Phospho-PTEN (Ser370) Antibody

What is Phospho-PTEN (Ser370) and why is it significant in research?

Phospho-PTEN (Ser370) refers to the phosphorylated form of PTEN (Phosphatase and tensin homolog) at serine residue 370, which is located within the C-terminal tail of the protein. PTEN functions as a dual-specificity phosphatase with both lipid and protein phosphatase activities, and is one of the most commonly lost tumor suppressors in human cancer . The phosphorylation at Ser370 is particularly significant as it contributes to the regulation of PTEN's enzymatic activity, membrane association, and protein stability.

The importance of Phospho-PTEN (Ser370) in research stems from its role in modulating PTEN's function as a negative regulator of the PI3K/AKT pathway. PTEN dephosphorylates phosphatidylinositol-3,4,5-trisphosphate (PIP3), thereby antagonizing PI3K signaling and suppressing cell proliferation and survival signals . Phosphorylation at Ser370 appears to reduce PTEN's lipid phosphatase activity without necessarily affecting its protein phosphatase activity, creating a complex regulatory mechanism .

Research has shown that PTEN is constitutively phosphorylated on multiple residues in the C-terminal tail, including Ser370, under normal conditions . This phosphorylation contributes to PTEN conformational changes that affect its subcellular localization and function, influencing its tumor suppressor capabilities.

What detection methods and experimental techniques are available for studying PTEN phosphorylation at Ser370?

Several complementary techniques can be employed to detect and quantify PTEN phosphorylation at Ser370:

Western Blotting (WB):

• Optimal dilutions typically range from 1:500-1:2000 for phospho-specific antibodies

• Expected molecular weight of phospho-PTEN is approximately 54-58 kDa

• Sample preparation should include phosphatase inhibitors to preserve phosphorylation status

• Multiple cell lines demonstrate detectable levels, including MCF-7, HeLa, NIH/3T3, and SHSY5Y

Immunohistochemistry (IHC):

• Recommended dilutions range from 1:50-1:300

• Antigen retrieval using sodium citrate buffer (pH 6.0) with microwave treatment for 8-15 minutes is typically required

• Counterstaining with hematoxylin provides context for phospho-PTEN localization

Immunofluorescence (IF):

• Dilutions typically range from 1:50-1:200

• Cell fixation with 4% paraformaldehyde for 15 minutes, followed by permeabilization with 0.25% Triton X-100 is an effective protocol

• Can be combined with cytoskeletal markers to study relationships between PTEN phosphorylation and cellular structures

Cell-Based ELISA:

• Allows high-throughput, lysate-free quantification of phospho-PTEN (Ser370)

• Particularly useful for screening compounds that affect PTEN phosphorylation

• Can simultaneously measure total PTEN and phospho-PTEN levels for normalization

Antibody Validation Techniques:

• Phosphatase treatment: Lambda phosphatase treatment eliminates phospho-specific signal, confirming antibody specificity

• Antibody-peptide competition: Only the phosphopeptide corresponding to PTEN (pS370) should block the antibody signal

• PTEN mutant expression: S370A mutants can serve as negative controls while S370D mutants may serve as positive controls for some functional aspects

How does phosphorylation at Ser370 affect PTEN's enzymatic activities and functions?

Phosphorylation of PTEN at Ser370 has distinct impacts on its dual enzymatic activities and cellular functions:

Effects on Lipid Phosphatase Activity:

• Ser370 phosphorylation reduces PTEN's lipid phosphatase activity toward PIP3

• S370D PTEN (phosphomimetic mutant) increases cellular PIP3 levels, indicating decreased PIP3 dephosphorylation

• At physiologically relevant low concentrations of PIP3 (<1 mol%), the reduced activity of phosphorylated PTEN is particularly significant

Effects on Protein Phosphatase Activity:

• Unlike phosphorylation at Thr366 or cluster sites (Ser380-385), Ser370 phosphorylation appears to have distinct effects on protein phosphatase function

• S370D PTEN increases PIP3 levels but does not induce F-actin depolymerization on its own, unlike T366D PTEN

• This suggests Ser370 phosphorylation may selectively inhibit lipid phosphatase activity while potentially preserving some protein phosphatase functions

Impact on Membrane Association:

• Phosphorylation at Ser370, along with other C-terminal phosphorylations, significantly reduces PTEN's affinity for membrane phospholipids

• This decreased membrane binding limits access to membrane-bound PIP3 substrate, further reducing effective catalytic activity

• Phosphorylated PTEN requires higher concentrations of anionic lipids (PS, PIP2) to achieve membrane association compared to unphosphorylated PTEN

Conformational Changes:

• Ser370 phosphorylation contributes to a conformational compaction of PTEN

• This likely involves intramolecular interactions between the phosphorylated C-terminal tail and the C2 domain

• This closed conformation further restricts membrane interaction and substrate access

Cellular Consequences:

How can researchers distinguish between effects of phosphorylation at Ser370 versus other PTEN phosphorylation sites?

Distinguishing the specific effects of Ser370 phosphorylation from other phosphorylation sites requires multiple complementary approaches:

Site-Specific Phospho-Antibodies:

• Use antibodies specifically targeting individual phosphorylation sites (Ser370, Thr366, cluster sites)

• This approach allows monitoring of site-specific phosphorylation patterns in response to various stimuli

• Western blotting with these antibodies enables temporal resolution of phosphorylation events

• For example, leptin treatment rapidly increases phosphorylation at multiple PTEN sites including Ser370

Phosphomimetic and Phospho-Deficient Mutants:

• Comparative analysis of PTEN mutants reveals site-specific functional consequences:

  • S370D and T366D both increase PIP3 levels, but only T366D reduces F-actin levels

  • S370A and T366A both enhance PIP3 phosphatase activity

  • These differential effects highlight unique roles for each phosphorylation site

Functional Readouts:

• Multiple downstream measures should be assessed to capture site-specific effects:

  • PIP3 levels (measured by PIP3 antibody staining)

  • F-actin depolymerization (using phalloidin staining)

  • PI3K-dependency (using inhibitors like LY294002)

  • Membrane association (using fractionation or imaging techniques)

Combined Site Mutations:

• Creating PTEN with multiple phosphomimetic or phospho-deficient mutations helps determine hierarchical relationships

• For example, comparing single S370D versus combined S370D/T366D mutations can reveal additive or synergistic effects

Kinase Manipulation:

• Different kinases may target specific PTEN phosphorylation sites:

  • CK2 is implicated in phosphorylating Ser370 and cluster sites

  • GSK3β may primarily phosphorylate Thr366

  • Selective kinase inhibition can help isolate site-specific effects

Quantitative Mass Spectrometry:

• Enables measurement of phosphorylation stoichiometry at multiple sites simultaneously

• Can reveal temporal relationships between phosphorylation events

• Helps identify which sites are co-regulated versus independently regulated

The complexity of distinguishing site-specific effects is highlighted by research showing that while in vitro assays indicate no alteration in enzyme activity for T366D and S370D mutants, cellular studies show these mutations increase PIP3 levels, with differential effects on downstream processes like F-actin depolymerization .

What are the methodological challenges when using phosphomimetic PTEN mutants (S370D) versus studying actual phosphorylation?

Phosphomimetic mutations like S370D present several methodological challenges for researchers attempting to model physiological phosphorylation:

Chemical and Physical Limitations:

• Aspartic acid (D) provides a single negative charge (-1), while phosphoserine carries a stronger charge (-2 at physiological pH)

• The molecular structure of aspartate differs from phosphoserine, potentially affecting protein folding and interaction surfaces

• These differences may explain discrepancies between in vitro and cellular studies of S370D PTEN

Constitutive "Phosphorylation" Effects:

• Phosphomimetic mutations represent 100% constitutive "phosphorylation" at all times

• Native phosphorylation is typically dynamic and may occur at varying stoichiometries

• S370D PTEN increases PIP3 levels in cells but doesn't completely replicate the effects of native phosphorylation

Experimental Validation Requirements:

• Phospho-deficient mutants (S370A) should be studied alongside phosphomimetic ones

• Antibody-peptide competition assays using both phosphopeptide and non-phosphopeptide controls are essential

• Lambda phosphatase treatment provides verification that actual phosphorylation is being studied

Data from Phosphomimetic Mutants:

This table demonstrates how different PTEN phospho-variants produce distinct cellular phenotypes, highlighting the complex relationship between phosphorylation status and function.

Alternative Approaches:

• Generate semisynthetic site-specifically phosphorylated PTEN using expressed protein ligation

• Develop phospho-specific "intrabodies" that recognize only the phosphorylated form

• Use quantitative phosphoproteomics to measure endogenous phosphorylation stoichiometry

• Employ temporal control of kinase activity to better mimic physiological phosphorylation dynamics

How does phosphorylation at Ser370 affect PTEN's membrane association and what techniques can measure this?

Phosphorylation at Ser370 significantly impacts PTEN's membrane association through several mechanisms that can be measured using various techniques:

Mechanisms of Reduced Membrane Association:

• Conformational Closure:

  • Phosphorylation promotes conformational compaction of PTEN involving interactions between the phosphorylated C-terminal tail and the C2 domain

  • This closed conformation masks membrane-binding surfaces on the C2 domain

• Competition Mechanism:

  • Evidence indicates a direct competition between membrane phospholipids and the phosphorylated PTEN tail for binding to the C2 domain

  • This competition provides a molecular basis for phosphorylation-regulated membrane recruitment

• Altered Lipid Binding Kinetics:

  • Phosphorylated PTEN shows increased Ks values for PIP3-containing vesicles, indicating reduced binding affinity

  • This reduction is most significant at physiologically relevant low PIP3 concentrations

Measurement Techniques:

• Vesicle Sedimentation Assays:

  • Phosphorylated PTEN shows markedly reduced binding to vesicles containing anionic lipids like PS and PIP2

  • Both phosphorylated and unphosphorylated PTEN show increased binding with higher concentrations of these lipids, but phosphorylated PTEN requires significantly higher concentrations to achieve similar association

• Enzyme Kinetic Analysis:

  • Determine Ks (substrate binding constant) for phosphorylated versus unphosphorylated PTEN with PIP3-containing vesicles

  • At 1% PIP3, phosphorylated PTEN shows approximately threefold higher Ks value compared to unphosphorylated PTEN

• Fluorescence Microscopy:

  • Immunofluorescence analysis using phospho-PTEN (Ser370) antibodies can reveal subcellular distribution patterns

  • Comparison with total PTEN staining indicates relative membrane association

• FRET-Based Biosensors:

  • Construct FRET sensors with fluorophores positioned to detect PTEN conformational changes

  • Changes in FRET efficiency indicate alterations in domain proximity related to open/closed states

• Subcellular Fractionation:

  • Separate membrane and cytosolic fractions to quantify distribution of phosphorylated versus total PTEN

  • Western blotting with phospho-specific antibodies enables quantification of phospho-PTEN in each fraction

Lipid Dependencies:

Experimental data shows that increasing the concentration of anionic lipids like phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP2) enhances membrane binding of both phosphorylated and unphosphorylated PTEN, but phosphorylated PTEN consistently shows reduced membrane association across all conditions .

What kinases phosphorylate PTEN at Ser370 and how can researchers experimentally manipulate this phosphorylation?

Several kinases have been implicated in phosphorylating PTEN at Ser370, with various experimental approaches available for manipulation:

Identified Kinases:

• CK2 (Casein Kinase 2): Most consistently implicated in phosphorylating PTEN at Ser370 and cluster sites under normal conditions

• GSK3β: While primarily associated with Thr366 phosphorylation, may influence Ser370 phosphorylation through priming mechanisms

• MAST1, MAST2, MAST3, and STK11: Have been reported to phosphorylate PTEN at various sites in vitro

Physiological Stimuli:

• Leptin: Rapidly (<5 min) increases phosphorylation at Ser370 through a PI3K-independent pathway

• Insulin: Also reported to affect PTEN phosphorylation status

Experimental Manipulation Approaches:

• Pharmacological Inhibitors:

  • CK2 inhibitors: CX-4945, TBB, DMAT

  • GSK3β inhibitors: SB216763, CHIR99021

  • Control experiments should include specificity validation and dose-response curves

• Genetic Manipulation:

  • siRNA or shRNA knockdown of candidate kinases

  • CRISPR/Cas9-mediated knockout

  • Overexpression of constitutively active or dominant-negative kinase mutants

• In Vitro Kinase Assays:

  • Recombinant PTEN incubated with purified kinases

  • Mass spectrometry to identify and quantify phosphorylation sites

  • ATP analogs for specific kinase labeling

• Phosphatase Treatment:

  • Lambda phosphatase treatment removes phosphate groups

  • Useful for confirming antibody specificity and establishing baseline unphosphorylated state

• Phosphomimetic/Phospho-Deficient Mutants:

  • S370D mimics constitutive phosphorylation

  • S370A prevents phosphorylation at this site

  • These can be expressed in cells to study functional consequences

Validation Methods for Phosphorylation Status:

• Phospho-Specific Antibodies:

  • Western blotting with antibodies that specifically recognize PTEN phosphorylated at Ser370

  • Antibody specificity should be validated through peptide competition and phosphatase treatment

• Antibody-Peptide Competition:

  • Preincubate antibody with the phosphopeptide immunogen (should block signal)

  • A non-phosphorylated version of the same peptide (should not block signal)

  • An unrelated phospho-peptide (should not block signal)

• Mass Spectrometry:

  • Quantitative phosphoproteomics to determine stoichiometry of phosphorylation

  • Can simultaneously measure multiple phosphorylation sites

How can researchers measure and interpret conformational changes induced by Ser370 phosphorylation?

Phosphorylation of PTEN at Ser370 is thought to induce conformational changes that affect its function. Several techniques can measure these conformational alterations:

Biophysical Techniques:

Functional Assays Reflecting Conformation:

• Membrane Binding Assays:

  • Vesicle sedimentation assays show reduced membrane binding for phosphorylated PTEN

  • This reduction reflects conformational changes that mask membrane-binding surfaces

• PIP3 Phosphatase Activity:

  • Enzyme kinetic analysis reveals increased Ks values for phosphorylated PTEN

  • These changes in substrate binding reflect altered access to the active site

• Protein-Protein Interaction Studies:

  • Pull-down assays or co-immunoprecipitation to identify proteins that interact differentially with phosphorylated versus unphosphorylated PTEN

  • Yeast two-hybrid screening with phosphomimetic versus phospho-deficient PTEN as bait

Interpreting Conformational Data:

• Evidence indicates phosphorylated PTEN undergoes conformational compaction via an intramolecular interaction between its phosphorylated C-terminal tail and the C2 domain

• This closed conformation reduces membrane binding by masking membrane-interaction surfaces

• There appears to be competition between membrane phospholipids and the phosphorylated tail for binding to the C2 domain

• These conformational changes affect both catalytic activity and subcellular localization

From research with semisynthetic site-specifically phosphorylated PTEN, phosphorylation (including at Ser370) leads to reduced catalytic activity and membrane affinity through conformational compaction . This mechanism provides a molecular basis for regulation of PTEN tumor suppressor function through phosphorylation.

How does leptin signaling regulate PTEN phosphorylation at Ser370 and what are the downstream consequences?

Leptin signaling demonstrates a significant regulatory effect on PTEN phosphorylation, with specific impacts on Ser370:

Leptin-Induced Phosphorylation Dynamics:

• Leptin (10 nM) rapidly (<5 min) increases PTEN phosphorylation at multiple sites including Ser370 in hypothalamic and pancreatic β-cells

• This phosphorylation is sustained for at least 60 minutes

• The effect occurs in leptin-responsive cell lines such as N29/4 hypothalamic cells

Signaling Pathway Characteristics:

• Leptin-induced PTEN Ser370 phosphorylation is not blocked by the PI3K inhibitor LY294002 (10 μM)

• This indicates the involvement of a PI3K-independent pathway, distinguishing it from insulin signaling mechanisms

• CK2 has been implicated as a potential kinase mediating this phosphorylation

Functional Consequences:

Experimental Evidence:

• Overexpression of S370A PTEN (phospho-deficient mutant) prevents leptin-induced F-actin reduction

• S370D PTEN increases PIP3 levels but does not affect F-actin in the absence of leptin

• Unlike the G129E PTEN mutant (lipid phosphatase dead, protein phosphatase active), S370D PTEN does not block leptin-mediated F-actin depolymerization

Proposed Model:

What implications does PTEN Ser370 phosphorylation have for cancer research and potential therapeutic approaches?

PTEN Ser370 phosphorylation has significant implications for cancer research and therapeutic development:

Cancer-Relevant Mechanisms:

• PTEN is one of the most commonly lost tumor suppressors in human cancer

• Phosphorylation at Ser370 reduces PTEN's lipid phosphatase activity, potentially promoting oncogenic PI3K/AKT signaling

• The conformational changes induced by phosphorylation affect PTEN's subcellular localization and function

• Unlike genetic loss, phosphorylation provides a reversible mechanism of PTEN regulation that could potentially be targeted therapeutically

Experimental Approaches for Cancer Research:

• Immunohistochemical analysis of PTEN (pS370) in cancer tissues versus normal tissues

• Comparison of phosphorylation status across cancer types and correlation with clinical outcomes

• Analysis of kinase expression/activity (particularly CK2) in relation to PTEN phosphorylation status

• Functional studies using phosphomimetic and phospho-deficient PTEN mutants in cancer models

Potential Therapeutic Strategies:

• Kinase Inhibition:

  • CK2 inhibitors could potentially reduce PTEN phosphorylation at Ser370 and restore tumor suppressor function

  • Selectivity and off-target effects present significant challenges

• Conformation-Targeting Compounds:

  • Small molecules that bind to PTEN and prevent phosphorylation-induced conformational closure

  • Compounds that disrupt the interaction between the phosphorylated C-tail and C2 domain

• Phosphatase Activators:

  • Molecules that enhance PTEN activity even in the phosphorylated state

  • May require structure-based drug design approaches

• Combination Approaches:

  • Combining PI3K/AKT/mTOR inhibitors with agents targeting PTEN phosphorylation

  • May overcome resistance mechanisms to current targeted therapies

Biomarker Applications:

• Phospho-PTEN (Ser370) levels could serve as biomarkers for:

  • PI3K pathway activation status

  • Potential responsiveness to PI3K/AKT/mTOR inhibitors

  • Prognosis or disease progression

• Clinically applicable detection methods include immunohistochemistry and ELISA-based approaches

The development of Phospho-PTEN (Ser370) Colorimetric Cell-Based ELISA Kits enables high-throughput screening of compounds that might affect PTEN phosphorylation status, facilitating drug discovery efforts in this area .

How does PTEN Ser370 phosphorylation interact with other post-translational modifications and what methods can study these relationships?

PTEN undergoes multiple post-translational modifications (PTMs) that can interact with Ser370 phosphorylation, creating complex regulatory networks:

Interactions with Other Phosphorylation Sites:

• C-terminal Cluster (Ser380, Thr382, Thr383, Ser385):

  • Most cellular PTEN appears to be phosphorylated on both Ser370 and the C-terminal cluster sites

  • These sites are phosphorylated under normal conditions, likely by CK2

  • This multi-site phosphorylation contributes to PTEN conformational closure and reduced membrane binding

• Thr366 Phosphorylation:

  • Thr366 is another key regulatory phosphorylation site, reportedly targeted by GSK3β

  • While both T366D and S370D PTEN mutants increase PIP3 levels, they have different effects on F-actin dynamics

  • This suggests distinct but potentially complementary functions

• Other Phosphorylation Sites:

  • PTEN is phosphorylated on multiple additional serine, threonine, and tyrosine residues

  • Tyrosine phosphorylation at Tyr336 by FRK/PTK5 affects PTEN stability by inhibiting its binding to NEDD4

  • Thr319 and Thr321 phosphorylation in the C2 domain has also been reported

Additional PTM Interactions:

• Ubiquitination:

  • Phosphorylation status affects PTEN recognition by E3 ubiquitin ligases

  • Phosphorylation can protect PTEN from ubiquitin-mediated degradation

  • This creates a regulatory network connecting phosphorylation and protein stability

• SUMOylation and Acetylation:

  • PTEN undergoes additional modifications including SUMOylation and acetylation

  • These modifications may work in concert with or opposition to Ser370 phosphorylation

  • The interplay between these modifications affects PTEN localization and function

Experimental Methods to Study PTM Crosstalk:

• Mass Spectrometry-Based PTM Mapping:

  • Quantitative analysis of multiple PTMs on the same PTEN molecule

  • Can identify mutually exclusive or co-occurring modifications

  • Approaches include:

    • Enrichment of phosphopeptides prior to MS analysis

    • Multiplexed PTM enrichment strategies

    • Top-down proteomics of intact PTEN

• Sequential Modification Assays:

  • In vitro modification of recombinant PTEN with one enzyme followed by another

  • Determines if one modification facilitates or inhibits another

• Combinatorial Mutant Analysis:

  • Create PTEN variants with mutations at multiple PTM sites

  • Example combinations might include:

    • S370D/T366D double mutants

    • S370D combined with mutations of ubiquitination sites

    • Phosphomimetic mutations combined with acetylation-mimetic mutations

• Proximity Ligation Assays:

  • Detect co-occurrence of different PTMs on the same PTEN molecule in situ

  • Provides spatial information about modification patterns

PTM-Specific Tools:

• Antibodies that recognize specific combinations of modifications

• Mass spectrometry methods optimized for detection of multiple PTMs

• Genetic code expansion to incorporate modified amino acids at specific positions

• Semisynthetic approaches to generate PTEN with defined PTM patterns

Understanding these complex PTM interactions will provide insights into the fine-tuning of PTEN function in different cellular contexts and may reveal new therapeutic opportunities for diseases where PTEN function is dysregulated.