependent on both Tyr559 and Tyr807 additionally requires Src family kinase (SFK) pathways . This differential pathway engagement suggests opportunities for selective therapeutic targeting in different disease contexts.

Ligand-independent activity considerations:
The observation that mutation Y807AB exhibits CSF-1-independent proliferation raises important concerns for therapeutic development. Inhibitors designed to block ligand binding or dimerization might be ineffective against receptors with constitutive activity driven by Tyr809-dependent mechanisms.

Current CSF1R inhibitors like PLX3397 and PLX5622 (used at 15 μM in experimental systems) target the kinase domain broadly. Future drug development might benefit from understanding the specific conformational changes associated with Tyr559 and Tyr809 phosphorylation to design more selective agents that modulate specific aspects of receptor function rather than blocking all activity.

What emerging technologies are advancing our understanding of CSF1R Tyr809 phosphorylation dynamics?

Several emerging technologies are providing unprecedented insights into CSF1R Tyr809 phosphorylation dynamics:

Advanced mass spectrometry approaches:
Phosphoproteomics technologies like PhosphoScan® are enabling more comprehensive detection and quantification of CSF1R phosphorylation sites, including Tyr809 . These approaches allow researchers to monitor multiple phosphorylation events simultaneously and track their dynamics following receptor activation.

Live-cell imaging of phosphorylation:
Genetically encoded biosensors for tyrosine phosphorylation can be adapted to study CSF1R Tyr809 phosphorylation in living cells. These approaches provide real-time visualization of phosphorylation events at the single-cell level, revealing spatial and temporal dynamics that are inaccessible to biochemical methods.

Single-molecule studies of receptor aggregation:
Advanced microscopy techniques allow visualization of CSF1R aggregation in relationship to phosphorylation status. This is particularly relevant given the discovery that TNFAIP2 augments CSF-1–mediated activation and phosphorylation of CSF1R at Tyr809 by enhancing receptor aggregation .

Computational modeling of receptor dynamics:
Molecular dynamics simulations and systems biology approaches are providing mechanistic insights into how Tyr809 phosphorylation affects receptor conformation and signaling. These computational methods help integrate experimental data into coherent models of receptor function that can guide future research and therapeutic development.

These technological advances promise to deepen our understanding of how CSF1R Tyr809 phosphorylation integrates into broader signaling networks and how its dysregulation contributes to pathological states, ultimately informing more precise therapeutic interventions.