Phospho-AR (Ser650) Antibody

What is the biological significance of AR Ser-650 phosphorylation?

AR Ser-650 phosphorylation is located in the hinge region of the receptor and plays several critical regulatory roles. Studies using phosphospecific AR antibodies have demonstrated that this phosphorylation site mediates AR nuclear export . Research shows that protein phosphatase 1 (PP1) selectively dephosphorylates AR at Ser-650, with PP1 inhibition dramatically increasing phosphorylation at this site .

This phosphorylation appears to be constitutive in certain AR isoforms, particularly the 110 kDa isoform that appears immediately after AR synthesis . The Ser-650 site is part of a phosphorylation sequence (T-T-S(p)-P-T) that is important for AR function . Notably, when PP1 is inhibited, there is a marked decrease in nuclear localization of wild-type AR, but no alteration in nuclear levels of the S650A mutant AR (where serine is replaced with alanine), confirming this site's importance in subcellular distribution .

What experimental applications are appropriate for Phospho-AR (Ser650) antibodies?

Phospho-AR (Ser650) antibodies have been validated for multiple experimental approaches:

For immunofluorescence applications, cells should be fixed in 3.7% formaldehyde for 10 minutes, washed in PBS, and permeabilized in 0.1% Triton X-100 for 3 minutes. After blocking with 1% bovine serum albumin, incubate with primary antibody (1:200) for 2 hours followed by secondary antibody (1:400) for 2 hours .

How should researchers validate the specificity of Phospho-AR (Ser650) antibodies?

Validation of phospho-specific antibodies is critical for accurate experimental interpretation. Multiple approaches are recommended:

• Peptide competition assays: Pre-incubate the antibody with the immunizing phosphopeptide. This should block specific binding, resulting in loss of signal .

• Phosphatase treatment: Treat samples with phosphatases (like lambda phosphatase) to remove phosphorylation. This should abolish specific signal from phospho-AR (Ser650) antibodies .

• Genetic validation: Compare antibody reactivity between wild-type AR and the S650A mutant. The antibody should not detect the S650A mutant where the phosphorylation site is eliminated .

• Kinase/phosphatase manipulation: Treat cells with phosphatase inhibitors (like okadaic acid) to increase phosphorylation, or overexpress PP1 to decrease phosphorylation, and confirm corresponding changes in antibody signal .

• Fractionation controls: Since Ser-650 phosphorylation affects nuclear-cytoplasmic distribution, compare staining patterns in nuclear versus cytoplasmic fractions .

How is AR Ser-650 phosphorylation regulated in cells?

AR Ser-650 phosphorylation is regulated through a complex interplay of kinases and phosphatases:

• Dephosphorylation: Protein phosphatase 1 (PP1) directly dephosphorylates AR at Ser-650. Co-precipitation experiments have demonstrated a direct AR-PP1 interaction .

• Phosphorylation: While specific kinases for Ser-650 aren't explicitly identified in the search results, research suggests that stress kinase signaling through p38 and JNK kinases plays a role in Ser-650 phosphorylation and subsequent nuclear export .

• Cross-regulation with other phosphosites: Mutation studies reveal that phosphorylation at Ser-515 may influence Ser-650 phosphorylation. The S515A mutation resulted in an unphosphorylated form of the peptide containing Ser-650, suggesting cross-talk between different phosphorylation sites .

• Hormone regulation: Androgen stimulation leads to nuclear translocation of both AR and PP1, allowing for dynamic regulation of Ser-650 phosphorylation .

• Growth factor signaling: In prostate cancer cell lines, treatment with forskolin, epidermal growth factor, and phorbol-12-myristate-13-acetate enhances AR-Ser(P)-650 phosphorylation .

How does AR Ser-650 phosphorylation differ between cancer types and disease stages?

Phosphorylation patterns of AR Ser-650 show significant differences between cancer types and stages:

In breast cancer:

• Nuclear AR-Ser(P)-650 expression is decreased in tumors compared to benign tissue by 1.9-fold (p<0.0001)

• Cytoplasmic AR-Ser(P)-650 expression is decreased in tumors compared to benign tissue by 1.7-fold (p<0.0001)

Between breast cancer subtypes:

• Compared to invasive lobular carcinoma, invasive ductal carcinoma exhibits increased cytoplasmic AR-Ser(P)-650 expression (3.2-fold, p<0.0001)

• In ER-negative breast cancers, nuclear AR-Ser(P)-650 is decreased (1.4-fold, p=0.005) while cytoplasmic AR-Ser(P)-650 is increased (1.4-fold, p=0.003) compared to ER-positive cancers

In metastatic versus primary breast cancer:

• Cytoplasmic AR-Ser(P)-650 is increased in metastatic lesions compared to primary tumors (1.5-fold, p=0.003)

These findings suggest that AR Ser-650 phosphorylation may serve as a prognostic marker, particularly in aggressive breast cancer subtypes and metastases. The shift from nuclear to cytoplasmic localization in aggressive cancers suggests that AR nuclear export facilitated by Ser-650 phosphorylation may play a role in disease progression.

What is the relationship between AR Ser-650 phosphorylation and other AR post-translational modifications?

AR undergoes multiple post-translational modifications that function in concert to regulate its activity. Research indicates complex interrelationships between these modifications:

This interconnected nature of AR post-translational modifications highlights the importance of studying modifications in combination rather than in isolation.

How can researchers effectively design experiments to study the dynamic regulation of AR Ser-650 phosphorylation?

To study the dynamic regulation of AR Ser-650 phosphorylation, researchers should consider multi-faceted experimental approaches:

• Phosphatase manipulation:

  • Use specific inhibitors: fostriecin (PP2A inhibitor), tautomycin (PP1 inhibitor)

  • siRNA against the PP1α catalytic subunit to specifically inhibit PP1 activity

  • PP1α overexpression to enhance dephosphorylation

• Mutational analysis:

  • Compare wild-type AR with S650A mutant responses to stimuli

  • Include phospho-mimetic mutations (S650D or S650E) to simulate constitutive phosphorylation

  • Investigate cross-talk with other sites using combination mutants (e.g., S515A/S650A)

• Signaling pathway activation:

  • Stress kinase activation: UV irradiation or anisomycin treatment to activate p38/JNK pathways

  • Growth factor signaling: EGF, forskolin, or phorbol ester treatment

• Hormone manipulation:

  • Compare AR phosphorylation in the presence and absence of androgens (DHT, R1881)

  • Use time-course experiments to track phosphorylation changes after hormone addition

• Subcellular fractionation and imaging:

  • Nuclear-cytoplasmic fractionation to quantitatively assess AR distribution

  • Live-cell imaging with phospho-specific fluorescent reporters to track real-time changes

  • Proximity ligation assays to detect interactions between phosphorylated AR and regulatory partners

• Detection methods:

  • Use multiple detection methods (western blot, immunofluorescence, mass spectrometry)

  • Employ phospho-specific antibodies validated against appropriate controls

  • Consider using reversed-phase HPLC to separate differentially phosphorylated forms for detailed analysis

What methodological considerations are important when performing immunohistochemistry with Phospho-AR (Ser650) antibodies?

Immunohistochemistry with phospho-specific antibodies requires careful attention to experimental details:

• Fixation and antigen retrieval:

  • Use formalin fixation (10% neutral buffered formalin or 3.7% formaldehyde)

  • Perform heat-induced epitope retrieval using 10mM citrate buffer in a microwave oven

  • Treat with 3% H₂O₂ to block endogenous peroxidase activity

• Blocking and antibody incubation:

  • Block with 1% bovine serum albumin to reduce non-specific binding

  • Use optimal antibody dilutions (typically 1:50-1:300 for IHC)

  • Include appropriate incubation times (2 hours for primary antibody is common)

• Controls:

  • Positive control: Include AR-expressing tissues known to contain phosphorylated Ser-650

  • Negative controls:

    • Primary antibody omission

    • Pre-incubation with immunizing phosphopeptide

    • Phosphatase-treated sections

• Scoring methods:

  • Consider both nuclear and cytoplasmic staining separately, as they may have different biological significance

  • Use a standardized histoscore method to semi-quantitatively assess expression levels

  • Document both intensity and percentage of positive cells

• Interpretation challenges:

  • Be aware that phosphorylation patterns may differ between cell types within the same tissue

  • Consider hormone status of the tissue, as this affects AR phosphorylation

  • Phospho-epitopes may be particularly sensitive to pre-analytical variables (ischemia time, fixation duration)

How does the functional impact of AR Ser-650 phosphorylation compare in different experimental systems?

Research indicates that the functional consequences of AR Ser-650 phosphorylation may vary depending on the experimental system:

• Cell type-dependent effects:

  • Protein phosphatase inhibitors show cell type-dependent effects on AR expression

  • In 293T, CV1, and PC3 cells, phosphatase inhibition increases AR protein levels

  • In HeLa cells and prostate cancer cell lines (LNCaP, CWR22Rv1), phosphatase inhibition decreases AR expression

• Functional assays:

  • Despite its importance for nuclear export, mutation of Ser-650 to alanine (S650A) showed activities identical to wild-type AR in multiple functional analyses:

    • Transactivation assays

    • N- and C-terminal-domain interaction assays

    • Co-activation by transcriptional intermediary factor 2 (TIF2)

  • This was consistent across four different cell lines, suggesting compensatory mechanisms may exist

• Cancer type differences:

  • In breast cancer, AR-Ser(P)-650 shows distinctive patterns between ductal and lobular carcinomas

  • Differential subcellular distribution is observed between ER-positive and ER-negative breast cancers

What are the latest methodological approaches for studying the kinetics of AR Ser-650 phosphorylation?

Advanced methodological approaches for studying AR Ser-650 phosphorylation kinetics include:

• Mass spectrometry-based approaches:

  • Quantitative MS using isotope labeling to track phosphorylation changes over time

  • Targeted selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for sensitive detection of specific phosphopeptides

  • Analysis of tryptic peptides separated by reversed-phase HPLC coupled to electrospray ionization MS and MS/MS

• Advanced imaging techniques:

  • FRET-based biosensors to monitor AR phosphorylation in living cells

  • Super-resolution microscopy to track AR nuclear-cytoplasmic shuttling with nanometer precision

  • Fluorescence recovery after photobleaching (FRAP) to assess how phosphorylation affects AR mobility

• Computational modeling:

  • Kinetic modeling of phosphorylation/dephosphorylation reactions

  • Integration of multiple phosphorylation sites into predictive models of AR function

  • Molecular dynamics simulations to understand how phosphorylation alters AR conformation

• Single-cell analysis:

  • Single-cell western blotting to detect cell-to-cell variability in phosphorylation status

  • Mass cytometry (CyTOF) with phospho-specific antibodies to analyze heterogeneity in cell populations

  • Single-cell sequencing combined with phosphoproteomics

• In vivo models:

  • Generation of phospho-mimetic or phospho-deficient knock-in mouse models

  • In vivo imaging of phosphorylation dynamics using genetically encoded biosensors

  • Patient-derived xenografts to study phosphorylation patterns in response to therapies

These advanced approaches offer researchers powerful tools to understand the complex regulation and functional consequences of AR Ser-650 phosphorylation in both normal physiology and disease states.