Phospho-PRKD1 (Ser910) Antibody

What is PRKD1 and what is the significance of Ser910 phosphorylation?

PRKD1 (Protein Kinase D1), also known as PKD, PKC-mu, PKCM, or PRKCM, is a serine/threonine protein kinase involved in multiple cellular processes including protein secretion, proliferation, cytoskeletal reorganization, Golgi function, immune function, and apoptosis . It has a molecular weight of approximately 102-115 kDa and contains structural domains including cysteine-rich domains and a pleckstrin homology (PH) domain .

Ser910 phosphorylation represents an important regulatory site on PRKD1. Research indicates that Ser910 conforms to a type I PDZ domain-binding motif (S/T-X-φ, where X is any amino acid and φ is a hydrophobic amino acid), and its phosphorylation disrupts PRKD1's docking interaction with PDZ domain-containing scaffolding proteins . Additionally, Ser910 plays a role in structuring the kinase core for some aspects of catalysis, as studies have shown that a S910A substitution abrogates PRKD1 autophosphorylation at Ser742 and prolongs in vivo PRKD1 signaling responses .

Proper storage and handling are crucial for maintaining antibody functionality:

• Store at -20°C or -80°C depending on the manufacturer's recommendation

• Avoid repeated freeze-thaw cycles to maintain antibody activity

• Some products are supplied in glycerol (typically 50%) with buffer components such as PBS, sodium azide (0.02%), and stabilizers like BSA (0.5%)

• Upon receipt, aliquoting is recommended for long-term storage

• For continuous use, undiluted antibody can be stored at 2-8°C for up to a week

• Spin the vial before opening to ensure complete recovery of contents

How do I optimize detection of phosphorylated PRKD1 in Western blot applications?

For optimal Western blot results when detecting Phospho-PRKD1 (Ser910):

• Sample preparation: Use fresh lysates from appropriate cell lines (A431 cells have been validated in multiple studies)

• Blocking conditions: Block membranes using 5% nonfat dry milk in PBS (pH 7.2)

• Antibody incubation: Incubate for ≥2 hours with diluted antibody (1:500-1:2000 range, optimize for your specific experiment)

• Detection system: Use enhanced chemiluminescence with horseradish peroxidase-conjugated secondary antibodies

• Controls: Include appropriate controls to validate specificity:

  • Non-phosphorylated protein samples

  • Phosphatase-treated samples

  • Blocking with the phosphopeptide (several manufacturers show the antibody signal is blocked when the phosphopeptide is included)

• Expected molecular weight: Look for a band at approximately 102-117 kDa (calculated molecular weight is 102 kDa, but observed weight may be 110-117 kDa due to post-translational modifications)

What cell stimulation conditions promote PRKD1 Ser910 phosphorylation?

PRKD1 Ser910 phosphorylation can be induced by various stimuli, which is valuable information when designing experiments:

• Growth factors and bioactive lipids: PKD1 can be activated by various growth factors and bioactive lipids

• Oxidative stress: PRKD1 is involved in resistance to oxidative stress through activation of NF-kappa-B

• Cell surface receptor activation: Cross-linking of B- and T-cell receptors and some G-protein coupled receptors (GPCR) can activate PRKD1

• Cell wounding: In intestinal epithelial cells, wounding can induce rapid PKD1 phosphorylation, though studies have focused more on Ser916 than Ser910 phosphorylation in this context

• Membrane translocation: PRKD1 is located mainly in the cytoplasm in unstimulated cells but migrates to the membrane when activated

How can I verify the specificity of a Phospho-PRKD1 (Ser910) antibody?

Verifying antibody specificity is crucial for reliable results:

• Phosphopeptide competition: The signal should be blocked when the antibody is pre-incubated with the phosphopeptide immunogen (peptide sequence around phosphorylation site of serine 910, typically R-V-S(p)-I-L)

• Non-phosphopeptide competition: The signal should remain when the antibody is pre-incubated with the non-phosphorylated peptide

• Phosphatase treatment: Treating samples with phosphatases (such as λ phosphatase) should eliminate the signal

• Genetic approaches: Using PRKD1 knockout cells or PRKD1-S910A mutant-expressing cells can provide definitive confirmation of specificity

• Cross-reactivity assessment: Most manufacturers have purified the antibodies to remove non-phospho specific antibodies through chromatography using non-phosphopeptide

What is the relationship between PRKD1 Ser910 phosphorylation and kinase activity?

The relationship between Ser910 phosphorylation and PRKD1 activity is complex and context-dependent:

• Disconnect between phosphorylation and activity: Several laboratories have described agonist-dependent increases in PKD1 activation loop phosphorylation and catalytic activity that are not accompanied by increased PKD1-Ser910 phosphorylation

• Autophosphorylation vs. trans-phosphorylation: PRKD1-K612W (a catalytically inactive form) can still be phosphorylated at Ser910 in trans by endogenous PKD1 or other enzymes with Ser910 kinase activity

• Privileged catalytic reaction: PKD1-Ser910 autophosphorylation is a privileged catalytic reaction that:

  • Proceeds at exceedingly low ATP concentrations

  • Does not require prior PKD1 phosphorylation at Ser738/Ser742

  • Is not necessarily accompanied by increased PKD1 activity toward heterologous protein substrates

• Unreliable activity marker: These findings "seriously undermine the assumption that immunoblotting studies that track PKD1-Ser910 phosphorylation provide a reliable measure of PKD1 activity under all experimental conditions"

How do Ser910 and Ser916 phosphorylation sites differ in function and detection?

Understanding the differences between these two phosphorylation sites is important for experimental design:

• Functional differences:

  • Ser910 phosphorylation disrupts PDZ domain interactions and is required for Ser742 autophosphorylation

  • Ser916 is a well-established autophosphorylation site that serves as a marker for activation in some contexts

• Differential regulation:

  • Ser910 phosphorylation may not always correlate with activation loop phosphorylation

  • Ser916 phosphorylation has been observed as early as 45 seconds after cell wounding, reaching maximum levels after 3 minutes

• Detection considerations:

  • Both sites can be detected using specific phospho-antibodies

  • Ser916 antibodies are available from multiple vendors including Proteintech (catalog numbers 80080-2-PBS, 80080-2-RR)

  • Researchers should choose the appropriate phospho-site antibody based on their specific research question

What roles does PRKD1 Ser910 phosphorylation play in cell migration and wound healing?

PRKD1 phosphorylation is implicated in cell migration and wound healing processes:

• Migration promotion: PKD1 signaling is required to promote migration of intestinal epithelial cells into denuded areas of wounds

• Rapid activation: In intestinal epithelial cells (IEC-18), wounding induces a striking increase in phospho-specific immunoreactivity in cells at the wound edge

• Inhibitor sensitivity: PKD inhibitors like kb NB 142-70 and CRT0066101 can prevent phosphorylation and reduce wound-induced migration

• Localization changes: PRKD1 is mainly cytoplasmic in unstimulated cells but migrates to the membrane when activated, which is required for kinase activity

• Research approach: When studying cell migration, researchers should consider using both siRNA targeting PRKD1 and pharmacological inhibitors to validate findings

How can discrepancies in PRKD1 phosphorylation data be reconciled across different experimental systems?

Researchers may encounter conflicting results when studying PRKD1 phosphorylation. Here are strategies to reconcile such discrepancies:

• Consider context-dependent regulation: PRKD1 phosphorylation may be regulated differently depending on:

  • Cell type (epithelial cells, immune cells, etc.)

  • Stimulation conditions (growth factors, oxidative stress, cell wounding)

  • Temporal dynamics (immediate vs. sustained responses)

• Assess technical variables:

  • Antibody specificity and lot-to-lot variation

  • Sample preparation methods

  • Detection sensitivity differences between techniques

• Evaluate the relationship between phosphorylation sites:

  • Ser910 phosphorylation does not always correlate with activation loop (Ser738/Ser742) phosphorylation

  • Ser910 and Ser916 phosphorylation may have different kinetics and requirements

• Use multiple approaches:

  • Combine phospho-specific antibodies with in vitro kinase assays

  • Validate with genetic approaches (phospho-mimetic and phospho-deficient mutants)

  • Consider mass spectrometry-based approaches for unbiased phosphorylation analysis

What are common problems when using Phospho-PRKD1 (Ser910) antibodies and how can they be resolved?

How can I distinguish between different PRKD isoforms when studying phosphorylation?

PRKD1 belongs to a family that includes PRKD2 and PRKD3. Distinguishing between these isoforms requires careful experimental design:

• Sequence comparison: Understand the sequence similarities and differences around Ser910:

  • The antibodies are typically generated against a peptide sequence around the phosphorylation site of serine 910 (R-V-S(p)-I-L) derived from Human PKD/PKCμ

  • Cross-reactivity with other PRKD family members should be considered

• Isoform-specific approaches:

  • Use isoform-specific antibodies alongside phospho-specific antibodies

  • Employ genetic approaches (siRNA knockdown of specific isoforms)

  • Consider the molecular weight differences (though these can be subtle)

• Expression patterns:

  • Different cell types express different levels of PRKD isoforms

  • The antibody primarily recognizes PRKD1, which is the major PKD family isoform in many cell types, although signals from PRKD2 can sometimes be detected

• Validation strategies:

  • Immunoprecipitate specific isoforms followed by phospho-detection

  • Use recombinant proteins of each isoform as controls

What emerging technologies may improve the study of PRKD1 phosphorylation?

As research on PRKD1 continues to evolve, several emerging technologies show promise for advancing our understanding:

• Phospho-proteomics: Mass spectrometry-based approaches allow for unbiased, quantitative assessment of multiple phosphorylation sites simultaneously

• Live-cell imaging: Phospho-specific biosensors based on FRET (Förster Resonance Energy Transfer) technology can monitor PRKD1 phosphorylation dynamics in real-time

• Single-cell analysis: Technologies that assess phosphorylation at the single-cell level can reveal heterogeneity in PRKD1 signaling within cell populations

• CRISPR-based approaches: Precise genome editing to create endogenous tagged versions of PRKD1 or phospho-mutants can provide more physiologically relevant models

• Structural biology: Advanced techniques like cryo-EM may reveal how Ser910 phosphorylation affects PRKD1 conformation and interaction with binding partners

What are key unanswered questions about PRKD1 Ser910 phosphorylation?

Despite extensive research, several important questions about PRKD1 Ser910 phosphorylation remain unanswered:

• Regulatory enzymes: What are the complete set of kinases and phosphatases that regulate Ser910 phosphorylation in different cellular contexts?

• Functional consequences: How does Ser910 phosphorylation affect the complete interactome of PRKD1, particularly with regard to PDZ domain-containing proteins?

• Disease relevance: What role does aberrant Ser910 phosphorylation play in pathological conditions like cancer, inflammation, or metabolic disorders?

• Temporal dynamics: What is the precise timing of Ser910 phosphorylation relative to other phosphorylation events on PRKD1 during signaling?

• Spatial regulation: How does the subcellular localization of PRKD1 influence Ser910 phosphorylation and vice versa?

• Cross-talk: How does Ser910 phosphorylation influence other post-translational modifications on PRKD1, such as ubiquitination or SUMOylation?