Phospho-AKT1 (Tyr326) Antibody

What is Phospho-AKT1 (Tyr326) Antibody and what does it specifically detect?

Phospho-AKT1 (Tyr326) Antibody is a research reagent designed to detect endogenous levels of AKT1 protein only when phosphorylated at tyrosine 326. This antibody specifically recognizes the phosphorylated form at this particular residue, allowing researchers to study this specific post-translational modification of AKT1 . For example, commercially available antibodies like STJ90503 are typically generated against synthetic peptides derived from the human AKT region surrounding the phosphorylation site of Tyr326, covering approximately amino acids 292-341 .

What is the biological significance of AKT1 Tyr326 phosphorylation in cellular signaling?

Tyr326 phosphorylation represents a distinct regulatory mechanism for AKT1 activation that operates independently of the classical PI3K pathway. According to research findings, the SH3 domain of tyrosine kinase Src interacts with a PXXP motif of AKT and activates it by phosphorylating Tyr315 and Tyr326 . These phosphorylation events are thought to occur prior to the conventional phosphorylation at Thr308 and Ser473 . This creates a PI3K-independent activation pathway that may be particularly relevant in cancer contexts where PI3K inhibitors might be ineffective due to alternative activation mechanisms .

How does Tyr326 phosphorylation differ from other well-known AKT1 phosphorylation sites?

While Thr308 and Ser473 are the most extensively studied phosphorylation sites of AKT1, Tyr326 represents a distinct regulatory mechanism. The phosphorylation profile comparison is as follows:

Research has demonstrated that mutations affecting both Tyr315 and Tyr326 residues, while retaining Thr308 phosphorylation capability, result in the loss of ability to phosphorylate downstream substrates upon EGF stimulation .

What experimental techniques can be used with Phospho-AKT1 (Tyr326) Antibody?

Phospho-AKT1 (Tyr326) Antibody is verified for use in multiple experimental techniques:

Research has demonstrated that this antibody maintains its specificity even after various treatments, but researchers should validate phosphatase treatment controls to confirm specificity of phosphorylation-dependent signals .

How can I validate the specificity of Phospho-AKT1 (Tyr326) Antibody in my experiments?

Validating antibody specificity is crucial for obtaining reliable results. Several approaches are recommended:

• Phosphatase treatment: Treat half of your samples with phosphatase to demonstrate loss of signal

• Stimulation experiments: Compare unstimulated cells with those stimulated with growth factors known to activate AKT1 signaling (e.g., insulin, PDGF)

• Dot blot analysis: Test antibody against phosphorylated and non-phosphorylated peptides

• Peptide competition assay: Pre-incubate antibody with phosphorylated peptide to block specific binding

• Genetic approaches: Use AKT1 knockout cells or AKT1 Tyr326 mutant (Y326F) to demonstrate specificity

Research has shown that phosphatase treatment of membrane strips at 37°C for 1 hour with 150 U/ml of phosphatase can effectively demonstrate phospho-specificity in Western blot applications .

What are the recommended sample preparation methods for optimal detection of phosphorylated AKT1 (Tyr326)?

Detecting phosphorylated proteins requires careful sample preparation to preserve the phosphorylation state:

• Cell lysis buffer composition: Use buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) and protease inhibitors

• Rapid sample processing: Minimize time between cell harvest and protein denaturation

• Cold processing: Keep samples on ice during preparation

• Protein concentration determination: Bradford or BCA assay compatible with phosphatase inhibitors

• Storage: Aliquot and store at -80°C to avoid freeze-thaw cycles

For Western blot applications specifically, include 50 mM DTT in sample buffer and heat samples at 60°C rather than boiling to preserve phospho-epitopes .

How does phosphorylation at Tyr326 affect AKT1 substrate selectivity compared to other phosphorylated forms?

Recent research indicates that different phosphorylated forms of AKT1 display distinct substrate selectivity profiles . A comprehensive study demonstrated that:

• AKT1 phosphorylated at Thr308 (pAKT1 T308) exhibits preferential activity toward certain substrates and promotes faster cell proliferation compared to other phospho-forms

• AKT1 phosphorylated at Ser473 (pAKT1 S473) shows selective ability to increase phospho-GSK-3β levels

• Doubly phosphorylated AKT1 (pAKT1 T308,S473) displays yet another substrate profile

While less extensively characterized than Thr308 and Ser473 phosphorylation, Tyr326 phosphorylation likely contributes to a distinct substrate selection profile, potentially explaining its role in cancer contexts where alternative AKT1 activation pathways are critical .

Can Phospho-AKT1 (Tyr326) Antibody distinguish between different AKT isoforms?

The ability to distinguish between AKT isoforms (AKT1, AKT2, AKT3) depends on the specific antibody design. Based on the available information:

• Antibodies like STJ90503 are designed against the human AKT sequence around Tyr326 (amino acids 292-341)

• Sequence alignment of AKT isoforms shows high conservation in this region, potentially leading to cross-reactivity

If isoform-specific detection is critical, researchers should:

• Perform validation experiments using recombinant AKT1, AKT2, and AKT3 proteins

• Use isoform-specific knockout or knockdown models as controls

• Consider complementary approaches like mass spectrometry-based phosphoproteomics

The development of assays like the capillary-based immunoassay described in source has enabled simultaneous detection and quantification of all three AKT isoforms and their phosphoforms using isoform-specific antibodies.

How does O-GlcNAcylation interact with phosphorylation of AKT1 at Tyr326?

Cross-talk between different post-translational modifications on AKT1 creates complex regulatory networks. Research has demonstrated that:

• O-GlcNAcylation at Thr305 and Thr312 inhibits activating phosphorylation at Thr308 by disrupting the interaction between AKT1 and PDPK1

• O-GlcNAcylation at Ser473 interferes with phosphorylation at this site

• The proximity of these modifications to Tyr326 suggests potential cross-talk mechanisms

This interplay creates a complex regulatory system where multiple post-translational modifications collectively determine AKT1 activity and substrate selectivity. Researchers investigating Tyr326 phosphorylation should consider the potential influence of these other modifications, particularly in metabolic contexts where O-GlcNAcylation is prominent .

What are common challenges in detecting Phospho-AKT1 (Tyr326) and how can they be overcome?

Detection of phosphorylated AKT1 at Tyr326 can present several challenges:

Proper controls are essential, including both positive controls (cells treated with growth factors known to activate AKT) and negative controls (phosphatase-treated samples) .

How can I optimize detection of Phospho-AKT1 (Tyr326) in different sample types?

Different sample types require specific optimization strategies:

• Cell lines:

  • Serum starvation (overnight) followed by stimulation with growth factors (insulin, PDGF) enhances phosphorylation signal

  • Cell density affects basal phosphorylation levels; standardize plating density

  • Cell lysis directly in SDS buffer preserves phosphorylation state

• Tissue samples:

  • Flash freeze immediately after collection

  • Use specialized tissue lysis buffers with phosphatase inhibitors

  • Consider phospho-protein enrichment techniques prior to analysis

• Patient-derived samples:

  • Process rapidly to preserve phosphorylation status

  • Use small volume techniques as demonstrated in source , which enabled measurement of activated AKT from protein produced from as few as 56 cells

The assay sensitivity can be optimized to detect phosphorylated AKT1 from very small samples, allowing accurate evaluation of patient responses to drugs targeting activated PI3K-AKT pathways using scarce clinical specimens .

What considerations should be taken when designing experiments to study the interaction between Src and AKT1 phosphorylation at Tyr326?

When investigating the Src-AKT1 phosphorylation relationship:

• Experimental design considerations:

  • Include Src inhibitors (e.g., dasatinib) to demonstrate Src-dependent phosphorylation

  • Use constitutively active Src mutants (e.g., Src-Y527F) to enhance AKT1 phosphorylation

  • Design AKT1 mutants (Y326F) to prevent phosphorylation at this specific site

  • Employ proximity ligation assays to detect Src-AKT1 interactions in situ

• Pathway analysis tools:

  • Investigate PTEN status, as Src inhibits PTEN, reducing dephosphorylation of PIP3 and increasing AKT phosphorylation

  • Assess PI3K-independence using PI3K inhibitors like LY294002

  • Monitor the timeline of phosphorylation events at Tyr326, Thr308, and Ser473

• Advanced techniques:

  • Phosphoproteomic analysis to comprehensively identify all phosphorylation sites

  • CRISPR-Cas9 genome editing to introduce specific mutations at the endogenous AKT1 locus

  • Live-cell imaging with phospho-specific biosensors to monitor temporal dynamics

Research has demonstrated that Src interacts with AKT1 through its SH3 domain and a proline-rich motif (PXXP) in the C-terminal regulatory region of AKT1 , providing a molecular framework for designing interaction studies.

How do different phospho-forms of AKT1 (including Tyr326) contribute to cancer progression and therapy resistance?

Recent research has revealed distinct roles for different AKT1 phospho-forms in cancer:

• Hyperphosphorylation at either or both regulatory sites (Thr308, Ser473) is associated with poor survival outcomes in many human cancers

• PI3K-independent activation mechanisms, including Tyr326 phosphorylation by Src, represent potential resistance mechanisms to PI3K inhibitors in clinical use

• Differential substrate selectivity of various phospho-forms suggests distinct oncogenic programs

Particularly noteworthy is research demonstrating that cells transduced with TAT-pAKT1 T308 grew significantly faster than those with other pAKT1 variants, highlighting the specific contribution of this phospho-form to proliferation . Understanding the distinct roles of each phosphorylation site could enable more targeted therapeutic approaches for AKT1-dependent cancers.

What novel methods are being developed to study the dynamics of AKT1 phosphorylation at multiple sites simultaneously?

Emerging technologies are enabling more comprehensive analysis of AKT1 phosphorylation dynamics:

• Capillary-based immunoassay systems: Enable measurement of AKT isoforms and phosphoforms from very small sample volumes (as few as 56 cells)

• Phospho-specific cell-based ELISA kits: Allow detection of phosphorylated proteins in intact cells without lysate preparation

• Genetic code expansion: Production of AKT1 with programmed phosphorylation at specific sites using phosphoseryl-tRNA synthetase (SepRS) and tRNA

• Cell-penetrating peptide delivery: TAT-tagged AKT1 phospho-variants enable controlled introduction of specific phospho-forms into cells

• Mass spectrometry-based phosphoproteomics: Allow unbiased detection of all phosphorylation sites and their stoichiometry

These techniques collectively provide researchers with unprecedented tools to dissect the complex roles of multiple phosphorylation events in regulating AKT1 function in normal and disease states.

How can researchers effectively integrate data from antibody-based detection of Phospho-AKT1 (Tyr326) with global phosphoproteomic analyses?

Integrating targeted antibody-based approaches with global phosphoproteomics requires careful experimental design:

• Validation strategies:

  • Use antibody-based detection to validate specific phosphorylation sites identified in phosphoproteomic screens

  • Employ phosphoproteomic analysis to identify additional phosphorylation sites on AKT1 not covered by commercial antibodies

• Quantitative considerations:

  • Calibrate antibody-based signals using recombinant phosphorylated standards

  • Normalize phosphoproteomic data against total protein abundance

  • Consider stoichiometry of phosphorylation at multiple sites

• Data integration approaches:

  • Develop computational models incorporating data from both approaches

  • Use pathway analysis tools to contextualize findings

  • Consider temporal dynamics of phosphorylation events

• Experimental design:

  • Include identical sample processing conditions for both antibody-based and phosphoproteomic analyses

  • Collect samples at multiple time points to capture dynamics

  • Include appropriate controls (phosphatase treatment, kinase inhibitors)

By combining the specificity of antibody-based detection with the comprehensive coverage of phosphoproteomics, researchers can gain deeper insights into the complex regulatory mechanisms governing AKT1 function.