Phospho-IRS1 (Ser307) Antibody

What is Phospho-IRS1 (Ser307) Antibody and what does it detect?

Phospho-IRS1 (Ser307) Antibody is a specialized immunological reagent designed to specifically recognize and bind to insulin receptor substrate 1 (IRS-1) protein only when phosphorylated at serine residue 307 in mice (equivalent to Ser312 in humans) . These antibodies are available in both polyclonal and monoclonal formats, with the monoclonal antibody clone 24.6.2 being commonly used for research applications . The antibody detects a protein of approximately 170 kDa representing phosphorylated IRS-1 at Ser307 in various cell types, including 3T3-L1 cells treated with insulin . The antibody does not cross-react with unphosphorylated IRS-1 or with other phosphorylation sites, making it a valuable tool for studying this specific post-translational modification in insulin signaling pathways .

How does Ser307 in mouse IRS1 correspond to human IRS1?

Ser307 in mouse IRS1 corresponds to Ser312 in human IRS1 due to slight differences in the amino acid sequence between species . Despite this numerical difference, the phosphorylation site serves similar functions in both species and is located in a conserved region of the protein . The synthetic phosphopeptide immunogen used to generate many commercial antibodies corresponds to residues E(301)SITAT(pS)PASMVGGK(315) of human IRS-1, which is 100% conserved in rats and mice . When designing experiments or interpreting research literature, it's critical to note this species-specific numbering difference to avoid confusion, especially when translating findings between animal models and human studies .

What techniques can Phospho-IRS1 (Ser307) Antibody be used in?

Phospho-IRS1 (Ser307) Antibody has been validated for multiple experimental techniques:

When using these techniques, researchers should include appropriate controls, such as dephosphorylation with alkaline phosphatase, to confirm antibody specificity . The antibody has been successfully used to detect changes in IRS-1 Ser307 phosphorylation in response to various stimuli, including insulin, TNF-α, and other inflammatory cytokines .

What is the role of IRS-1 in insulin signaling?

IRS-1 functions as a critical signaling adapter protein in the insulin signaling cascade. Upon insulin binding to its receptor, the insulin receptor undergoes autophosphorylation on multiple tyrosine residues . IRS-1 then binds to the insulin receptor through its phosphotyrosine binding (PTB) domain, which interacts with phosphorylated tyrosine 972 in the juxtamembrane region of the insulin receptor . Once bound, IRS-1 becomes phosphorylated on multiple tyrosine residues by the insulin receptor kinase activity.

These phosphorylated tyrosines on IRS-1 then serve as docking sites for downstream signaling molecules containing SH2 domains, including:

• The p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K)

• Growth factor receptor-bound protein 2 (GRB2)

• SH2 domain-containing protein tyrosine phosphatase (SHP2)

Through these interactions, IRS-1 activates multiple signaling pathways:

• The PI3K/Akt pathway - critical for metabolic effects of insulin

• The Ras/Raf/MEK/MAPK cascade - important for growth and proliferation

Additionally, IRS-1 is phosphorylated on various serine and threonine residues, which modulate its function in both positive and negative ways depending on the specific residue and cellular context .

What are the applications of Phospho-IRS1 (Ser307) Antibody in diabetes research?

Phospho-IRS1 (Ser307) Antibody has become an essential tool in diabetes research:

• Assessing insulin resistance mechanisms: The antibody allows researchers to monitor changes in Ser307 phosphorylation in various tissues (liver, muscle, adipose) in response to diet, exercise, or genetic manipulations .

• Inflammatory pathway analysis: Researchers use the antibody to investigate how inflammatory mediators like TNF-α influence IRS-1 serine phosphorylation and subsequent insulin resistance .

• Drug development: The antibody helps evaluate whether potential insulin-sensitizing compounds can modulate Ser307 phosphorylation, providing mechanistic insights into their mode of action .

• Biomarker development: Phospho-IRS1 (Ser307) levels are being explored as potential biomarkers for insulin resistance severity or progression .

• Cell signaling studies: The antibody enables researchers to examine the temporal dynamics of IRS-1 phosphorylation in response to insulin and other stimuli, particularly using HTRF (homogeneous time-resolved fluorescence) cell-based assays that quantify phosphorylated IRS-1 without requiring Western blotting .

These applications have significantly advanced our understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes .

How has our understanding of Ser307 phosphorylation in IRS1 evolved over time?

Our understanding of Ser307 phosphorylation in IRS1 has undergone a remarkable evolution:

Early 2000s - Inhibitory Paradigm: Initial studies using cell culture models suggested that Ser307 phosphorylation primarily inhibited insulin signaling by disrupting the interaction between IRS-1 and the insulin receptor . This view emerged from observations that inflammatory stimuli induced both Ser307 phosphorylation and insulin resistance.

2010 - Paradigm Shift: Copps et al. published a groundbreaking study generating knock-in mice in which Ser307 was replaced with alanine (A/A), preventing phosphorylation at this site . Unexpectedly, these mice developed more severe insulin resistance when fed a high-fat diet compared to control mice. This finding directly contradicted the prevailing view, suggesting that Ser307 phosphorylation might serve a protective role in vivo.

2014-2020 - Context-Dependent Model: Further research revealed that the role of Ser307 phosphorylation depends on:

• Tissue type (liver vs. muscle vs. adipose)

• Acute vs. chronic phosphorylation

• Concurrent phosphorylation at other sites

• The specific kinase involved in the phosphorylation

2024 - Structural Insights: Recent work has characterized the PIR (phosphorylation insulin resistance) domain in IRS-1, which includes Ser307 . This domain enhances binding to the insulin receptor when unphosphorylated but loses binding capacity when phosphorylated. This research suggests that Ser307 phosphorylation may influence insulin signaling by modulating the protective effect of IRS-1 against insulin receptor dephosphorylation .

This evolution highlights the complex, context-dependent roles of Ser307 phosphorylation in insulin signaling regulation .

What are the mechanisms by which Ser307 phosphorylation affects insulin resistance?

The mechanisms by which Ser307 phosphorylation affects insulin resistance are multifaceted and context-dependent:

Molecular Mechanisms of Inhibition:

• Phosphorylation at Ser307 was initially thought to sterically hinder the interaction between the IRS-1 PTB domain and the insulin receptor .

• It may induce conformational changes in IRS-1 that affect its ability to be tyrosine phosphorylated by the insulin receptor.

• Ser307 phosphorylation might create binding sites for negative regulators of insulin signaling.

Recent Mechanistic Discoveries:

• The IRS-1 PIR (phosphorylation insulin resistance) domain containing Ser307 enhances binding to the insulin receptor when unphosphorylated, with a measured binding affinity (KD) of approximately 40 nM .

• When serines in the PIR domain (including Ser307) are phosphorylated, this binding affinity decreases significantly to around 320 nM .

• Phosphorylation of Ser307 reduces the protective effect of IRS-1 against insulin receptor dephosphorylation by protein tyrosine phosphatase 1B (PTP1B) .

Physiological Integration:

• In knockout mouse models, mutation of Ser307 to alanine (preventing phosphorylation) paradoxically worsens insulin resistance during high-fat feeding .

• In liver-specific models, mice lacking hepatic IRS-2 but retaining a single knock-in allele of Ser307Ala showed profound insulin resistance compared to control mice .

• These in vivo findings suggest that transient Ser307 phosphorylation may be part of normal insulin signaling regulation, while chronic phosphorylation in pathological states may contribute to insulin resistance .

These mechanisms help explain the seemingly contradictory roles of Ser307 phosphorylation observed in different experimental systems .

How do different kinases contribute to Ser307 phosphorylation?

Multiple kinases can phosphorylate IRS-1 at Ser307, each activated by different stimuli and potentially leading to distinct functional outcomes:

These kinases may have overlapping yet distinct roles:

• JNK and IKK are primarily activated during inflammatory conditions and may mediate inflammation-induced insulin resistance .

• mTOR/S6K pathway activation represents a feedback mechanism during chronic insulin stimulation .

• Casein Kinase II may be involved in normal insulin signaling regulation .

The specific kinase responsible for Ser307 phosphorylation determines the context and functional outcome, which explains some of the complexity surrounding this phosphorylation site's role in insulin signaling .

What is the PIR domain in IRS-1 and how does phosphorylation at Ser307 affect its function?

The PIR (phosphorylation insulin resistance) domain is a recently characterized region within IRS-1 that plays a crucial role in insulin signaling regulation:

Structural characteristics:

• The PIR domain is located directly C-terminal to the PTB (phosphotyrosine binding) domain of IRS-1 .

• It encompasses numerous serine phosphorylation sites, including Ser307 (mouse)/Ser312 (human), Ser315, and Ser323 .

• These serine residues are targets for multiple kinases including JNK and IKK .

Functional significance:

• The unphosphorylated PIR domain works cooperatively with the PTB domain to enhance IRS-1 binding to the insulin receptor .

• Surface plasmon resonance studies have demonstrated that the PTB-PIR fragment binds to the insulin receptor with higher affinity (KD = 40 nM) than the PTB domain alone .

• When serine residues within the PIR domain are phosphorylated or mutated to phosphomimetic glutamate residues, binding to the insulin receptor is significantly reduced (KD = 320 nM) .

Mechanistic insights:

• Hydrogen-deuterium exchange mass spectrometry experiments indicate that the PIR domain interacts with the N-terminal lobe and hinge regions of the insulin receptor kinase domain .

• This interaction appears to protect the insulin receptor from dephosphorylation by protein tyrosine phosphatase 1B (PTP1B) .

• Cells expressing IRS-1 with phosphomimetic mutations (serines changed to glutamates) in the PIR domain show decreased phosphorylation of insulin receptor, IRS-1, and AKT compared to wild-type IRS-1 .

This integrated understanding of the PIR domain provides new insights into how Ser307 phosphorylation affects insulin signaling through modulation of the IRS-1-insulin receptor interaction .

How does Ser307 phosphorylation influence insulin receptor protection from PTP1B-mediated dephosphorylation?

Recent research has revealed a novel mechanism by which unphosphorylated Ser307 contributes to insulin signaling through protection of the insulin receptor from dephosphorylation:

Experimental evidence:

• In vitro dephosphorylation experiments show that IRS-1 PTB-PIR complexes with insulin receptor significantly delay the dephosphorylation of insulin receptor phosphotyrosines by PTP1B compared to PTB domain alone .

• When following the phosphorylation status of insulin receptor activation loop (pY1162/1163) during PTP1B treatment:

  • IR:PTB complex: Rapidly dephosphorylated to 20% of initial pY levels within 5 minutes and 5% within 20 minutes

  • IR:PTB-PIR complex: Protected with 65% of pY remaining after 5 minutes and 15% after 20 minutes

  • IR:PTB-PIR 4E complex (phosphomimetic mutations): Dephosphorylation rate similar to IR:PTB complex, indicating loss of protection

Proposed mechanism:

• Insulin binding to insulin receptor triggers autophosphorylation of tyrosine residues

• IRS-1 is recruited through cooperative binding of both the PTB domain to pY972 and the unphosphorylated PIR domain to the kinase domain

• The affinity between IRS-1 PTB-PIR and insulin receptor (KD = 40 nM) is higher than that between PTP1B and insulin receptor (KD > 100 nM)

• This allows IRS-1 to outcompete PTP1B for binding to activated insulin receptor, protecting it from dephosphorylation

• When serine residues in the PIR domain (including Ser307) become phosphorylated by kinases like JNK1, the affinity decreases (KD = 320 nM)

• The decreased affinity allows PTP1B to bind preferentially to insulin receptor, leading to dephosphorylation and attenuated insulin signaling

This mechanism represents a significant advancement in our understanding of how Ser307 phosphorylation contributes to insulin resistance, suggesting that it's the loss of protection rather than direct inhibition that impairs insulin signaling .

What are the methodological considerations when using Phospho-IRS1 (Ser307) Antibody in ex vivo or in vivo experiments?

When designing experiments with Phospho-IRS1 (Ser307) Antibody, researchers should consider several critical methodological aspects:

Sample collection and preparation:

• Phosphorylation states change rapidly; samples must be collected and processed quickly

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

• For tissue samples, flash freeze in liquid nitrogen immediately after collection

• Homogenization buffers should contain detergents suitable for membrane protein extraction (e.g., 0.1% Triton X-100)

Antibody validation:

• Confirm specificity using dephosphorylation controls (e.g., alkaline phosphatase treatment)

• Consider using blocking peptides (e.g., synthetic phosphopeptides corresponding to residues E(301)SITAT(pS)PASMVGGK(315)) to verify specificity

• Include positive controls, such as 3T3-L1 cells treated with insulin for 10 minutes or cells treated with TNF-α

Western blotting considerations:

• Optimal dilution ranges from 1:500-1:2000 depending on the specific antibody and sample

• SDS-PAGE conditions are critical as IRS-1 is a large protein (~170-180 kDa)

• Always normalize phospho-IRS1 signals to total IRS-1 levels to account for changes in protein expression

• Be aware that some treatments may affect total IRS-1 stability and turnover

Experimental design:

• Include time course analyses as phosphorylation can be transient

• For in vivo studies, control for feeding/fasting state as this significantly affects insulin signaling

• Consider both acute (minutes to hours) and chronic (days to weeks) treatments

• Compare effects across different tissues (liver, muscle, adipose) as responses may vary

Alternative detection methods:

• Consider HTRF (homogeneous time-resolved fluorescence) cell-based assays that use two labeled antibodies (donor and acceptor fluorophores) to detect phosphorylated IRS-1 without Western blotting

• These assays are plate-based, don't require gel electrophoresis, and can provide quantitative results with higher throughput

These methodological considerations ensure robust and reproducible results when studying IRS-1 Ser307 phosphorylation in complex biological systems .

How do inflammatory pathways influence Ser307 phosphorylation and subsequent insulin resistance?

Inflammatory pathways significantly influence Ser307 phosphorylation, providing a molecular link between inflammation and insulin resistance:

TNF-α signaling pathway:

• TNF-α potently induces Ser307 phosphorylation in multiple cell types

• This effect operates primarily through activation of:

  • JNK (c-Jun N-terminal kinase)

  • IKK (IκB kinase)

• TNF-α-induced Ser307 phosphorylation can be detected using phosphospecific antibodies within 30 minutes of treatment

• Studies using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser307 demonstrated that TNF-α-induced phosphorylation parallels markers for IKK activation

Direct IKK-IRS1 interaction:

• Co-immunoprecipitation experiments have confirmed direct interactions between endogenous IRS-1 and IKK in intact cells

• This interaction in the basal state decreases upon IKK activation and increased serine phosphorylation of IRS-1

• The phosphorylation of human IRS-1 at Ser312 in response to TNF-α is significantly reduced in cells:

  • Pretreated with the IKK inhibitor 15-deoxy-prostaglandin J2

  • Derived from IKK knockout mice

• In vitro kinase assays using recombinant IRS-1 confirm it as a direct substrate for IKK with multiple serine phosphorylation sites including Ser312 (human)

Cytokine integration:

• Pro-inflammatory cytokine cocktails (10 ng/mL TNF-α + 10 ng/mL IL-1β) induce Ser307 phosphorylation in 3T3-L1 adipocytes within 30 minutes

• The HTRF assay shows higher pharmacological windows for detecting this effect compared to Western blot

• This inflammatory response operates through distinct mechanisms compared to insulin-stimulated Ser307 phosphorylation

Clinical relevance:

• In obesity and type 2 diabetes, chronic low-grade inflammation in metabolic tissues leads to persistent activation of these pathways

• The sustained serine phosphorylation of IRS-1 contributes to insulin resistance characteristic of these conditions

• Therapeutic approaches targeting these inflammatory pathways may help improve insulin sensitivity

These findings establish multiple inflammatory mechanisms that converge on Ser307 phosphorylation as a critical mediator of inflammation-induced insulin resistance .

What are the latest findings on the role of Ser307 phosphorylation in type 2 diabetes models?

Recent research has substantially refined our understanding of Ser307 phosphorylation in type 2 diabetes models:

Ser307 as a positive regulatory site:

• Contrary to earlier cell-based studies, knock-in mice in which Ser307 was replaced with alanine (A/A) developed more severe insulin resistance when fed a high-fat diet compared to control mice

• This unexpected finding was accompanied by enhanced pancreatic compensation and impaired muscle insulin signaling

• These results suggest Ser307 serves as a positive regulatory site that moderates insulin resistance severity by maintaining proximal insulin signaling

Liver-specific effects:

• Chow-fed mice lacking hepatic IRS-2 but retaining a single knock-in allele (A/lox::LKO2) were profoundly insulin resistant compared to control mice (+/lox::LKO2)

• Hepatocytes from these mice showed impaired insulin signaling ex vivo

• Adenoviral delivery of mutant A307 IRS1 only partially restored insulin responses in mice lacking hepatic IRS1 and IRS2

Mechanistic advancements:

• The PIR domain of IRS-1 (containing Ser307) has been shown to interact with the insulin receptor kinase domain

• Surface plasmon resonance studies confirm the PTB-PIR fragment binds stronger to insulin receptor than just the PTB domain

• Phosphorylation at Ser307 and other serines within the PIR domain abrogates this binding

• The unphosphorylated PIR domain protects insulin receptor from PTP1B-mediated dephosphorylation, while phosphorylation at Ser307 reduces this protection

Integrated model:

• Initially in insulin signaling, IRS-1 binds to insulin receptor through both PTB and PIR domains

• This cooperative binding outcompetes PTP1B for insulin receptor binding, protecting insulin receptor phosphorylation

• In pathological states with elevated inflammatory signals, JNK1 and other kinases phosphorylate serines in the PIR domain

• This phosphorylation decreases IRS-1 affinity for insulin receptor, allowing PTP1B to bind preferentially

• PTP1B then dephosphorylates insulin receptor, downregulating insulin signaling

These findings represent a significant paradigm shift in our understanding of Ser307 phosphorylation in type 2 diabetes, suggesting more nuanced therapeutic approaches may be necessary to effectively target insulin resistance .