Phospho-SGK223 (Y413) Antibody

What is SGK223 and why is the Y413 phosphorylation site significant?

SGK223 (also known as Pragmin or Tyrosine-Protein Kinase SgK223) is a pseudokinase and scaffolding protein that plays important roles in oncogenic tyrosine kinase signaling pathways . Despite lacking catalytic activity, it functions as a critical scaffold for protein-protein interactions. The Y413 phosphorylation site represents a key regulatory site that appears to be differentially phosphorylated in cancer cell lines, particularly in pancreatic ductal adenocarcinoma (PDAC) . Mass spectrometry-based phosphoproteomic profiling has revealed that this site, along with Y159 and Y411, exhibits altered phosphorylation patterns across different PDAC cell lines, suggesting its importance in cancer-related signaling .

What are the specifications and validated applications for the Phospho-SGK223 (Y413) antibody?

The Phospho-SGK223 (Y413) antibody (ABIN3182135) is a rabbit polyclonal antibody that specifically detects endogenous levels of Pragmin protein only when phosphorylated at tyrosine residue 413 . It has been validated for Western Blotting (WB), ELISA, and Immunohistochemistry (IHC) applications . The antibody was generated using a synthesized peptide derived from human Pragmin around the phosphorylation site of Y413 as an immunogen, and was affinity-purified from rabbit antiserum by affinity-chromatography using this epitope-specific immunogen . It shows cross-reactivity with human, mouse, and rat samples .

How should researchers validate the specificity of this phospho-specific antibody?

Validation should include:

• Positive controls: Cell lysates from cell lines known to express phosphorylated SGK223 (Y413), such as AsPC-1 or BxPC-3 pancreatic cancer cells which show high levels of SGK223 phosphorylation

• Negative controls: Phosphatase-treated samples and SGK223 knockout/knockdown samples

• Specificity tests: Compare detection signals between stimulated vs. unstimulated conditions

• Cross-validation: Confirm phosphorylation status using orthogonal methods such as mass spectrometry-based phosphoproteomics

What is the recommended protocol for detecting phosphorylated SGK223 by Western blotting?

Based on research protocols using phospho-SGK223 antibodies:

• Sample preparation:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation status

  • Standardize protein loading to 20-50μg total protein per lane

  • Ensure proper sample denaturation at 95°C for 5 minutes in reducing sample buffer

• Electrophoresis and transfer:

  • Use 7.5-10% SDS-PAGE gels (SGK223 has an expected molecular weight of approximately 170 kDa)

  • Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer containing 20% methanol

• Antibody incubation:

  • Block with 5% BSA in TBST for 1 hour at room temperature

  • Incubate with Phospho-SGK223 (Y413) antibody at 1:1000 dilution overnight at 4°C

  • Wash 3× with TBST and incubate with HRP-conjugated secondary antibody

• Controls:

  • Include treated/untreated samples to demonstrate phosphorylation changes

  • Consider parallel blotting with total SGK223 antibody to normalize phosphorylation signals

How can researchers effectively study SGK223 phosphorylation patterns in different cancer models?

Research on SGK223 phosphorylation has employed several effective approaches:

• Cell line selection: Utilize a diverse panel of cancer cell lines with varying SGK223 expression and phosphorylation levels. The search results reference phosphoproteomic profiling across PDAC cell lines that identified subgroups with distinct SGK223 phosphorylation patterns :

  • Group 1: High Y159, low Y411 phosphorylation (MiaPaca2, Panc10.05, PL45)

  • Group 2: High Y411 phosphorylation (8 cell lines including AsPC-1 and BxPC-3)

• Experimental models:

  • Overexpression systems: Retroviral transduction of SGK223 in human pancreatic ductal epithelial (HPDE) cells

  • Knockout models: CRISPR/Cas9-generated SGK223 knockout cell lines

  • Domain deletion mutants: ΔCH, ΔPK, and ΔN constructs to study structure-function relationships

• Analytical techniques:

  • Quantitative phosphoproteomics to profile site-specific phosphorylation

  • Phosphosite-specific Western blotting for targeted analysis

  • Functional assays (migration, invasion) to correlate phosphorylation with phenotypic outcomes

How does SGK223 phosphorylation affect its interaction with the related pseudokinase SGK269?

SGK223 and SGK269 form both homo- and heterotypic complexes through their CH (C-terminal homology) regions and PK (pseudokinase) domains . Research has demonstrated that:

• Complex formation: Co-immunoprecipitation experiments show that wild-type SGK223 and SGK269 readily associate with each other in multiple cell types including MCF-10A cells and MDA-MB-231 breast cancer cells .

• Domains mediating interaction:

  • Deletion mutant studies revealed that both the CH region and PK domain are critical for efficient SGK223-SGK269 interaction

  • Wild-type and ΔN (N-terminal deletion) mutants of both proteins co-immunoprecipitated, but ΔCH or ΔPK mutants showed severely reduced or abolished association

• Phosphorylation relevance:

  • While the exact role of Y413 phosphorylation in these interactions isn't explicitly detailed in the search results, the differential phosphorylation patterns observed across cancer cell lines suggest that phosphorylation may regulate these interactions

  • The complex formation between SGK223 and SGK269 appears to be functionally important, as SGK269's ability to promote cell migration was significantly compromised in SGK223 knockout cells

The data suggests a model where phosphorylation status of SGK223 could modulate its scaffolding function by affecting its ability to form complexes with SGK269 and other signaling proteins.

What is the relationship between SGK223 phosphorylation and JAK1-STAT3 signaling?

Research has revealed a significant connection between SGK223 and the JAK1-STAT3 pathway:

• STAT3 activation: Overexpression of SGK223 in HPDE cells led to increased STAT3 Tyr705 phosphorylation and enhanced STAT3 transcriptional activity .

• JAK1 dependence:

  • SGK223-overexpressing cells exhibited increased JAK1 activation

  • Selective inhibitors demonstrated that the increased STAT3 signaling driven by SGK223 was JAK-dependent

  • Pharmacological inhibition of STAT3 revealed that STAT3 activation was required for the enhanced motility and invasion of SGK223-overexpressing cells

• Independence from other pathways:

  • The enhanced STAT3 activation was not mediated by Src family kinases (SFKs), as evidenced by:

    • Lower phosphorylation of Src Y416 and enhanced phosphorylation of the negative regulatory site Y527 in SGK223-overexpressing cells

    • Treatment with the selective Src inhibitor saracatinib did not affect STAT3 phosphorylation

  • The effect was also independent of EGFR signaling, as erlotinib treatment did not affect STAT3 activation despite inhibiting EGFR phosphorylation

This JAK1-STAT3 signaling axis represents a key mechanism through which phosphorylated SGK223 may promote cancer progression, particularly in terms of cell migration and invasion.

How can researchers distinguish between SGK223 homo- and heterotypic associations in experimental settings?

Based on the research methodologies described in the search results, several approaches can be used:

• Co-immunoprecipitation with differentially tagged proteins:

  • Co-express HA- and FLAG-tagged versions of SGK223 to detect homotypic interactions

  • Co-express differently tagged SGK223 and SGK269 to detect heterotypic interactions

  • Compare IP results between wild-type proteins and domain deletion mutants (ΔCH, ΔPK)

• GST pulldown assays:

  • Use purified GST-fusion proteins of SGK223 domains immobilized on beads to pull down potential interaction partners from solution

  • This approach helped demonstrate that the CH-PK region of SGK223 directly interacts with the corresponding region of SGK269

• Knockout/knockdown strategies:

  • Compare interaction patterns in wild-type cells versus SGK223 or SGK269 knockout/knockdown cells

  • Perform rescue experiments with wild-type or mutant constructs

• Functional readouts:

  • Cell migration assays in the presence or absence of SGK223/SGK269 can help determine the functional consequences of different complex formations

  • SGK269-driven migration was compromised in SGK223 knockout cells, suggesting the importance of heterocomplexes

What phosphorylation patterns of SGK223 are observed in pancreatic cancer, and what is their functional significance?

Mass spectrometry-based phosphoproteomic profiling across PDAC cell lines revealed distinct phosphorylation patterns:

The functional significance of these phosphorylation patterns includes:

• Expression correlation: SgK223 was overexpressed relative to non-transformed HPDE cells in almost all pancreatic cancer cell lines tested, particularly in AsPC-1 and BxPC-3 (members of the high Y411 phosphorylation subgroup) .

• Clinical relevance: Analysis of gene expression data from PDAC specimens revealed a marked increase in SgK223 expression compared to normal pancreatic tissue .

• Phenotypic effects: Overexpression of SgK223 in HPDE cells at levels comparable to those in PDAC cells:

  • Did not alter cell proliferation

  • Led to a more elongated morphology

  • Enhanced migration and invasion

  • Induced gene expression changes characteristic of a partial epithelial-mesenchymal transition (EMT)

• Signaling impact: While SgK223 overexpression did not affect activation of Erk or Akt, it led to:

  • Increased JAK1 activation

  • Enhanced STAT3 Tyr705 phosphorylation and transcriptional activity

  • Formation of SgK223-STAT3 complexes in vivo

These findings suggest that differential phosphorylation of SGK223 may contribute to PDAC progression by promoting an invasive phenotype through JAK1-STAT3 signaling.

How can researchers effectively model SGK223 phosphorylation-dependent functions in vitro?

Based on successful approaches in the literature:

• Overexpression systems:

  • Retroviral transduction of wild-type or phosphorylation site mutants (e.g., Y413F) of SGK223 in appropriate cell models such as HPDE cells

  • Expression levels should be titrated to match those observed in cancer cells for physiological relevance

• Domain-specific mutants:

  • Generation of deletion mutants (ΔCH, ΔPK, ΔN) to dissect structure-function relationships

  • Site-directed mutagenesis of specific phosphorylation sites to create phospho-deficient (Y to F) or phospho-mimetic (Y to E/D) mutants

• Pathway manipulation:

  • Selective inhibition of JAK1, STAT3, or other pathway components to determine signaling dependencies

  • Combinatorial approaches with SGK223 overexpression/knockdown and pathway inhibitors

• Functional assays:

  • Cell migration and invasion assays to assess metastatic potential

  • Gene expression analysis to monitor EMT marker changes

  • Co-immunoprecipitation to assess protein-protein interactions

  • STAT3 reporter assays to measure transcriptional activity

• Genetic approaches:

  • CRISPR/Cas9-mediated knockout of SGK223 in cancer cell lines followed by rescue with wild-type or mutant constructs

  • siRNA-mediated knockdown for transient reduction of SGK223 expression

What are common pitfalls when detecting phosphorylated SGK223, and how can they be addressed?

Common challenges and their solutions include:

• Phosphorylation lability:

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) in all buffers

  • Maintain samples at 4°C throughout processing

  • Avoid repeated freeze-thaw cycles of lysates

• Antibody specificity issues:

  • Validate antibody specificity using SGK223 knockout/knockdown samples

  • Include phosphatase-treated samples as negative controls

  • Pre-absorb antibody with non-phosphorylated peptide to reduce background

• Low signal strength:

  • Enrich for phosphorylated proteins using phosphotyrosine immunoprecipitation before Western blotting

  • Use enhanced chemiluminescence detection systems

  • Consider signal amplification methods for IHC applications

• Cell type variability:

  • The search results indicate different phosphorylation patterns across cell lines

  • Characterize baseline SGK223 expression and phosphorylation in your experimental system

  • Include appropriate positive controls (e.g., BxPC-3 cells for Y411 phosphorylation)

• Non-specific bands:

  • Use gradient gels for better resolution around the expected molecular weight (~170 kDa)

  • Optimize antibody dilution and incubation conditions

  • Consider using monoclonal antibodies for higher specificity when available

How should researchers interpret changes in SGK223 phosphorylation in the context of complex formation and signaling?

Interpreting SGK223 phosphorylation data requires consideration of several factors:

• Complex formation context:

  • SGK223 forms both homo- and heterotypic complexes with SgK269, mediated by CH and PK domains

  • Changes in phosphorylation may affect complex formation, altering downstream signaling

  • Always normalize phospho-SGK223 signals to total SGK223 expression levels

• Pathway integration:

  • Consider the JAK1-STAT3 pathway as a primary readout for SGK223 activity

  • Monitor both direct (STAT3 phosphorylation) and functional (migration, invasion) outcomes

  • Use pathway inhibitors to validate signaling connections

• Data interpretation framework:

  • Compare phosphorylation changes with phenotypic outcomes

  • Consider both absolute phosphorylation levels and the ratio of different phosphorylation sites

  • Map phosphorylation changes to known protein interaction domains

• Experimental design considerations:

  • Time course experiments may reveal transient phosphorylation events

  • Dose-response studies with pathway activators/inhibitors can establish causality

  • Simultaneous monitoring of multiple phosphorylation sites may reveal interdependence

• Functional validation:

  • Complement phosphorylation data with migration/invasion assays

  • Use gene expression analysis to monitor EMT marker changes as functional readouts

  • Correlate changes with clinical data when available for translational relevance