CDK16 Antibody

What is CDK16 and what are its primary cellular functions?

CDK16 (Cyclin Dependent Kinase 16) is a serine/threonine protein kinase belonging to the CMGC Ser/Thr protein kinase family. It plays critical roles in vesicle-mediated transport processes and exocytosis. In humans, the canonical protein has 496 amino acid residues and a mass of approximately 55.7 kDa. CDK16 exhibits subcellular localization in cell membranes, cytoplasmic vesicles, and cytoplasm. Its expression is notably prominent in pancreatic islets . Recent studies have revealed its involvement in cancer progression through mechanisms including p53 phosphorylation and degradation .

What are the common synonyms for CDK16 in scientific literature?

When reviewing literature for CDK16 research, it's important to search for multiple designations. CDK16 is frequently referenced under several synonyms:

• PCTAIRE1

• PCTGAIRE

• PCTK1

• PCTAIRE-motif protein kinase 1

• Cell division protein kinase 16

This nomenclature variability reflects the protein's historical characterization and functional classification within the CDK family.

What factors should be considered when selecting a CDK16 antibody for specific applications?

When selecting a CDK16 antibody, researchers should evaluate multiple parameters:

• Application compatibility: Verify the antibody has been validated for your intended application (WB, IHC, IF, ELISA, FCM). Evidence shows considerable variation in antibody performance across applications .

• Species cross-reactivity: Common CDK16 antibodies demonstrate reactivity with human, mouse, and rat orthologs. When working with other species, specific validation is necessary .

• Epitope location: Consider whether the antibody recognizes specific domains or post-translational modifications of CDK16.

• Validation data: Prioritize antibodies with published validation data, including positive/negative controls and knockdown verification .

• Clone type: Consider whether monoclonal specificity or polyclonal broad epitope recognition better suits your experimental needs.

How can I validate a CDK16 antibody for immunohistochemistry applications?

For rigorous IHC validation of CDK16 antibodies, implement this methodological approach:

• Positive tissue controls: Prioritize pancreatic islet tissue, where CDK16 is notably expressed . For cancer research, include TNBC or lung cancer tissues known to overexpress CDK16 .

• Negative controls: Employ tissues with CDK16 knockdown or those known to have minimal expression.

• Blocking peptide validation: Preincubate the antibody with a CDK16-specific blocking peptide to confirm binding specificity.

• Standardized protocol: For optimal results, implement antigen retrieval with boiling sodium citrate buffer (10 mM, pH 6.0), followed by blocking with 10% goat serum. Incubate with primary CDK16 antibody overnight at 4°C, then with HRP-conjugated secondary antibody for 30 minutes at room temperature .

• Quantification method: Adopt a semi-quantitative scoring system such as H-score (HS, range 0-300), calculated by multiplying intensity score (0-3) by distribution score (1-100%). This approach was successfully employed in CDK16 expression analysis in cancer tissue microarrays .

What are the established methods for studying CDK16 kinase activity?

Several approaches have been validated for assessing CDK16 kinase function:

• Kinase-inactive mutant comparisons: Generate the D304A mutant of CDK16 as a kinase-inactive control. Studies demonstrate that while wild-type CDK16 rescues cell proliferation defects in knockdown cells, the D304A mutant fails to restore this function, confirming kinase activity dependency .

• Substrate phosphorylation assays: Monitor phosphorylation of established CDK16 substrates, particularly p53, which is directly phosphorylated by CDK16 leading to its degradation .

• Inhibitor studies: Evaluate kinase activity in the presence of known CDK16 inhibitors, including type I (Dabrafenib) and type II (Rebastinib) inhibitors that have demonstrated efficacy .

• Cyclin Y/14-3-3 complex interaction: Assess the interaction between CDK16 and the cyclin Y/14-3-3 complex, which is critical for CDK16 kinase activity .

What are the recommended methods for CDK16 knockdown in experimental systems?

For effective CDK16 knockdown, multiple validated approaches have been documented:

• shRNA sequences: The following shRNA sequences have demonstrated effective CDK16 knockdown:

  • CDK16-sh1: 5′-GACCTACATTAAGCTGGACAA-3′

  • CDK16-sh2: 5′-CGAGGAGTTCAAGACATACAA-3′

  • CDK16-sh3: 5′-GCTCTCATCACTCCTTCACTT-3′

  • Cdk16-sh (mouse): 5′-GCACTAAAGGAGGTACAGCTA-3′

• Rescue experiments: Design knockdown targeting the 3′-UTR of CDK16 to allow subsequent rescue experiments with shRNA-resistant wild-type or mutant CDK16 constructs .

• Vector systems: For optimal delivery, the pLKO.1-GFP backbone has been successfully employed for shRNA expression .

• Validation approach: Confirm knockdown efficiency through both qPCR and western blot analysis, as demonstrated in multiple cancer cell models and patient-derived organoids .

How does CDK16 contribute to tumor progression in different cancer types?

CDK16 demonstrates oncogenic activity across multiple cancer types through several mechanisms:

What methods are recommended for studying CDK16 in patient-derived cancer models?

For translational research on CDK16, these methodological approaches have been validated:

• Patient-derived organoids (PDOs):

  • Dissociate tumor cells from patient samples

  • Infect with lentivirus carrying scramble or shCDK16 constructs with GFP tags

  • Isolate GFP+ cells by FACS

  • Culture in 3D system for organoid generation

  • Assess organoid formation efficiency and growth through size and number quantification

  • Evaluate proliferation via Ki67 staining

• Patient-derived xenografts (PDX):

  • Inoculate tumor fragments into immunodeficient mice (e.g., SCID-Beige recipients)

  • Once stable PDX lines are established, isolate tumor cells

  • Perform viral transduction for CDK16 manipulation

  • Sort transduced cells and re-xenograft into nude recipients

  • Monitor tumor occurrence, growth, and progression

• Clinical correlation analysis:

  • Utilize tissue microarrays for CDK16 immunohistochemical analysis

  • Implement H-score calculation: intensity score (0-3) × distribution score (1-100%)

  • Stratify scoring: HS<80 (score 0), 80<HS<120 (score 1), 120<HS<200 (score 2), HS>200 (score 3)

  • Correlate expression with clinical parameters and survival outcomes

How does the UBA1-CDK16 chimeric RNA influence immune function?

The UBA1-CDK16 chimeric RNA represents an emerging area of CDK16 research with sex-specific implications:

• Expression pattern: This chimeric RNA shows enrichment in myeloid lineage cells, including red blood cells and CD11b+ myeloid cells, compared to lymphoid lineage cells (NK, T, and B cells) .

• Developmental regulation: While undetectable in CD34+ hematopoietic stem cells (HSCs), UBA1-CDK16 expression progressively increases during myeloid differentiation induced by SCF, Flt3L, IL-3, IL-6, and GM-CSF .

• Functional impact: Knockdown of UBA1-CDK16 with junction-specific shRNAs leads to:

  • Increased CD11b+ myeloblast population

  • Elevated TLR8 expression

  • Upregulation of inflammatory response genes, particularly those in TNF-α/NF-κB signaling

• Sex specificity: This female-specific chimeric transcript appears to serve as a checkpoint against excessive myeloid differentiation and may contribute to sex-biased immunity by regulating inflammatory responses .

What structural insights inform the development of specific CDK16 inhibitors?

Structural analysis of CDK16 has revealed critical insights for inhibitor development:

• Kinase domain architecture: CDK16 exhibits the classical bi-lobal architecture with short insertions that contribute to its characteristic folding .

• Active site configuration: CDK16 shares higher structural similarity with active conformations of CDK1, CDK2, and CDK5 compared to inactive states, with particular alignment between the PCTAIRE domain of CDK16 and the active PSTAIRE domain of CDK1 .

• Inhibitor compatibility: Experimental evidence confirms that CDK16 can be targeted by both:

  • Type I inhibitors (e.g., Dabrafenib) that bind to active conformations

  • Type II inhibitors (e.g., Rebastinib) that recognize inactive conformations

• Cyclin binding interface: The cyclin binding domains of PCTAIREs show overlapping geometries, particularly between CDK16 and CDK17, which may influence inhibitor specificity across this kinase subfamily .

• Functional implications: The interaction between CDK16 and the cyclin Y/14-3-3 complex is critical for kinase activity and may be mutually exclusive with the binding of specific inhibitors .

What are common challenges in CDK16 immunohistochemistry and how can they be addressed?

Researchers frequently encounter these challenges when performing CDK16 IHC:

• Nonspecific staining:

  • Problem: Background staining may obscure specific CDK16 signal

  • Solution: Optimize blocking with 10% goat serum albumin, ensure longer (overnight at 4°C) primary antibody incubation, and validate antibody specificity with positive and negative controls

• Antigen retrieval issues:

  • Problem: Insufficient retrieval can result in false negatives

  • Solution: Implement boiling sodium citrate buffer (10 mM, pH 6.0) retrieval method shown to be effective for CDK16 detection

• Quantification variability:

  • Problem: Subjective scoring leading to inconsistent results

  • Solution: Employ H-score system combining intensity (0-3) and distribution (1-100%) scores, with multiple blinded examiners to ensure reliability

• Distinguishing isoforms:

  • Problem: Antibodies may detect multiple CDK16 isoforms (up to 3 reported)

  • Solution: Select antibodies with validated specificity for the isoform of interest and confirm with western blotting

How can researchers optimize western blot protocols for CDK16 detection?

For reliable western blot detection of CDK16, consider these evidence-based optimization strategies:

• Sample preparation:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation status

  • For membrane-associated CDK16 fraction, incorporate appropriate detergents in extraction buffers

• Gel selection:

  • Use 10-12% gels to properly resolve the 55.7 kDa CDK16 protein

  • Consider gradient gels when analyzing CDK16 complexes

• Transfer conditions:

  • Optimize transfer time and voltage for proteins in this molecular weight range

  • Verify transfer efficiency with reversible staining methods

• Antibody selection and dilution:

  • Select antibodies specifically validated for western blot applications

  • Implement titration experiments to determine optimal antibody concentration

  • Include positive controls (overexpression lysates) and negative controls (knockdown lysates)

• Signal detection:

  • For quantitative analysis, remain within the linear range of detection

  • When studying CDK16 interactions (e.g., with p53), optimize co-immunoprecipitation conditions before western blot analysis