Recombinant Rat Misshapen-like kinase 1 (Mink1), partial

What is the molecular classification and function of MINK1?

MINK1 is a serine/threonine kinase and a member of the germinal center kinases family. It is known to regulate cytoskeletal organization, oncogene-induced cell senescence, and is essential for cytokinesis . MINK1 acts as a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex and plays critical roles in regulatory networks of protein kinases and phosphatases that govern cellular processes . It functions through phosphorylation of various substrates, including PRICKLE1 and LL5β, to modulate cellular responses such as cell migration and focal adhesion dynamics .

What are MINK1's primary substrate targets in cellular systems?

Through phosphoproteomic approaches and SILAC methodology, researchers have identified multiple MINK1 substrates. PRICKLE1, a component of the Wnt/planar cell polarity (PCP) pathway, is directly phosphorylated by MINK1 at threonine 370, which triggers its localization to the plasma membrane . Another important substrate is LL5β, a membrane scaffold molecule that anchors microtubules at the cell cortex. MINK1 phosphorylation of LL5β promotes its interaction with CLASP proteins, facilitating focal adhesion disassembly and cell migration . In a comprehensive phosphoproteomic analysis, researchers identified 44 peptides representing 37 proteins as potential MINK1 substrates, with differential phosphorylation levels observed when MINK1 was knocked down .

How is MINK1 involved in cellular division processes?

MINK1 plays a critical role in cytokinesis, particularly in the abscission phase. Research has shown that while MINK1-depleted cells can initiate furrow formation during cytokinesis, they fail to complete the abscission process . This leads to the formation of multinucleated cells, indicating a defect in the final separation of daughter cells. MINK1 functions within a protein complex that includes STRN4 (Zinedin), a regulatory subunit of protein phosphatase 2A (PP2A). The interplay between MINK1 kinase activity and PP2A phosphatase activity creates a regulatory network essential for proper abscission completion .

What methods are effective for studying MINK1 function in cell models?

To effectively study MINK1 function, researchers commonly employ RNA interference (siRNA or shRNA) techniques to downregulate MINK1 expression. In published studies, stable cell lines expressing shRNA against MINK1 have been generated using TNBC cell models like MDA-MB-231 cells . Additionally, pharmacological inhibition of MINK1 kinase activity using compounds such as KY05009 at 1μM concentration has proven effective in studying MINK1-dependent processes . For protein interaction studies, co-immunoprecipitation experiments with tagged versions of MINK1 and its binding partners (like PRICKLE1 and LL5β) have been successfully employed to elucidate complex formation and regulation .

How can researchers effectively measure MINK1 kinase activity?

Researchers can measure MINK1 kinase activity through several approaches:

• In vitro kinase assays: Using purified recombinant MINK1 and identified substrate proteins (such as PRICKLE1 or LL5β), kinase activity can be measured by detecting phosphorylation via:

  • Radioactive ATP incorporation

  • Phospho-specific antibodies in western blotting

  • Mass spectrometry analysis of phosphopeptides

• Cell-based assays: Monitoring phosphorylation status of endogenous MINK1 substrates in cells with and without MINK1 expression/activity:

  • Phospho-specific western blotting

  • Phosphoproteomic analysis using SILAC methodology followed by phosphopeptide enrichment

  • Functional readouts such as cellular migration assays which are affected by MINK1 activity

• Inhibitor studies: Using MINK1 inhibitors like KY05009 and measuring changes in substrate phosphorylation or cellular functions regulated by MINK1 .

What controls should be included in MINK1 knockdown experiments?

When designing MINK1 knockdown experiments, the following controls should be included:

• Non-targeting control siRNA/shRNA: Essential for distinguishing specific effects of MINK1 depletion from non-specific effects of the knockdown procedure .

• Validation of knockdown efficiency: Western blot analysis should confirm reduction of MINK1 protein levels, as demonstrated in phosphoproteomic studies where shRNA efficacy was verified before further analysis .

• Multiple siRNA/shRNA sequences: Using at least two independent targeting sequences helps confirm that observed phenotypes are due to specific MINK1 depletion rather than off-target effects .

• Rescue experiments: Expressing siRNA-resistant MINK1 constructs to restore function and confirm phenotype specificity.

• Kinase-dead mutants: Including catalytically inactive MINK1 variants helps distinguish between scaffold functions and kinase activity-dependent functions of MINK1 .

• Substrate expression controls: Verifying that total levels of MINK1 substrates (e.g., LL5β) remain unchanged upon MINK1 depletion, ensuring observed effects are due to changes in phosphorylation rather than protein expression .

How does MINK1 contribute to cancer cell migration?

MINK1 plays a critical role in cancer cell migration through a complex mechanism involving multiple substrates and interactions:

• PRICKLE1 phosphorylation: MINK1 phosphorylates PRICKLE1 at threonine 370, which promotes its localization to the plasma membrane at the leading edge of migratory cells . This localization is essential for proper cell migration.

• LL5β phosphorylation: At the membrane, the MINK1-PRICKLE1 complex associates with and phosphorylates LL5β. This phosphorylation enhances LL5β's interaction with CLASP proteins, which are involved in microtubule stabilization and tethering to focal adhesions .

• Focal adhesion dynamics: The MINK1-PRICKLE1-LL5β-CLASP complex promotes focal adhesion disassembly by facilitating local delivery of exocytotic proteins and extracellular matrix degradation. This releases integrin-matrix connections and promotes integrin internalization, a process critical for cell migration .

• Cytoskeletal reorganization: MINK1 inhibition (either by knockdown or kinase inhibition) leads to profound changes in cellular morphology and actin cytoskeleton organization, demonstrating its role in maintaining proper cytoskeletal dynamics required for migration .

Experimental data shows that inhibition of MINK1 catalytic activity with KY05009 significantly decreases both the cumulative distance and Euclidean distance traveled by TNBC cells, as well as reducing cell speed .

What is the relationship between MINK1 and the Wnt/PCP pathway in cancer?

The relationship between MINK1 and the Wnt/planar cell polarity (PCP) pathway in cancer, particularly in triple-negative breast cancer (TNBC), involves several key interactions:

• Wnt/PCP pathway upregulation: The Wnt/PCP pathway is upregulated in many cancers and is associated with cancer development at both early and late stages .

• PRICKLE1 and VANGL2 overexpression: These core Wnt/PCP components are overexpressed in TNBC and associated with poor prognosis .

• MINK1-PRICKLE1 phosphorylation axis: MINK1 phosphorylates PRICKLE1, triggering its localization to the plasma membrane, which is a key step for its function in the Wnt/PCP pathway .

• Prometastatic activity: Knockdown experiments have demonstrated that both MINK1 and PRICKLE1 contribute to TNBC cell motility and spreading both in vitro and in vivo .

• Prognostic significance: Analysis of gene expression data shows that concomitant upregulation of PRICKLE1 and LL5β (both MINK1 substrates) is associated with poor metastasis-free survival for TNBC patients .

This evidence suggests that MINK1 acts as a key regulator of the Wnt/PCP pathway in cancer, controlling cell migration and potentially metastasis through its kinase activity on specific substrates within this pathway.

What experimental models are most appropriate for studying MINK1 in cancer?

Based on the search results, the following experimental models have proven effective for studying MINK1 in cancer:

• Cell line models:

  • MDA-MB-231 TNBC cells: Used extensively for MINK1 knockdown studies, phosphoproteomic analysis, and migration assays

  • SUM149PT TNBC cells: Also shown to respond to MINK1 manipulation with similar phenotypes to MDA-MB-231

• Genetic manipulation approaches:

  • Stable shRNA expression: Generation of cell lines with stable MINK1 knockdown for long-term studies

  • siRNA transfection: For transient knockdown experiments

  • Tagged protein expression: GFP-PRICKLE1, FLAG-PRICKLE1, etc., for protein interaction studies

• Pharmacological inhibition:

  • KY05009 at 1μM concentration has been used to inhibit MINK1 catalytic activity

• Functional assays:

  • Single cell motility tracking: Measuring cumulative distance, Euclidean distance, speed, and directionality of cell movement

  • Wound healing assays: For studying collective cell migration

  • Immunofluorescence and confocal analysis: For examining subcellular localization of MINK1 and its interacting partners

• Animal models:

  • MINK1 knockout mice have been reported to be viable, suggesting potential for in vivo studies of MINK1 inhibition in cancer models

These models provide complementary approaches to understand MINK1's roles in cancer progression and evaluate its potential as a therapeutic target.

How does the STRIPAK complex regulation affect MINK1 function?

The STRIPAK (striatin-interacting phosphatase and kinase) complex plays a crucial role in regulating MINK1 function. Mass spectrometry analysis has shown that MINK1 is a component of the STRIPAK complex, where it directly interacts with STRN4 (Zinedin), a regulatory subunit of protein phosphatase 2A (PP2A) . This interaction creates a regulatory circuit that modulates MINK1 activity:

• Reciprocal regulation: Within the STRIPAK complex, STRN4 can reduce MINK1 activity in the presence of catalytic and structural subunits of PP2A . This suggests that PP2A-mediated dephosphorylation may counterbalance MINK1 kinase activity.

• Functional consequences: Similar to MINK1 depletion, STRN4 knockdown induces multinucleated cells and inhibits the completion of abscission during cytokinesis . This parallel phenotype suggests that the proper balance between kinase and phosphatase activities within the STRIPAK complex is essential for normal cellular function.

• Regulatory network: The STRIPAK complex creates a novel regulatory network of protein kinases and phosphatases that coordinate complex cellular processes such as cytokinesis .

Understanding the dynamics of the STRIPAK complex is crucial for researchers working with MINK1, as this complex likely influences experimental outcomes when studying MINK1 function in isolation.

What are the current limitations in MINK1 inhibitor development?

While the search results don't directly address all limitations in MINK1 inhibitor development, several challenges and considerations can be inferred:

• Limited specific inhibitors: KY05009 has been used to inhibit MINK1 activity in research settings , but there appears to be a need for more specific MINK1 inhibitors, as the text mentions "a finding that should stimulate the future development of specific MINK1 inhibitors" .

• Selectivity challenges: MINK1 belongs to the germinal center kinases family , which includes other kinases with similar structures. Developing inhibitors that selectively target MINK1 without affecting related kinases likely presents a significant challenge.

• Validation approaches: Researchers need robust methods to validate inhibitor specificity and efficacy. Current approaches include:

  • Monitoring phosphorylation status of known MINK1 substrates like PRICKLE1 and LL5β

  • Examining cellular phenotypes such as migration, focal adhesion dynamics, and protein complex formation

  • Comparing inhibitor effects with genetic knockdown of MINK1

• Physiological relevance: Understanding whether MINK1 inhibition in animal models recapitulates cellular phenotypes is important. The search results mention that MINK1 knockout mice are viable , suggesting potential for therapeutic targeting, but comprehensive in vivo studies may be lacking.

• Target validation: While MINK1 inhibition shows promise in TNBC cell models , additional validation across more cancer types and in vivo models would strengthen the case for MINK1 as a therapeutic target.

How can phosphoproteomic approaches enhance our understanding of MINK1 signaling networks?

Phosphoproteomic approaches have proven valuable for elucidating MINK1 signaling networks, as demonstrated in the search results:

• Substrate identification: Using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) methodology followed by phosphopeptide enrichment and identification, researchers identified 1,323 phosphopeptides differentially expressed between control and MINK1-knockdown cells . This approach revealed 44 peptides (representing 37 proteins) showing significant changes in phosphorylation status (log2 ratio difference above 1.5), indicating potential MINK1 substrates .

• Validation strategies: The phosphoproteomic approach allows for initial large-scale screening, which can then be validated through:

  • Western blot analysis to confirm protein expression levels remain unchanged while phosphorylation status changes

  • In vitro kinase assays to confirm direct phosphorylation by MINK1

  • Functional studies to assess the biological relevance of identified phosphorylation events

• Network analysis: Cross-analyzing phosphoproteomic datasets with protein interaction data (e.g., proteins associated with the MINK1-PRICKLE1 complex) enables identification of proteins that are both interactors and substrates of MINK1 . This integrated approach led to the identification of LL5β as a key MINK1 substrate within its signaling network.

• Pathway mapping: Phosphoproteomic data can reveal connections between MINK1 and established signaling pathways. For example, this approach helped establish MINK1's role in the Wnt/PCP pathway through its phosphorylation of PRICKLE1 and subsequent effects on downstream signaling events .

• Quantitative analysis: SILAC-based phosphoproteomics provides quantitative data on phosphorylation changes, allowing researchers to prioritize the most significantly affected sites for further investigation.

What are the critical quality control parameters for recombinant rat MINK1?

While the search results don't specifically address quality control for recombinant rat MINK1, based on standard practices for recombinant kinases and the information provided, the following quality control parameters would be critical:

• Purity assessment:

  • SDS-PAGE analysis to verify size and purity

  • Mass spectrometry to confirm protein identity and detect potential modifications

• Activity verification:

  • Kinase activity assays using known substrates like PRICKLE1 or LL5β

  • Phospho-specific antibody detection of substrate phosphorylation

  • ATP consumption assays

• Structural integrity:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Thermal shift assays to evaluate protein stability

  • Size exclusion chromatography to detect aggregation

• Post-translational modification status:

  • Phosphorylation status analysis, as MINK1 activity may be regulated by its own phosphorylation state

  • Mass spectrometry to map and quantify phosphorylation sites

• Functional validation:

  • Inhibitor sensitivity testing (e.g., response to KY05009)

  • Binding assays with known interactors like STRN4 or PRICKLE1

  • Confirmation that recombinant MINK1 can phosphorylate validated substrates in vitro

• Batch-to-batch consistency:

  • Comparative activity assays between batches

  • Consistent specific activity measurement

These parameters would ensure that recombinant rat MINK1 is suitable for research applications and provides reliable results in experimental settings.

How do truncated or partial MINK1 constructs differ functionally from the full-length protein?

• Domain-specific functions: MINK1, as a member of the germinal center kinases family, likely contains multiple functional domains beyond its kinase domain . A partial MINK1 construct might lack:

  • Regulatory domains that control kinase activity

  • Protein-protein interaction domains necessary for complex formation with partners like STRN4 or PRICKLE1

  • Localization signals that direct MINK1 to specific subcellular compartments

• Substrate specificity: Truncated versions of MINK1 may exhibit altered substrate specificity compared to the full-length protein. While the kinase domain alone might retain catalytic activity toward some substrates, the specificity and efficiency of phosphorylation could be compromised without additional substrate recognition elements found in the full-length protein.

• Regulatory mechanisms: Full-length MINK1 is subject to regulation by the STRIPAK complex through its interaction with STRN4 and PP2A . Partial constructs might escape this regulation, potentially exhibiting constitutive activity or altered responsiveness to cellular signals.

• Complex formation: MINK1 functions within multi-protein complexes, including the STRIPAK complex and the PRICKLE1-LL5β-CLASP complex . Truncated versions may fail to form these complexes properly, affecting downstream signaling events.

When working with partial MINK1 constructs, researchers should carefully consider these potential functional differences and validate that the construct retains the specific activities relevant to their research questions.

What are the most promising therapeutic applications of MINK1 inhibition?

Based on the search results, MINK1 inhibition shows particular promise for therapeutic applications in cancer, especially triple-negative breast cancer (TNBC):

• Anti-metastatic potential: MINK1 inhibition (both via knockdown and pharmacological approaches with KY05009) significantly reduces TNBC cell migration in vitro . Since metastasis is a major cause of cancer-related mortality, targeting MINK1 could potentially reduce cancer spread.

• Wnt/PCP pathway modulation: MINK1 is involved in the Wnt/PCP pathway, which is upregulated in many cancers and associated with cancer development at both early and late stages . Inhibiting MINK1 could provide a strategy to target this pathway in cancers where it is dysregulated.

• Potential for selective targeting: The viability of MINK1 knockout mice suggests that systemic MINK1 inhibition might be tolerated, potentially offering a therapeutic window for cancer treatment with manageable side effects.

• Combination therapy potential: Given MINK1's role in promoting cell migration through complex mechanisms involving focal adhesion dynamics , combining MINK1 inhibitors with other anti-cancer therapies targeting complementary pathways could enhance therapeutic efficacy.

• Biomarker-guided treatment: The finding that concomitant upregulation of MINK1 substrates (PRICKLE1 and LL5β) correlates with poor metastasis-free survival in TNBC patients suggests potential for biomarker-guided treatment strategies, where patients with high expression of these markers might particularly benefit from MINK1 inhibition.

The search results specifically highlight TNBC as a cancer type where MINK1 inhibition shows promise, stating that "our results suggest that MINK1 may represent a potential target in TNBC" .

How might emerging technologies advance our understanding of MINK1 biology?

While the search results don't explicitly discuss emerging technologies for MINK1 research, we can identify several cutting-edge approaches that could significantly advance understanding of MINK1 biology:

• CRISPR-Cas9 genome editing:

  • Generation of precise MINK1 mutants to study specific phosphorylation sites or domains

  • Creation of endogenously tagged MINK1 to study localization and dynamics without overexpression artifacts

  • Screening for synthetic lethal interactions to identify context-dependent vulnerabilities

• Advanced imaging technologies:

  • Live-cell super-resolution microscopy to visualize MINK1-containing complexes at the plasma membrane

  • FRET-based biosensors to monitor MINK1 activity in real-time in living cells

  • Lattice light-sheet microscopy to track MINK1 dynamics during processes like cell migration and cytokinesis

• Proteomics and interactomics:

  • Proximity labeling approaches (BioID, APEX) to identify context-specific MINK1 interaction partners

  • Thermal proteome profiling to identify proteins stabilized by MINK1 interactions

  • Advanced phosphoproteomics with enrichment strategies to capture low-abundance MINK1 substrates

• Structural biology approaches:

  • Cryo-EM to resolve structures of MINK1 within larger complexes like STRIPAK

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon activation or inhibition

  • AlphaFold2 and other AI-based structure prediction to model MINK1 interactions with substrates and partners

• Single-cell technologies:

  • Single-cell phosphoproteomics to capture cell-to-cell variation in MINK1 signaling

  • Single-cell RNA-seq to identify transcriptional consequences of MINK1 activity

  • Multiomics approaches integrating genetic, proteomic, and phenotypic data

These technologies could help resolve current knowledge gaps about MINK1, such as the full spectrum of its substrates, the structural basis of its interactions, and its context-dependent roles in different tissues and disease states.

What are the most significant unanswered questions in MINK1 research?

Based on the search results, several significant unanswered questions remain in MINK1 research: