Phospho-SMC1A (Ser957) Antibody

What is SMC1A and why is its phosphorylation at Ser957 significant in cellular processes?

SMC1A (Structural Maintenance of Chromosomes 1A) is a critical component of the cohesin complex that plays essential roles in chromosome cohesion during the cell cycle and DNA repair. It contains a myosin-like ATPase domain that serves as a molecular motor to help organize chromatin . Phosphorylation of SMC1A at Ser957 occurs primarily in response to DNA damage, particularly double-strand breaks (DSBs).

The significance of Ser957 phosphorylation lies in its role in DNA damage response pathways. Following DNA damage induced by chemical treatment or ionizing radiation, SMC1A is phosphorylated on Ser957 and Ser966 residues by the ATM (Ataxia Telangiectasia Mutated) kinase in the presence of DSBs . This phosphorylation is crucial for successful DNA repair and maintenance of genome stability. Non-phosphorylatable mutants of SMC1A show impaired DNA repair capacity, highlighting the importance of this post-translational modification.

How should researchers prepare positive controls for validating Phospho-SMC1A (Ser957) antibody specificity?

To properly validate Phospho-SMC1A (Ser957) antibody specificity, researchers should prepare appropriate positive controls:

• Cell treatment protocols: Nuclear extract of HeLa cells treated with etoposide (ETO) at 100 µM for 2 hours can serve as a reliable positive control . Alternatively, 293 cells treated with UV irradiation also show increased phosphorylation at Ser957 .

• Oxidative stress induction: Treatment with H₂O₂ increases SIRT2 expression and interaction with SMC1A, leading to enhanced SMC1A phosphorylation at Ser957 . This provides another methodological approach for generating positive controls.

• Phosphomimetic mutants: Using SMC1A S957D (phosphomimetic) mutants as a positive control can help validate antibody specificity in transfection experiments .

• Blocking peptide validation: For definitive confirmation of specificity, researchers should perform parallel experiments using a blocking peptide containing the phosphorylated Ser957 epitope to demonstrate signal abrogation .

The inclusion of these controls ensures that the detected signal is specific to phosphorylated SMC1A at Ser957 rather than non-specific binding or detection of non-phosphorylated forms.

What are the optimal sample preparation methods for detecting Phospho-SMC1A (Ser957) in different experimental systems?

Optimal sample preparation varies based on the experimental technique:

For Western Blot analysis:

• Extract proteins using a buffer containing phosphatase inhibitors (critical to prevent dephosphorylation)

• Use freshly prepared lysates when possible, or store at -80°C with protease/phosphatase inhibitors

• For cancer cell lines (e.g., HepG2, Bel7402, HCT116), standard RIPA buffer with phosphatase inhibitor cocktail yields good results

For Immunohistochemistry:

• Formalin-fixed paraffin-embedded (FFPE) tissue sections should undergo high-pressure antigen retrieval in citrate buffer (pH 6.0) for 2 minutes

• For tissue microarrays, nuclear immunoreactivity should be evaluated by calculating the percentage of positive-staining tumor cells over total tumor cells

• Signal intensity can be scored using 5% increments (0%, 5%, 10%...100%; with 10% = score of 1)

For Immunofluorescence:

• Mitotic cells should be labeled with anti-α-tubulin antibody to visualize the spindle

• Chromosomes should be counterstained with DAPI

• This allows comparison of SMC1A phosphorylation levels between mitotic and interphase cells

How does phosphorylation of SMC1A at Ser957 contribute to cancer progression mechanisms?

Research has revealed multiple mechanisms by which SMC1A phosphorylation at Ser957 contributes to cancer progression:

• Enhanced proliferation: Phosphorylation of SMC1A at Ser957 and Ser966 promotes cancer cell proliferation, as demonstrated in hepatocellular carcinoma (HCC) cells. Re-expressing phosphomimetic mutants (S957DS966D) in SMC1A knockdown HepG2 and Bel7402 cells significantly increased proliferation rates by 67% and 44.2%, respectively .

• Increased migration capacity: SMC1A phosphorylation dramatically increases the expression of MMP9 (by 36% in HepG2 cells and 51.8% in Bel7402 cells), enhancing the migration capacity of cancer cells .

• Resistance to cytotoxic agents: Cells expressing phosphomimetic SMC1A mutants (S957DS966D) exhibit significantly higher viability when exposed to doxorubicin compared to those expressing wild-type SMC1A .

• Tumor growth promotion: In vivo studies show that phosphorylated SMC1A promotes hepatocellular carcinoma growth, with S957DS966D mutant SMC1A demonstrating larger tumor size and greater tumor growth rate than wild-type SMC1A in xenograft models .

• Prognostic significance: Overexpression of phosphorylated SMC1A is significantly associated with worse prognostic outcomes in hepatocellular carcinoma and other cancer types .

The SIRT2-SMC1A axis further regulates this phosphorylation, with SIRT2-mediated deacetylation of SMC1A at K579 promoting phosphorylation at Ser957/Ser966, which enhances cell proliferation and tumor growth .

What is the relationship between SMC1A phosphorylation and chromatin dynamics during mitosis?

The phosphorylation status of SMC1A at Ser957 plays a critical role in regulating chromatin dynamics during mitosis:

• Mitotic localization pattern: Phosphorylated SMC1A shows increased levels in cells in the mitotic phase compared to surrounding interphase cells, indicating cell cycle-dependent regulation .

• Cohesin complex association with chromatin: The phosphorylated form on Ser957 and Ser966 associates with chromatin during G1/S/G2 phases but not during M phase, suggesting that phosphorylation does not regulate cohesin function during mitosis itself .

• Bipolar spindle formation: Phosphorylation of Ser957 and Ser966 of SMC1A stimulates binding to RNA export factor 1 (Rae1) during mitosis, which is required for proper bipolar spindle formation. Disruption of this phosphorylation impedes SMC1A-Rae1 interaction, leading to spindle multipolarity characteristic of cancer cells .

• Checkpoint regulation: SMC1A phosphorylation is involved in G2-M checkpoint regulation. Loss of this phosphorylation causes higher mitotic index under oxidative stress conditions, and only wild-type or phosphomimetic mutants (S957DS966D), but not non-phosphorylatable mutants (S957AS966A), can rescue this phenotype .

• Cross-talk with acetylation: A negative cross-talk exists between acetylation at K579 and phosphorylation of SMC1A at Ser957. SMC1A K579 acetylation decreases in mitotic cells, while phosphorylation increases, suggesting coordinated regulation of these post-translational modifications during cell division .

How can researchers accurately quantify changes in SMC1A Ser957 phosphorylation levels?

For accurate quantification of SMC1A Ser957 phosphorylation levels, researchers should consider several methodological approaches:

• Western blot quantification:

  • Always run total SMC1A as a normalization control alongside phospho-specific detection

  • Use semiquantitative analysis with densitometry software

  • In comparative studies (e.g., wild-type vs. mutant), express results as fold-change relative to control conditions

• Immunohistochemistry scoring:

  • Evaluate nuclear immunoreactivity by calculating the percentage of positive-staining tumor cells over total tumor cells

  • Use standardized scoring with 5% increments (0%, 5%, 10%...100%; with 10% = score of 1)

  • Have two independent observers score samples to ensure reproducibility

• Appropriate controls for normalization:

  • For treatments that induce phosphorylation (e.g., DNA damaging agents), include time-course experiments

  • Use phosphatase treatment of a portion of samples as a negative control

  • Include phosphomimetic (S957D) and non-phosphorylatable (S957A) mutants as reference points

• Cell cycle considerations:

  • Since phosphorylation levels change during cell cycle progression, synchronize cells when possible

  • For unsynchronized populations, use cell cycle markers (e.g., cyclin B1, H3-p) to correlate phosphorylation status with cell cycle phase

What are the critical factors affecting the specificity of Phospho-SMC1A (Ser957) antibody detection?

Several critical factors can affect the specificity and reliability of Phospho-SMC1A (Ser957) antibody detection:

• Antibody purification method: High-quality antibodies are purified by affinity-chromatography using epitope-specific phosphopeptides. Non-phospho specific antibodies should be removed by chromatography using non-phosphopeptide to ensure specificity .

• Epitope sequence consideration: The exact epitope sequence around the phosphorylation site (typically G-S-S(p)-Q-G for human SMC1A Ser957) should be verified in the species being studied, as sequence variations can affect antibody binding .

• Buffer composition:

  • Optimal buffers include phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4

  • Addition of 150mM NaCl, 0.02% sodium azide, and 50% glycerol improves stability

• Storage conditions:

  • Aliquot and store at -20°C to avoid freeze/thaw cycles

  • Some suppliers recommend storage at -80°C for longer term

• Cross-reactivity testing:

  • Test for cross-reactivity with non-phosphorylated SMC1A

  • Check reactivity with related phospho-proteins, particularly SMC3

  • Validate with phosphatase treatment to confirm signal specificity

How is Phospho-SMC1A (Ser957) used to investigate cancer mechanisms and potential therapeutic targets?

Phospho-SMC1A (Ser957) antibodies have proven valuable for investigating cancer mechanisms and identifying therapeutic targets:

• Colorectal cancer (CRC) studies:

  • SMC1A expression is significantly higher in CRC tissues than in normal tissues

  • Patients with high SMC1A expression had late stages, short survival, and poor prognosis

  • Phosphorylated SMC1A promotes hepatocellular carcinoma cells growth in vivo

• Hepatocellular carcinoma (HCC) research:

  • Phospho-SMC1A (Ser957) is highly expressed in HepG2 and Bel7402 cells

  • SMC1A knockdown reduces proliferation and migration

  • Phosphomimetic mutants (S957DS966D) enhance proliferation and migration

  • Expression of phosphorylated SMC1A is significantly higher in human HCC compared to benign hepatocytes

• Therapeutic targeting strategies:

  • Low doses of oxaliplatin or 5-FU (first-line therapeutic agents for colon cancer) significantly suppress the survival of cells with altered SMC1A phosphorylation

  • Combined cytotoxic and SMC1A-targeted therapy may achieve synergistic treatment effects

  • K579-acetylated SMC1A (which inhibits phosphorylation) may be a potential novel anticancer target

• Biomarker development:

  • Phosphorylated SMC1A may serve as a prognostic biomarker in various cancer types

  • Tissue microarray analysis with phospho-specific antibodies can stratify patient populations for targeted therapies

What is the role of SMC1A Ser957 phosphorylation in the DNA damage response pathway?

SMC1A Ser957 phosphorylation plays a crucial role in the DNA damage response (DDR) pathway:

• Kinase-mediated phosphorylation:

  • ATM kinase phosphorylates SMC1A at Ser957 and Ser966 in response to double-strand breaks (DSBs)

  • ATR kinase mediates phosphorylation after single-strand breaks

  • This phosphorylation occurs in an NBS1-dependent fashion

• Downstream effector function:

  • Phosphorylated SMC1A works as a downstream effector both in the ATM/NBS1 branch and in the ATR/MSH2 branch of S-phase checkpoint

  • It contributes to the intra-S phase checkpoint activation following DNA damage

• Chromosomal stability maintenance:

  • SMC1A phosphorylation is essential for maintaining chromosomal stability after DNA damage

  • SMC1A knockdown induces genome instability, and cells from Cornelia de Lange syndrome (CdLS) patients show a high degree of spontaneous chromosome aberrations

• Response to oxidative stress:

  • Oxidative stress (e.g., H₂O₂ treatment) increases the interaction between SIRT2 and SMC1A

  • This enhances SIRT2-dependent deacetylation of SMC1A, which is required for its phosphorylation at Ser957

  • The deacetylation-phosphorylation regulation of the SIRT2-SMC1A axis is critical for cellular responses to oxidative stress

• Methodology for studying DDR:

  • Researchers can use Phospho-SMC1A (Ser957) antibodies to monitor activation of the DDR pathway

  • Treatments like etoposide (100 µM for 2 hours) or UV irradiation serve as positive controls for inducing this phosphorylation

How can researchers design experiments to study the cross-talk between SMC1A acetylation and phosphorylation?

To effectively study the cross-talk between SMC1A acetylation and phosphorylation, researchers should design experiments with the following considerations:

• Sequential post-translational modification analysis:

  • Generate site-specific acetylation and phosphorylation antibodies for SMC1A

  • Use acetylation mimetic (K579Q) and phosphomimetic (S957D) mutants to study causal relationships

  • The SMC1A K579Q mutant significantly impairs SMC1A phosphorylation compared to wild-type and SMC1A K579R proteins

• Stress response experiments:

  • Apply oxidative stress (H₂O₂) treatment to induce SIRT2 expression and interaction with SMC1A

  • Monitor changes in both acetylation and phosphorylation levels over time

  • The negative correlation between K579 acetylation and phosphorylation of SMC1A in response to oxidative stress is concentration-dependent

• Deacetylase inhibition studies:

  • Use SIRT2 inhibitors like AGK2 to test effects on SMC1A phosphorylation

  • Apply general deacetylase inhibitors like TSA (trichostatin A) and NAM (nicotinamide)

  • These treatments decrease oxidative stress-induced SMC1A phosphorylation

• Immunofluorescence co-localization analysis:

  • Visualize the localization of SMC1A K579 acetylation and phosphorylation during mitosis

  • Label mitotic cells with anti-α-tubulin antibody to visualize the spindle

  • Counterstain chromosomes with DAPI

  • This reveals that SMC1A K579 acetylation decreases while phosphorylation increases in mitotic cells compared to interphase cells

• Protein-protein interaction studies:

  • Investigate how acetylation/phosphorylation affects SMC1A interactions with binding partners

  • The reexpression of acetylmimetic SMC1A K579Q significantly reduces SMC1A-Rae1 interaction, which is critical for proper spindle formation

What are the recommended approaches for comparing phosphorylated SMC1A levels across different cancer types?

When comparing phosphorylated SMC1A levels across different cancer types, researchers should employ the following approaches: