Phospho-PIK3R1 (Tyr607) Antibody

What is PIK3R1 and what role does phosphorylation at Tyr607 play in cellular signaling?

PIK3R1 encodes the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K), a crucial signaling protein that phosphorylates the inositol ring of phosphatidylinositol at the 3-prime position. The p85α subunit binds to activated (phosphorylated) protein-tyrosine kinases through its SH2 domain and functions as an adapter, mediating the association of the p110 catalytic unit to the plasma membrane . Phosphorylation at Tyrosine 607 represents a significant post-translational modification that regulates PI3K activity and signaling outcomes.

Recent research has revealed that phosphorylation at Tyr607 creates an interaction site with the N-terminal SH2 domain, supporting dimerization of the regulatory subunits . This phosphorylation event appears to be particularly significant in nuclear-enriched cell fractions, suggesting compartment-specific regulatory mechanisms that extend beyond the traditional understanding of PI3K signaling at the plasma membrane .

What are the specific characteristics of Phospho-PIK3R1 (Tyr607) antibodies?

Phospho-PIK3R1 (Tyr607) antibodies are highly specialized immunological reagents designed to detect endogenous levels of PI3K p85α only when phosphorylated at Tyrosine 607 . These antibodies recognize a specific motif (D-Q-Y-S-L) surrounding the phosphorylated tyrosine residue . The polyclonal versions are typically generated by immunizing rabbits with synthesized phosphopeptides derived from human PI3K p85α sequences surrounding the Tyr607 phosphorylation site .

The antibodies undergo rigorous purification processes, including sequential chromatography on phospho- and non-phospho-peptide affinity columns, to ensure high specificity for the phosphorylated form of the protein . This specificity is crucial for distinguishing between phosphorylated and non-phosphorylated states of the protein in experimental settings.

How should researchers validate the specificity of Phospho-PIK3R1 (Tyr607) antibodies?

Validation of Phospho-PIK3R1 (Tyr607) antibody specificity should involve multiple complementary approaches:

• Phosphatase treatment control: Treating samples with lambda phosphatase prior to immunoblotting should abolish the signal if the antibody is truly phospho-specific.

• Phospho-mimetic and phospho-dead mutants: Generate Y607F (phospho-dead) and, if possible, Y607E (phospho-mimetic) mutants of p85α to confirm antibody specificity through differential recognition patterns.

• Kinase activation and inhibition: Stimulate cells with factors known to induce Tyr607 phosphorylation (e.g., ACK activation) or inhibit relevant kinases to demonstrate dynamic changes in antibody reactivity .

• Cross-reactivity assessment: Test antibody reactivity with other PI3K regulatory subunit isoforms (p85β, p55α, p50α, p55γ) to determine isoform specificity or cross-reactivity . This is particularly important as p55γ lacks the specific motif recognized by some Phospho-PIK3R1 (Tyr607) antibodies .

How can researchers effectively use Phospho-PIK3R1 (Tyr607) antibodies across different experimental techniques?

Phospho-PIK3R1 (Tyr607) antibodies demonstrate versatility across multiple experimental techniques, each requiring specific optimization:

Western Blotting (WB):

• Optimal sample preparation should include phosphatase inhibitors

• Recommended dilution ranges: 1:500-1:2000

• Sample loading: 20-40 μg of total protein per lane

• Detection systems: Enhanced chemiluminescence provides sufficient sensitivity

Immunohistochemistry (IHC) and Immunofluorescence (IF):

• Fixation: 4% paraformaldehyde is preferable for preserving phosphoepitopes

• Antigen retrieval: Citrate buffer (pH 6.0) heat-induced epitope retrieval

• Blocking: 5% BSA with phosphatase inhibitors

• Recommended dilution: 1:50-1:200

Immunocytochemistry (ICC):

• Cell fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 for 5 minutes

• Counterstaining: DAPI for nuclear visualization to assess nuclear vs. cytoplasmic distribution

What is the relationship between ACK-mediated phosphorylation of PI3K regulatory subunits and cell proliferation?

Recent research has demonstrated that ACK (Activated Cdc42-associated Kinase) directly binds to all five PI3K regulatory subunit isoforms and phosphorylates p85α, p85β, p50α, and p55α specifically at Tyr607 (or analogous residues in other isoforms) . This phosphorylation event has significant functional consequences:

• Proliferation effects: Phosphorylation of p85β at the Tyr607-equivalent residue promotes cell proliferation in HEK293T cells, while mutation of this residue decreases proliferation .

• Nuclear localization: ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also predominantly resides .

• Novel dimerization mechanism: Phosphorylation at Tyr607 facilitates an interaction with the N-terminal SH2 domain, supporting dimerization of the regulatory subunits .

• p110-independent functions: Evidence suggests that ACK targets p110-independent p85, potentially revealing novel nuclear functions of regulatory subunit dimers that have been previously unexplored .

This interaction between ACK and PI3K regulatory subunits represents an additional layer of regulation for PI3K signaling, particularly in nuclear compartments, which may have implications for understanding cell proliferation control in both normal and pathological contexts.

How do mutations in PIK3R1 contribute to immunodeficiency disorders?

Mutations in PIK3R1 have been linked to primary immunodeficiency disorders through several mechanisms:

• Structural protein alterations: Mutations affecting splice sites in PIK3R1 can lead to deletion of exon 10, producing a shortened p85α protein that lacks part of the PI3K p110-binding domain .

• Deregulated signaling: The loss of p85α-mediated inhibition of p110 activity results in elevated phosphorylation of downstream signaling kinases like AKT in patient T cell blasts .

• Immune cell defects: Patients with PIK3R1 mutations exhibit:

  • Low naive T and memory B cell counts

  • Enhanced activation-induced cell death in T cells

  • Weak B lymphocyte proliferation in response to B cell receptor and TLR9 activation

  • Hypogammaglobulinemia and recurrent infections

• Therapeutic insights: T cell defects in patients with PIK3R1 mutations can be corrected by addition of PI3Kδ inhibitors like IC87114, suggesting potential therapeutic avenues .

The phenotype observed in patients with PIK3R1 splice site mutations resembles that of patients carrying gain-of-function mutations in PIK3CD, underscoring the critical importance of tightly regulated PI3K activity in T and B lymphocytes .

What experimental approaches can distinguish between p110-dependent and p110-independent functions of phosphorylated p85α?

Distinguishing between p110-dependent and p110-independent functions of phosphorylated p85α requires strategic experimental design:

• Subcellular fractionation studies: Since phosphorylated p85α at Tyr607 appears to be predominantly nuclear, comparing cytoplasmic versus nuclear fractions can help isolate p110-independent functions .

• Co-immunoprecipitation with phospho-specific antibodies: Using Phospho-PIK3R1 (Tyr607) antibodies for immunoprecipitation followed by mass spectrometry to identify interacting partners can reveal p110-dependent versus independent complexes.

• Mutational analysis: Creating p85α mutants that cannot bind p110 but retain Tyr607 phosphorylation sites can isolate p110-independent functions.

• Proximity labeling techniques: BioID or APEX2 fused to p85α can identify proteins in proximity to phosphorylated p85α in different cellular compartments.

• Correlation with dimerization status: Since phosphorylation at Tyr607 promotes regulatory subunit dimerization, techniques that assess dimerization status alongside functional readouts can reveal dimerization-dependent functions .

What are the technical considerations for detecting differential phosphorylation across PI3K regulatory subunit isoforms?

The PI3K regulatory subunit family includes multiple isoforms (p85α, p85β, p55α, p50α, and p55γ) with varying phosphorylation patterns and antibody reactivity profiles:

• Motif differences: While p85α, p85β, p50α, and p55α contain analogous phosphorylation sites to Tyr607, p55γ lacks the specific motif (D-Q-Y-S-L) recognized by some anti-p85 pTyr607 antibodies .

• Isoform-specific detection strategies:

  Isoform	Phosphorylation Site	Antibody Detection	Verification Method
  p85α	Tyr607	Direct with anti-pTyr607	Western blot, MS
  p85β	Analogous to Tyr607	Direct with anti-pTyr607	Western blot, MS
  p50α	Tyr307 (equivalent to Tyr607)	Direct with anti-pTyr607	Western blot, MS
  p55α	Analogous to Tyr607	Direct with anti-pTyr607	Western blot, MS
  p55γ	Tyr341 (equivalent)	Not detected by anti-pTyr607; requires pan-pTyr antibody	MS confirmation

• Mass spectrometry verification: In vitro kinase assays followed by mass spectrometry analysis have confirmed that ACK phosphorylates p50α only at Tyr307 (equivalent to Tyr607 in p85α) and does not phosphorylate p55γ at the equivalent site .

How might Phospho-PIK3R1 (Tyr607) status serve as a biomarker for disease or therapeutic response?

The phosphorylation status of PIK3R1 at Tyr607 may serve as a potential biomarker in several contexts:

• Immunodeficiency disorders: Given the role of PI3K signaling in immune function and the link between PIK3R1 mutations and immunodeficiency, phosphorylation at Tyr607 might serve as a functional readout of pathway integrity .

• Insulin resistance and metabolic disorders: Since PI3K plays a critical role in insulin signaling and mutations in PIK3R1 have been associated with insulin resistance, Tyr607 phosphorylation status could provide insights into metabolic dysfunction .

• Proliferative disorders: The observation that phosphorylation of p85β at the Tyr607-equivalent residue promotes cell proliferation suggests potential relevance to proliferative disorders or cancer .

• Therapeutic response prediction: Monitoring Tyr607 phosphorylation in patients receiving PI3K pathway inhibitors might help predict or monitor therapeutic responses, particularly for PI3Kδ inhibitors in immunological disorders .

What are the emerging research questions regarding nuclear functions of phosphorylated PIK3R1?

The discovery that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where phosphorylated Tyr607 also predominantly resides, opens several intriguing research directions:

• Nuclear PI3K signaling regulation: How does nuclear phosphorylated p85α modulate nuclear phosphoinositide signaling, if at all?

• Transcriptional regulation: Does phosphorylated p85α interact with transcription factors or chromatin modifiers to regulate gene expression?

• Cell cycle control: What is the relationship between nuclear p85α phosphorylation, dimerization, and cell cycle progression?

• Stress responses: How might nuclear p85α phosphorylation respond to cellular stresses like DNA damage?

• Therapeutic targeting: Could selective targeting of nuclear versus cytoplasmic p85α phosphorylation provide new therapeutic opportunities?

Understanding these nuclear functions could revolutionize our understanding of PI3K regulatory subunits beyond their canonical role as adapters for p110 catalytic subunits .

What best practices should researchers follow when working with Phospho-PIK3R1 (Tyr607) antibodies?

To ensure reliable and reproducible results when working with Phospho-PIK3R1 (Tyr607) antibodies, researchers should:

• Proper sample handling: Maintain samples on ice and include phosphatase inhibitors in all buffers to preserve phosphorylation status.

• Include appropriate controls: Use samples from cells treated with phosphatase, kinase inhibitors, and kinase activators to validate phospho-specificity.

• Validate with multiple techniques: Confirm findings using complementary approaches such as Western blotting, immunoprecipitation, and mass spectrometry.

• Consider subcellular localization: Given the nuclear enrichment of phosphorylated p85α, include proper subcellular fractionation procedures and controls.

• Isoform awareness: Be cognizant of the different PI3K regulatory subunit isoforms present in your experimental system and their differential reactivity with phospho-specific antibodies.

• Report detailed methods: Document antibody catalog information, dilutions, incubation conditions, and validation approaches to enhance reproducibility.