STK16 Antibody

What is STK16 and why is it significant for cell biology research?

STK16, also known as serine/threonine kinase 16 (alternative names include KRCT, MPSK, TSF1, or PKL12), is a 305 amino acid lipid-anchored membrane protein that plays a crucial role in cellular signaling pathways. Its significance stems from its unique function as an actin-binding protein located in the Golgi apparatus. STK16 undergoes post-translational modifications, particularly phosphorylation, which is vital for regulating various cellular processes including cell division, differentiation, and response to DNA damage . The phosphorylation of serine and threonine residues by STK16 can activate or deactivate target proteins, thereby influencing critical functions within the cell. Moreover, STK16 is the first identified serine/threonine kinase residing in the Golgi that can bind to actin directly and regulate actin dynamics, making it a unique research target for understanding Golgi-cytoskeletal interactions .

How should I validate STK16 antibody specificity in my experimental system?

To validate STK16 antibody specificity, implementing multiple controls is essential:

• RNAi knockdown validation: Perform siRNA knockdown of STK16 and demonstrate reduced signal with your antibody. To ensure knockdown specificity, construct a stable cell line with an RNAi-resistant mutant containing silent mutations at the siRNA target region. This mutant should express wild-type STK16 protein but escape siRNA knockdown effects .

• RT-PCR verification: When antibody detection is suboptimal, verify STK16 knockdown efficiency at the mRNA level using RT-PCR .

• Protein detection quantification: Measure STK16 protein levels using tagged versions (such as GFP or Flag tags) if direct antibody detection is problematic. In previous studies, researchers observed around 50% and 45% reduction in protein levels as measured by GFP and Flag antibodies in knockdown experiments .

• Functional validation: Confirm biological effects of STK16 perturbation, such as changes in F-actin levels or Golgi morphology, which should be rescued in the RNAi-resistant mutant cell line .

How does STK16 regulate Golgi structure and what techniques can reveal this function?

STK16 regulates Golgi structure primarily through its effects on actin dynamics. To investigate this relationship:

• Time-course experiments: Treat cells with STK16 inhibitors (such as STK16-IN-1) and monitor both actin polymer status and Golgi complex integrity over time. Research has shown that 1 μM STK16-IN-1 begins to depolymerize actin as early as 30 minutes, while the Golgi complex integrity shows no significant change until after 1 hour . With 10 μM STK16-IN-1, actin puncta formation occurs within 15 minutes, while Golgi effects appear only after 4-6 hours .

• Co-localization studies: Use Golgi markers such as Giantin (an integral membrane protein residing in the Golgi complex), M6PR, or TGN46 alongside STK16 visualization to track structural changes. This approach is particularly valuable during mitosis when the Golgi undergoes fragmentation and reorganization .

• Actin visualization techniques: Combine STK16 perturbation (via RNAi or inhibitors) with actin visualization to establish the causal relationship between actin depolymerization and Golgi structural changes .

The experimental evidence suggests that STK16 first affects actin dynamics, which subsequently leads to Golgi structural changes, indicating that STK16 may regulate Golgi complex integrity through its effects on the actin cytoskeleton .

What are the critical autophosphorylation sites in STK16 and how do they affect its function?

Three key autophosphorylation sites have been identified in STK16: Threonine185, Serine197, and Tyrosine198. Their functional significance can be studied using site-directed mutagenesis approaches:

• Phosphomimetic and phospho-deficient mutations: Replace these residues with glutamate (E) to mimic the phosphorylated state or with alanine (A) to mimic the unphosphorylated state .

• Single vs. multiple site mutations: Create single mutants (T185E, T185A, S197E, S197A, Y198E, Y198A) as well as combined mutants (2E: S197E-S198E, 2A: S197A-Y198A, 3E: T185E-S197E-Y198E, 3A: T185A-S197A-Y198A) .

Research has shown differential effects of these mutations:

• Single mutations of T185 and S197 (both E and A variants) had no effect on STK16 subcellular localization or Golgi structure .

• Y198 mutations (both Y198E and Y198A) resulted in altered subcellular localization of STK16, suggesting this site is particularly critical for proper localization and function .

These findings indicate that Tyr198 is an essential autophosphorylation site for STK16 function, particularly regarding its proper localization to the Golgi apparatus.

How does STK16 impact cell cycle progression and what methods can best detect these effects?

STK16 plays critical roles in cell cycle regulation, particularly affecting G2 phase, mitotic entry, mitotic progression, and cytokinesis. To investigate these effects:

• Flow cytometry: This provides initial evidence of cell cycle disruption, showing increased G2/M phase population upon STK16 knockdown or inhibition .

• Immunofluorescence with cell cycle markers:

  • Cyclin B1 staining identifies G2 phase cells (STK16 knockdown increases this population by approximately 3-fold)

  • Phospho-Histone H3 staining identifies mitotic cells

  • Quantify cells in each mitotic phase (prophase through cytokinesis) to pinpoint specific effects

• Cell synchronization experiments: Synchronize cells with double thymidine block and track their progression through the cell cycle following STK16 perturbation. This reveals detailed timing effects:

  • STK16 inhibition delays mitotic entry

  • Both STK16 knockdown and kinase inhibition prolong mitosis (from ~3 hours in control cells to 5-6 hours in STK16-perturbed cells)

  • Quantification reveals increased cell populations in both prometaphase and cytokinesis

• Chromosome bridge analysis: STK16 perturbation increases telophase cells with chromosome bridges by 3-4 fold, indicating mitotic defects .

• Actin cytoskeleton analysis in late mitosis: STK16 knockdown reduces actin spikes and membrane blebbing, which are critical for furrow ingression and abscission during cytokinesis .

The experimental evidence demonstrates that STK16 regulates both actin dynamics and Golgi assembly, with critical roles in mitotic entry, progression, and cytokinesis .

How can I overcome the limitations of commercial STK16 antibodies for certain applications?

Given that commercial STK16 antibodies have shown limitations in certain applications, researchers can implement several alternative strategies:

• Tagged expression systems: Generate stable cell lines expressing STK16 with epitope tags such as GFP and FLAG. This approach has been successfully used for both localization studies and functional assessments .

• RNAi with rescue experiments: Combine STK16 knockdown with expression of RNAi-resistant STK16 variants. This approach not only validates knockdown specificity but also allows for structure-function studies using STK16 mutants .

• RT-PCR for expression analysis: When protein-level detection is problematic, use RT-PCR to examine STK16 mRNA levels in cells .

• Inhibitor-resistant mutants: For pharmacological studies, generate inhibitor-resistant mutants (such as F100C for STK16-IN-1 resistance) to validate inhibitor specificity and separate on-target from off-target effects .

• Multiple cell lines validation: Test STK16 perturbation effects across different cell lines (such as HeLa, NIH-3T3, and MCF-7) to establish the generality of your findings .

These approaches collectively help circumvent antibody limitations while maintaining experimental rigor in studying STK16 function.

What are the optimal experimental designs for studying STK16's role in actin dynamics?

To effectively study STK16's role in actin dynamics:

• Temporal analysis following STK16 perturbation:

  • Short-term (minutes to hours) and long-term (hours to days) effects should be distinguished

  • Research shows actin changes precede Golgi structural alterations (actin effects within 15-30 minutes; Golgi effects after 4-6 hours)

• Multiple visualization techniques:

  • F-actin staining in fixed cells

  • Live-cell imaging with actin reporters

  • Quantification of F-actin to G-actin ratio in biochemical assays

• Combined genetic and pharmacological approaches:

  • RNAi knockdown of STK16

  • STK16 inhibitors (such as STK16-IN-1)

  • Expression of dominant-negative mutants

• Cell type considerations: Effects have been observed in multiple cell lines (HeLa, NIH-3T3, MCF-7), suggesting broad relevance but potentially different magnitudes of effect depending on cell type .

• Cell cycle phase specificity: Given STK16's role in mitosis, actin dynamics should be studied both in interphase and throughout different mitotic stages, with particular attention to cytokinesis where STK16 knockdown shows striking effects on actin spikes and membrane blebbing .

How can I resolve discrepancies between phenotypes observed with STK16 knockdown versus inhibition?

When facing discrepancies between STK16 knockdown and inhibition results:

• Timeline considerations: Inhibition typically produces more rapid effects than knockdown. For example, STK16-IN-1 causes actin depolymerization within minutes, while RNAi effects develop over days as protein levels gradually decrease .

• Specificity validation:

  • For RNAi: Use multiple siRNA sequences and rescue experiments with RNAi-resistant constructs

  • For inhibitors: Use structurally different inhibitors targeting STK16 or inhibitor-resistant mutants (such as F100C for STK16-IN-1)

• Kinase-dependent vs. scaffolding functions: Inhibitors typically affect only kinase activity while preserving protein interactions, whereas knockdown eliminates both catalytic and scaffolding functions. This can help distinguish which STK16 functions depend on its kinase activity versus its physical presence .

• Concentration/dosage effects: Test multiple inhibitor concentrations or varying levels of knockdown efficiency to establish dose-response relationships.

• Combined approaches: Apply both methods simultaneously to determine if effects are additive, synergistic, or redundant.

By systematically addressing these considerations, researchers can resolve apparent discrepancies and gain deeper insights into STK16's multifaceted functions.

What controls should be included when studying STK16's phosphorylation targets?

When investigating STK16's phosphorylation targets:

• Kinase-dead controls: Include kinase-inactive mutants of STK16 (K49M has been used) to distinguish between phosphorylation events dependent on STK16's catalytic activity versus those resulting from scaffolding effects or indirect associations.

• Phosphosite mutants: For putative target proteins, mutate the suspected phosphorylation sites (Ser/Thr residues) to alanine (preventing phosphorylation) or to glutamate/aspartate (mimicking phosphorylation) to validate functional significance.

• In vitro kinase assays: Perform kinase assays with purified components to establish direct phosphorylation by STK16, rather than effects mediated through intermediate kinases.

• Phosphatase controls: Include phosphatase treatments to reverse STK16-mediated phosphorylation events and confirm specificity.

• Autophosphorylation distinction: Given STK16's known autophosphorylation sites (T185, S197, Y198), carefully distinguish between autophosphorylation and substrate phosphorylation .

• Phospho-specific antibodies: When available, use antibodies that specifically recognize phosphorylated forms of putative targets to directly monitor STK16-dependent phosphorylation in vivo.

How should I quantify and interpret Golgi morphology changes following STK16 perturbation?

Quantification and interpretation of Golgi morphology changes require systematic approaches:

• Standardized classification system:

  • Normal: Compact, perinuclear Golgi ribbon structure

  • Fragmented: Dispersed Golgi elements throughout the cytoplasm

  • Condensed: Abnormally compact Golgi structure

  • Vesiculated: Complete breakdown into vesicular structures

• Multiple Golgi markers: Use combinations of cis-, medial-, and trans-Golgi markers (e.g., GM130, Giantin, TGN46) to comprehensively assess Golgi structural changes .

• Quantification parameters:

  • Area of Golgi elements

  • Number of discrete Golgi fragments

  • Distance of Golgi elements from the nucleus

  • Fluorescence intensity distribution

• Temporal analysis: Establish the time course of Golgi changes relative to actin cytoskeleton alterations. Research shows that actin changes (15-30 minutes) precede Golgi structural alterations (4-6 hours) with STK16 inhibition .

• Statistical analysis: Apply appropriate statistical tests to quantified parameters across multiple cells (n>100) from independent experiments to establish significance.

• Rescue experiments: Confirm that reintroduction of wild-type STK16 or specific mutants (particularly focusing on Y198 status) can reverse observed Golgi phenotypes, providing causal evidence for STK16's role .

This methodical approach ensures reliable and reproducible assessment of STK16's effects on Golgi morphology.

What are the key considerations when interpreting cell cycle defects caused by STK16 perturbation?

When interpreting cell cycle defects resulting from STK16 perturbation:

• Primary vs. secondary effects: Determine whether observed defects stem directly from STK16 function or are secondary consequences of Golgi/actin disruption. The temporal sequence suggests STK16 first affects actin dynamics, which subsequently impacts Golgi structure and cell cycle progression .

• Phase-specific analysis: Quantify cells in specific cell cycle phases:

  • G2 phase: Cyclin B1-positive cells with intact nuclear envelope

  • Mitotic phases: Prophase, prometaphase, metaphase, anaphase, telophase

  • Cytokinesis: Analyzing bridge formation and resolution

• Context from synchronized populations: Use synchronized cell populations to accurately measure:

  • Time of mitotic entry (delayed by STK16 perturbation)

  • Duration of mitosis (prolonged from ~3 hours to 5-6 hours with STK16 disruption)

  • Specific phases affected (prometaphase and cytokinesis show greatest increases)

• Chromosome segregation analysis: Quantify chromosome bridges in telophase (increased 3-4 fold with STK16 perturbation), indicating mitotic fidelity issues .

• Actin-dependent effects in cytokinesis: Analyze actin spike formation and membrane blebbing during cytokinesis, which are critically reduced in STK16-knockdown cells .

This comprehensive analysis framework helps distinguish direct cell cycle regulatory functions of STK16 from indirect consequences of cytoskeletal and Golgi disruption.

How can I integrate STK16 research with broader studies of Golgi-cytoskeleton interactions?

To integrate STK16 research with broader Golgi-cytoskeleton studies:

• Comparative analysis with other Golgi kinases: Compare STK16's functions with other Golgi-associated kinases (e.g., PKD1/PKD2, Cdc42, PAK4) to establish unique versus overlapping roles. Unlike these other kinases, STK16 directly binds actin and regulates its dynamics rather than working through signaling intermediates .

• Golgi-actin visualization approaches:

  • Super-resolution microscopy to visualize Golgi-associated actin

  • Live-cell imaging with dual markers for Golgi and actin

  • Correlative light and electron microscopy (CLEM) for ultrastructural context

• Cargo trafficking studies: Investigate whether STK16's effects on actin and Golgi alter specific cargo trafficking pathways. Current evidence suggests STK16 is not critical for ER to Golgi or Golgi to PM transport of VSVG, but may affect other specialized trafficking routes .

• Relationship to Golgi fragmentation pathways: Connect STK16 function to known regulators of Golgi fragmentation during stress and mitosis, including GRASP proteins, Golgins, and mitotic kinases.

• Cross-comparison across cell types: Study STK16's role in specialized secretory cells with extensive Golgi networks (e.g., neurons, pancreatic beta cells) versus standard cell culture models.

This integrative approach positions STK16 research within the broader context of Golgi biology and cytoskeletal regulation.

What emerging technologies could advance our understanding of STK16 function?

Several cutting-edge technologies hold promise for deeper insights into STK16 function:

• Proximity labeling approaches: BioID or APEX2 fused to STK16 can identify proximal interacting proteins in living cells, potentially revealing the complete STK16 interactome at the Golgi.

• CRISPR/Cas9 genome editing:

  • Generate clean STK16 knockout cell lines

  • Create endogenously tagged STK16 (avoiding overexpression artifacts)

  • Introduce specific phosphosite mutations at the endogenous locus

• Phosphoproteomics: Global phosphoproteomic analysis comparing wild-type, STK16-knockout, and kinase-dead STK16 cells can identify the complete set of direct and indirect STK16-dependent phosphorylation events.

• High-content screening: Automated microscopy combined with machine learning for feature extraction can quantify subtle phenotypic changes in Golgi morphology, actin organization, and cell cycle progression across large populations.

• Optogenetic STK16 activation/inhibition: Light-controllable STK16 activity would allow precise temporal control and localized activation/inhibition within specific cellular regions.

• Cryo-electron tomography: This could visualize STK16's effects on actin organization at the Golgi at nanometer resolution in a near-native state.

These technological approaches promise to reveal new dimensions of STK16 biology beyond what conventional methods have achieved.

STK16 Antibody Detection Methods and Applications

STK16 Mutant Effects on Cellular Phenotypes