Phospho-STAT5B (Ser731) Antibody

What is the biological significance of STAT5B Ser731 phosphorylation?

Phosphorylation at serine 731 represents a critical post-translational modification in the transactivation domain of STAT5B. This modification significantly modulates STAT5B activity, particularly in conjunction with tyrosine phosphorylation at Y699. EGF stimulation has been demonstrated to enhance S731 phosphorylation as early as 5 minutes after treatment, with phosphorylation being sustained for up to 3 hours before gradually decreasing . S731 phosphorylation plays a crucial role in STAT5B's transcriptional activity and subsequent biological functions, including DNA synthesis in cancer cells .

How does Ser731 phosphorylation relate to other STAT5B phosphorylation sites?

STAT5B contains multiple phosphorylation sites that work in concert to regulate its function. While Y699 is the critical tyrosine phosphorylation site required for activation, S731 phosphorylation in the transactivation domain enhances activation and nuclear translocation . Additionally, STAT5B contains other phosphorylation sites including S715, Y725, Y740, and Y743. Notably, while Y740 and Y743 act as negative regulators of transcription by reducing Y699 phosphorylation, S731 works synergistically with Y699 to promote STAT5B activity .

What is the relationship between STAT5A Ser726 and STAT5B Ser731 phosphorylation?

STAT5A Ser726 and STAT5B Ser731 represent homologous phosphorylation sites in these highly related proteins. Despite their similarities, these phosphorylation events may have distinct regulatory mechanisms and biological outcomes. Some antibodies recognize both phosphorylation sites due to sequence similarity surrounding these serine residues . Both sites are located in the carboxy-terminal P(M)SP motifs and can undergo phosphorylation in response to cytokine stimulation, particularly in hematopoietic cells .

What are the recommended applications for Phospho-STAT5B (Ser731) antibodies?

Phospho-STAT5B (Ser731) antibodies have been validated for multiple experimental applications:

These applications allow researchers to detect and quantify phosphorylated STAT5B across various experimental systems .

What cell stimulation protocols effectively induce STAT5B Ser731 phosphorylation?

Several stimulation protocols have been validated for inducing STAT5B Ser731 phosphorylation:

• EGF stimulation: Demonstrated to significantly increase S731 phosphorylation as early as 5 minutes post-treatment .

• GM-CSF treatment: Effectively induces phosphorylation in TF-1 cells, a common model for hematopoietic signaling studies .

• IL-2 family cytokines: Tightly control Ser731 phosphorylation through rapamycin-sensitive mechanisms .

• Calyculin A (100 nM for 30 minutes): A phosphatase inhibitor that can be used to artificially increase phosphorylation levels for positive controls .

When designing experiments, it's critical to include appropriate positive controls and time-course analyses, as S731 phosphorylation can be rapid and transient .

How can researchers validate the specificity of phospho-STAT5B (Ser731) antibody signals?

Validation should employ multiple complementary approaches:

• Phosphatase treatment controls: Treating samples with lambda phosphatase should eliminate the specific signal.

• Phospho-blocking peptide competition: Pre-incubation of the antibody with phosphorylated peptide should block specific binding.

• Mutation analysis: Using S731A STAT5B mutants as negative controls provides definitive evidence of specificity, as demonstrated in published research .

• Stimulation time-course: The temporal pattern of phosphorylation following stimulation (rapid increase followed by gradual decrease) can help confirm signal authenticity.

• Inhibitor experiments: Using specific inhibitors of pathways known to regulate S731 phosphorylation (e.g., mTOR inhibitors) can validate signal regulation .

How does STAT5B Ser731 phosphorylation influence cancer development and progression?

STAT5B Ser731 phosphorylation has significant implications for cancer biology:

• Breast cancer: Increased S731 phosphorylation has been observed in breast cancer cell lines that overexpress EGFR and HER2 tyrosine kinases .

• Hematopoietic malignancies: Constitutive phosphorylation of Ser731 has been documented in several lymphoid tumor cell lines and primary leukemia and lymphoma patient samples .

• Transcriptional activity: S731 phosphorylation is required for maximal STAT5B transcriptional activity, which drives expression of genes involved in cell proliferation and survival .

• DNA synthesis: Experimental evidence indicates that S731 phosphorylation significantly impacts DNA synthesis rates in cancer cells, with S731A mutation decreasing proliferation below levels seen with wild-type STAT5B .

Understanding these mechanisms provides potential therapeutic targets for cancers with aberrant STAT5B signaling .

What is the relationship between STAT5B Ser731 phosphorylation and the Y740/743F mutations?

Research has revealed a complex interplay between these sites:

• The Y740/743F STAT5B mutant demonstrates increased basal and EGF-induced S731 phosphorylation compared to wild-type STAT5B .

• This mutant also shows enhanced Y699 phosphorylation, increased transcriptional activity, and elevated DNA synthesis .

• Critically, mutation of S731 (S731A) in the context of Y740/743F abrogates these effects, demonstrating that S731 phosphorylation is necessary for the increased activity of the Y740/743F mutant .

• These findings indicate that S731, Y699, Y740, and Y743 form an interconnected regulatory network that precisely controls STAT5B function .

This relationship highlights potential mechanisms for designing therapeutics that target STAT5B activity in cancer cells .

What signaling pathways regulate STAT5B Ser731 phosphorylation?

Multiple upstream pathways converge on STAT5B Ser731 phosphorylation:

• EGF/EGFR pathway: EGF stimulation enhances S731 phosphorylation, implicating receptor tyrosine kinase signaling .

• mTOR signaling: Inducible S731 phosphorylation is sensitive to mTOR inhibitors, indicating mTOR as a potential upstream kinase .

• Protein phosphatase 2A (PP2A): Inhibition of PP2A induces S731 phosphorylation, suggesting this phosphatase actively regulates the phosphorylation state .

• MAPK family: Kinases including ERKs, JNK, p38 MAPK, and PAK have been associated with serine phosphorylation of STAT proteins, potentially including S731 .

• IL-2 family cytokines: These cytokines tightly control S731 phosphorylation through rapamycin-sensitive mechanisms, further supporting the role of mTOR .

Understanding these regulatory pathways may reveal novel therapeutic approaches for diseases with aberrant STAT5B activity .

How can researchers distinguish between phosphorylated STAT5A and STAT5B in experimental systems?

Distinguishing between phosphorylated STAT5A and STAT5B requires careful experimental design:

• Isoform-specific antibodies: Use antibodies that specifically recognize either phospho-STAT5A (Ser726) or phospho-STAT5B (Ser731), though some antibodies detect both due to sequence similarity .

• Immunoprecipitation approach: Perform immunoprecipitation with isoform-specific antibodies followed by phospho-detection.

• Molecular weight discrimination: STAT5A and STAT5B have slightly different molecular weights (94 kDa vs. 92 kDa), which can sometimes be resolved by extended SDS-PAGE runs.

• Genetic approaches: In cell culture systems, siRNA knockdown of one isoform followed by phospho-detection can help attribute signals.

• Mass spectrometry: For definitive identification, phospho-peptide analysis by mass spectrometry can distinguish between the isoforms based on unique peptide sequences .

These approaches can be combined for more robust discrimination between the phosphorylated isoforms .

What are the optimal sample preparation methods for detecting phospho-STAT5B (Ser731)?

To maximize detection of phosphorylated STAT5B (Ser731):

• Rapid sample processing: Process samples quickly to prevent dephosphorylation by endogenous phosphatases.

• Phosphatase inhibitors: Include comprehensive phosphatase inhibitor cocktails in all buffers (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate, and calyculin A).

• Optimal lysis conditions: Use buffers containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris pH 7.4, and 10% glycerol.

• Protein denaturation: For western blotting, sample buffer should contain SDS to ensure complete protein denaturation.

• Sample concentration: For low abundance samples, consider immunoprecipitation before western blot analysis.

• Subcellular fractionation: Since S731 phosphorylation occurs primarily in the cytoplasmic compartment prior to nuclear translocation, subcellular fractionation may enhance detection sensitivity .

These optimizations help prevent signal loss and ensure reliable detection of phosphorylated STAT5B .

How do different fixation and permeabilization protocols affect detection of phospho-STAT5B (Ser731) in immunofluorescence experiments?

Different protocols significantly impact phospho-epitope detection:

• Paraformaldehyde fixation (4%): Preserves phospho-epitopes while maintaining cellular architecture; recommended fixation time is 15-20 minutes at room temperature.

• Methanol fixation: Can improve detection of some phospho-epitopes but may reduce signal for others; test empirically for phospho-STAT5B (Ser731).

• Glutaraldehyde: Generally not recommended as it can mask phospho-epitopes through excessive cross-linking.

• Permeabilization agents:

  • Triton X-100 (0.1-0.5%): Provides good permeabilization but may extract some cellular components

  • Saponin (0.1-0.3%): Gentler permeabilization that preserves more cellular structures

  • Methanol: Acts as both fixative and permeabilizer

• Antigen retrieval: For tissue sections or strongly fixed samples, heat-induced epitope retrieval in citrate buffer (pH 6.0) may improve phospho-epitope accessibility.

Optimization is crucial, as phospho-STAT5B (Ser731) detection appears to be sensitive to fixation conditions .

What is the relevance of STAT5B Ser731 phosphorylation in normal versus pathological hematopoiesis?

STAT5B Ser731 phosphorylation exhibits distinct patterns in normal versus pathological hematopoiesis:

• Normal hematopoiesis: S731 phosphorylation is tightly regulated and transiently induced by cytokines such as IL-2 family members and growth factors .

• Leukemias and lymphomas: Constitutive phosphorylation of S731 has been observed in multiple lymphoid tumor cell lines and primary patient samples, suggesting dysregulation of this modification in hematological malignancies .

• Regulatory mechanisms: In normal cells, phosphatases like PP2A actively regulate S731 phosphorylation, whereas in cancer cells, this regulation may be impaired .

• Functional consequences: Aberrant S731 phosphorylation contributes to increased transcriptional activity and DNA synthesis, potentially promoting malignant transformation and progression .

These differences highlight S731 phosphorylation as a potential biomarker and therapeutic target in hematological malignancies .

How does STAT5B Ser731 phosphorylation interact with other post-translational modifications to form a regulatory code?

STAT5B function is regulated by a complex network of modifications:

• Tyrosine phosphorylation: S731 phosphorylation works in concert with Y699 phosphorylation, which is essential for dimerization and nuclear translocation .

• Additional serine phosphorylation: S715 phosphorylation may complement S731 in regulating STAT5B activity .

• Inhibitory tyrosine phosphorylation: Y740 and Y743 negatively regulate STAT5B by reducing Y699 phosphorylation; S731 appears to counteract this inhibition in the Y740/743F mutant .

• SUMOylation: This modification inhibits STAT5 phosphorylation and may compete with or complement S731 phosphorylation .

• Acetylation: Lysine acetylation promotes STAT5 phosphorylation and may work synergistically with S731 phosphorylation to enhance activity .

• O-GlcNAcylation: This modification enhances tyrosine phosphorylation and may interact with the S731 phosphorylation pathway .

This multifaceted regulatory network allows for precise control of STAT5B activity in response to diverse cellular signals .

What therapeutic strategies could target STAT5B Ser731 phosphorylation in cancer?

Several approaches could exploit S731 phosphorylation as a therapeutic target:

• mTOR inhibitors: Given the sensitivity of S731 phosphorylation to rapamycin, mTOR inhibitors may reduce aberrant STAT5B activation in cancer cells .

• Serine kinase inhibitors: Developing specific inhibitors of the kinases responsible for S731 phosphorylation could provide targeted therapy options.

• Phosphatase activators: Compounds that enhance PP2A activity might reduce constitutive S731 phosphorylation in cancer cells .

• Structure-based drug design: Developing small molecules that specifically bind to the region around S731 could prevent its phosphorylation or recognition by downstream effectors.

• Combination therapies: Targeting S731 phosphorylation in combination with inhibitors of Y699 phosphorylation could provide synergistic effects in cancers with aberrant STAT5B signaling .

Understanding the biological mechanisms regulating S731 phosphorylation is crucial for developing these potential therapeutic approaches .