Phospho-HNRNPK (Ser216) Antibody

What is the biological significance of hnRNP K Ser216 phosphorylation?

hnRNP K Ser216 phosphorylation plays crucial roles in transcriptional regulation and DNA damage response pathways. JNK phosphorylation of hnRNP K on Ser216 increases AP1-dependent transcriptional activities by enhancing DNA binding affinity and protein-protein interactions, particularly in UV-treated cells . This phosphorylation site is located in the linker region between KH domains and affects hnRNP K's affinity for RNAs or DNAs without significantly altering its structure .

Which kinases are responsible for phosphorylating hnRNP K at Ser216?

JNK (c-Jun N-terminal kinase) is the primary kinase identified to phosphorylate hnRNP K at Ser216, particularly in response to cellular stresses such as UV radiation . Unlike ERK-mediated phosphorylation which affects nuclear export and translation inhibition, JNK phosphorylation of Ser216 primarily influences transcriptional activities of hnRNP K .

How does Ser216 phosphorylation compare with other phosphorylation sites on hnRNP K?

hnRNP K contains at least 20 documented phosphorylation sites among its 72 potential phosphorylation sites (31 serine, 24 threonine, and 17 tyrosine residues) . While phosphorylation at Ser284 and Ser353 by ERK affects cytoplasmic accumulation, and Ser302 phosphorylation by PKC δ modulates protein interactions, Ser216 phosphorylation by JNK specifically enhances transcriptional activity without affecting nuclear export or RNA translation inhibition .

What are the key specifications of commercial Phospho-HNRNPK (Ser216) antibodies?

Most commercial Phospho-HNRNPK (Ser216) antibodies are rabbit polyclonal antibodies raised against synthetic phosphopeptides containing the Ser216 phosphorylation site. The immunogen typically consists of peptide sequences surrounding the phosphorylation site of serine 216 (S-E-S(p)-P-I) derived from human hnRNP K . These antibodies generally have cross-reactivity with human, mouse, and rat samples, and are suitable for Western blotting (1:500-1:3000 dilution) and ELISA (1:20000 dilution) .

How can I validate the specificity of Phospho-HNRNPK (Ser216) antibodies?

To validate antibody specificity, incorporate these controls:

• Phosphopeptide competition: Preincubate the antibody with the phosphopeptide used as immunogen to block specific binding .

• Phosphatase treatment: Treat lysate samples with lambda phosphatase to remove phosphate groups, which should eliminate antibody detection.

• Positive controls: Use lysates from cells treated with known inducers of Ser216 phosphorylation, such as UV radiation or JNK pathway activators .

• Ser216 mutants: Compare wild-type hnRNP K with S216A (non-phosphorylatable) mutants .

Western blot analysis of extracts from JurKat cells shows specific detection of phosphorylated Ser216 that can be blocked with competing phosphopeptide .

What are the optimal conditions for detecting Phospho-HNRNPK (Ser216) by Western blotting?

For optimal Western blot detection:

• Sample preparation: Lyse cells in buffer containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, and β-glycerophosphate).

• Protein amount: Load 20-50 μg of total protein per lane.

• Blocking: Use 5% BSA in TBST rather than milk, as milk contains phosphatases.

• Antibody dilution: Use at 1:500-1:3000 dilution in 5% BSA-TBST .

• Detection system: Enhanced chemiluminescence systems provide adequate sensitivity.

• Positive control: Include lysates from UV-treated or JNK-activated cells .

How can I induce and detect changes in hnRNP K Ser216 phosphorylation?

To study dynamic changes in Ser216 phosphorylation:

• UV treatment: Expose cells to UV radiation (40-100 J/m²), which activates JNK pathway and increases Ser216 phosphorylation .

• JNK pathway activators: Treat cells with anisomycin (10 μM, 30 min) or bacterial lipopolysaccharide .

• Serum stimulation: Serum addition after starvation can modulate hnRNP K phosphorylation patterns .

• Time course: Monitor phosphorylation changes at multiple timepoints (15, 30, 60, 120 min) after stimulation.

• JNK inhibitors: Use SP600125 or JNK-IN-8 as negative controls to confirm JNK-dependent phosphorylation.

Can Phospho-HNRNPK (Ser216) antibodies be used for immunoprecipitation or immunofluorescence?

While most commercial antibodies are validated for Western blot and ELISA applications , immunoprecipitation and immunofluorescence applications require additional optimization:

For immunoprecipitation:

• Use 2-5 μg antibody per 500 μg protein lysate

• Pre-clear lysates with protein A/G beads

• Include phosphatase inhibitors throughout the procedure

• Validate specificity using S216A mutants as negative controls

For immunofluorescence:

• Fix cells with 4% paraformaldehyde (avoid methanol fixation which can cause loss of phospho-epitopes)

• Use higher antibody concentrations (1:100-1:500)

• Include phosphatase inhibitors in all buffers

• Validate specificity using peptide competition and phosphatase treatment controls

How can I use phosphate-affinity electrophoresis to characterize hnRNP K phosphorylation states?

Phosphate-affinity electrophoresis provides superior resolution of different phosphorylated forms:

• Phos-tag™ SDS-PAGE: Incorporate Phos-tag™ acrylamide (50-100 μM) and MnCl₂ (100-200 μM) into polyacrylamide gels.

• 2D electrophoresis: Combine isoelectric focusing with phosphate-affinity SDS-PAGE for enhanced resolution of >20 distinct hnRNP K forms .

• Sample preparation: Include EDTA-free phosphatase inhibitor cocktail in lysis buffers.

• Comparison with standard SDS-PAGE: Run parallel gels with and without Phos-tag™ to identify mobility shifts.

• Mass spectrometry validation: Confirm phosphorylation sites by excising gel spots and performing LC-MS/MS analysis .

This approach revealed that nuclear fractions contain more than 20 spots of endogenous hnRNP K on 2D maps, with phosphorylation states correlating with subcellular localization and alternative splicing patterns .

How can I study the relationship between Ser216 phosphorylation and hnRNP K function in cancer?

To investigate the role of Ser216 phosphorylation in cancer:

• Site-directed mutagenesis: Generate S216A (non-phosphorylatable) and S216D/E (phosphomimetic) mutations in hnRNP K expression constructs .

• Rescue experiments: Knock down endogenous hnRNP K using siRNA targeting the 3'UTR, then express shRNA-resistant wild-type or mutant hnRNP K .

• Cell migration assays: Compare migration capabilities of cells expressing wild-type versus S216A/D/E mutants using Boyden chamber or wound healing assays .

• RNA-binding analysis: Perform RNA immunoprecipitation (RIP) to assess how Ser216 phosphorylation affects RNA target binding .

• Subcellular fractionation: Examine nuclear versus cytoplasmic distribution of wild-type and mutant hnRNP K .

Research suggests cytoplasmic accumulation of hnRNP K is crucial for its role in metastasis, which may be regulated by specific phosphorylation events .

What is the role of hnRNP K Ser216 phosphorylation in DNA damage response?

hnRNP K is phosphorylated in an ATM-dependent manner in response to DNA damage . While ATM primarily phosphorylates hnRNP K at S121, T174, T370, and T440, JNK-mediated Ser216 phosphorylation may also contribute to DNA damage responses :

• DNA damage induction: Treat cells with ionizing radiation (2-10 Gy) or etoposide (10-50 μM).

• ATM inhibition: Use KU-55933 (10 μM) to block ATM kinase activity .

• Co-immunoprecipitation: Assess interaction between phosphorylated hnRNP K and p53 using Phospho-Ser216 antibodies.

• Chromatin immunoprecipitation: Examine recruitment of phosphorylated hnRNP K to p53 target gene promoters.

• Gene expression analysis: Measure expression of p53 target genes (p21, GADD45α) in cells expressing wild-type versus S216A mutant hnRNP K .

What are common issues when working with Phospho-HNRNPK (Ser216) antibodies and how can they be resolved?

How should I select between different commercially available Phospho-HNRNPK (Ser216) antibodies?

Consider these factors when selecting the appropriate antibody:

• Validation data: Look for comprehensive validation including phosphopeptide competition, phosphatase treatment, and mutant controls .

• Host species: Consider rabbit polyclonal for higher sensitivity or mouse monoclonal for consistency between lots.

• Applications: Confirm validation for your intended application (WB, ELISA, IP, IF) .

• Species reactivity: Verify cross-reactivity with your experimental model (human, mouse, rat) .

• Purification method: Antibodies purified by epitope-specific affinity chromatography typically show higher specificity .

How can I quantify changes in hnRNP K Ser216 phosphorylation levels?

For accurate quantification:

• Normalization strategies:

  • Normalize phospho-signal to total hnRNP K detected on separate blots or after stripping

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls

• Image analysis software:

  • Use ImageJ, Image Lab, or similar software for densitometry

  • Apply background subtraction and consistent ROI selection

• Multiplexed detection systems:

  • Consider fluorescently-labeled secondary antibodies for simultaneous detection of phospho and total proteins

  • Use two-color infrared imaging systems (LI-COR Odyssey) for improved quantitative range

• Alternative quantitative approaches:

  • ELISA-based phosphoprotein quantification

  • Mass spectrometry with SILAC or TMT labeling for absolute quantification

What is the role of hnRNP K Ser216 phosphorylation in viral infections?

Recent studies suggest hnRNP K functions in viral replication processes:

• HIV-1 and HTLV-1 interactions: hnRNP K promotes internal ribosome entry site (IRES) activity in HIV-1 and HTLV-1 .

• Post-translational modifications: PTMs of hnRNP K, potentially including Ser216 phosphorylation, modulate its ability to stimulate viral IRES-mediated translation .

• Experimental approaches:

  • Use luciferase reporter assays with viral IRES elements

  • Compare wild-type and S216A/D/E mutant effects on viral protein synthesis

  • Assess colocalization of phosphorylated hnRNP K with viral components

How does Ser216 phosphorylation intersect with other post-translational modifications of hnRNP K?

hnRNP K undergoes multiple PTMs including:

• Methylation: Primarily at arginine residues by protein arginine methyltransferases (PRMTs) .

• Sumoylation: Modifies hnRNP K to function as a transcriptional coactivator of p53 .

• Ubiquitylation: Regulates hnRNP K stability and cytoplasmic levels .

Potential cross-talk between phosphorylation and other PTMs can be studied by:

• Sequential immunoprecipitation with phospho-specific and other PTM-specific antibodies

• Mass spectrometry analysis of differently modified hnRNP K populations

• Generation of multi-site mutants affecting different PTM types

• Using specific inhibitors for different PTM-generating enzymes in combination

What techniques are emerging for single-cell analysis of hnRNP K phosphorylation?

Advanced techniques for phosphorylation analysis at single-cell resolution include:

• Phospho-flow cytometry: Using fluorescently labeled Phospho-HNRNPK (Ser216) antibodies.

• Mass cytometry (CyTOF): Metal-conjugated antibodies for multi-parameter analysis.

• Proximity ligation assay: In situ detection of phosphorylated hnRNP K interactions with binding partners.

• Single-cell western blotting: Microfluidic platforms for protein analysis in individual cells.

• Live-cell biosensors: FRET-based reporters for real-time phosphorylation dynamics.

These approaches allow correlation of phosphorylation status with cellular phenotypes at the individual cell level, revealing heterogeneity within populations.

How should I interpret changes in hnRNP K Ser216 phosphorylation in different subcellular fractions?

When analyzing subcellular distribution:

• Fractionation quality control: Verify fraction purity using markers (lamin A/C for nucleus, GAPDH for cytoplasm).

• Normalization approach: Use fraction-specific loading controls rather than total protein.

• Comparative analysis: Calculate nuclear:cytoplasmic ratios of phosphorylated versus total hnRNP K.

• Correlation analysis: Connect phosphorylation patterns with alternative splicing or target gene expression .

Research indicates subcellular localization correlates with phosphorylation states and alternative splicing patterns, with multiple forms differentially modulated in response to external stimuli like lipopolysaccharide or serum .

What are the implications of altered hnRNP K Ser216 phosphorylation in disease models?

In various disease contexts:

• Cancer: Cytoplasmic accumulation of hnRNP K, potentially regulated by phosphorylation, is crucial for its role in metastasis . Phosphorylation status may serve as a biomarker for cancer progression or therapeutic response.

• DNA damage response: ATM-dependent phosphorylation of hnRNP K, including potential cross-talk with Ser216 phosphorylation, is required for p53-mediated transcriptional responses to genotoxic stress .

• Viral infections: Post-translational modifications of hnRNP K modulate its ability to stimulate IRES-mediated translation in HIV-1 and HTLV-1 .

When analyzing disease models, consider:

• Temporal dynamics of phosphorylation changes

• Correlation with disease progression markers

• Therapeutic interventions that modulate the responsible kinases

• Potential for combination therapies targeting multiple PTMs

What are promising approaches for targeting hnRNP K Ser216 phosphorylation therapeutically?

Potential therapeutic strategies include:

• JNK pathway inhibitors: Target the kinase responsible for Ser216 phosphorylation.

• Phosphatase activators: Enhance dephosphorylation of Ser216.

• Peptide-based approaches: Develop cell-penetrating peptides that compete with hnRNP K for JNK binding.

• Structure-based drug design: Design small molecules that specifically recognize the phosphorylated Ser216 region.

• Combination approaches: Target multiple PTMs simultaneously to synergistically modulate hnRNP K function.

What novel techniques might advance our understanding of hnRNP K Ser216 phosphorylation dynamics?

Emerging technologies with potential applications include:

• Optogenetic control: Light-inducible kinase systems to manipulate Ser216 phosphorylation with spatiotemporal precision.

• CRISPR-based approaches: Base editing to introduce phosphomimetic mutations at the endogenous locus.

• Interactome profiling: BioID or APEX proximity labeling to identify protein interactions specific to phosphorylated Ser216.

• Cryo-EM and structural studies: Visualize conformational changes induced by Ser216 phosphorylation.

• Single-molecule imaging: Track individual hnRNP K molecules and their interactions in living cells.