Recombinant Xenopus tropicalis Serine/threonine-protein kinase greatwall (mastl), partial

What is Greatwall kinase and what is its function in Xenopus tropicalis?

Greatwall kinase, encoded by the mastl gene (NM_001127074.1) in Xenopus tropicalis, is a serine/threonine protein kinase belonging to the AGC family of kinases. It serves as a critical regulator of mitotic entry and maintenance by indirectly inhibiting protein phosphatase 2A (PP2A) with the B55 regulatory subunit (PP2A-B55) . During mitosis, Greatwall phosphorylates downstream substrates including endosulfine (ENSA) and cAMP-regulated phosphoprotein 19 (Arpp19), which then bind to and inhibit PP2A-B55, the principal phosphatase for Cdk-phosphorylated substrates . This inhibition creates a permissive environment for Cdk1-driven mitotic phosphorylation events to accumulate, enabling proper mitotic progression.

How does Greatwall kinase differ between Xenopus laevis and Xenopus tropicalis?

While Greatwall kinase serves similar functions in both Xenopus species, studying the protein in Xenopus tropicalis offers distinct advantages. Xenopus tropicalis possesses a diploid genome (unlike the pseudotetraploid X. laevis) and has a shorter generation time, making it more suitable for genetic studies . The Xenopus tropicalis mastl gene produces a protein that maintains the core functional domains and regulatory sites found in X. laevis Greatwall, but differences in sequence may affect antibody recognition and potentially protein-protein interactions. For research requiring genetic manipulation and multi-generational studies, X. tropicalis offers significant advantages despite its smaller eggs and embryos compared to X. laevis .

What is the relationship between Greatwall kinase and the cell cycle in Xenopus?

Greatwall kinase functions within an autoregulatory loop with maturation promoting factor (MPF, composed of cyclin B and Cdk1) during mitosis . Experimental evidence demonstrates that:

• Greatwall is activated during mitosis through phosphorylation, with MPF serving as an upstream kinase

• Depletion of Greatwall from mitotic extracts rapidly reduces MPF activity due to accumulation of inhibitory phosphorylations on Cdk1

• Greatwall depletion prevents cycling extracts from entering M phase

• The effects of Greatwall depletion can be rescued by adding either wild-type Greatwall or a non-inhibitable form of Cdk1 kinase

In G2 phase oocytes, Greatwall binds to active PP2A/B55 but dissociates from it when progesterone-treated oocytes reach M phase . This spatial and temporal regulation of Greatwall is essential for proper mitotic timing and completion.

What are the recommended approaches for expressing recombinant Xenopus tropicalis Greatwall kinase?

For recombinant expression of Xenopus tropicalis Greatwall kinase, researchers should consider the following methodological approach:

• cDNA Clone Selection: Utilize expression-ready ORF clones based on the NM_001127074.1 accession . Select a vector system compatible with your expression needs (prokaryotic vs. eukaryotic expression).

• Expression Systems:

  • For biochemical characterization: Baculovirus-insect cell expression systems are preferred as they maintain proper post-translational modifications

  • For structural studies: E. coli expression systems may be used for protein domains, though full-length protein often requires eukaryotic expression

  • For cellular studies: Mammalian expression systems using CMV or other strong promoters

• Purification Strategy:

  • Incorporate affinity tags (His6, FLAG, or GST) for simplified purification

  • Include a TEV protease cleavage site to remove tags if necessary for activity assays

  • Employ size exclusion chromatography as a final purification step to ensure homogeneity

• Protein Stabilization:

  • Add phosphatase inhibitors during purification to maintain phosphorylation status

  • Consider co-expression with interacting partners for stability

  • Optimize buffer conditions (pH 7.5-8.0, with 10-15% glycerol) for long-term storage

How can I generate transgenic Xenopus tropicalis expressing modified Greatwall kinase?

Transgenic Xenopus tropicalis expressing modified Greatwall kinase can be generated using restriction enzyme mediated integration (REMI), which involves three primary steps :

• Preparation of High-Speed Egg Extracts:

  • These extracts facilitate replacement of protamines in sperm nuclei with nucleosomes

  • The extracts decondense chromatin of sperm nuclei to prepare for DNA integration

• Sperm Nuclei Isolation:

  • Isolate high-quality sperm nuclei from male X. tropicalis using established protocols

  • Maintain nuclei in sperm dilution buffer to preserve integrity

• Nuclear Transplantation:

  • Mix sperm nuclei, restriction enzyme, and high-speed extract in vitro

  • Transplant the treated nuclei into unfertilized eggs

  • This procedure generates non-mosaic transgenic embryos with high efficiency

For mastl-specific modifications, design your construct with:

• Tissue-specific or inducible promoters for controlled expression

• Fluorescent tags (C-terminal preferred to avoid disrupting kinase function)

• Point mutations of interest (e.g., phosphorylation sites or catalytic residues)

What antibody tools are available for studying Xenopus tropicalis Greatwall kinase?

Several monoclonal antibodies have been developed specifically for Xenopus Greatwall kinase that are applicable for multiple experimental techniques :

When selecting antibodies, consider:

• The specific epitope recognized (N-terminal vs. C-terminal vs. phospho-specific)

• The experimental application (Western blotting typically requires different antibodies than immunoprecipitation)

• Whether functional inhibition or simple detection is required

How does Greatwall kinase regulate the spindle assembly checkpoint in Xenopus?

Greatwall kinase plays a critical role in maintaining the spindle assembly checkpoint (SAC) through regulation of protein phosphorylation states:

• MPS1 Regulation: Mastl is required for multi-site phosphorylation of MPS1, an essential SAC kinase, as well as for robust MPS1 kinase activity in mitosis .

• PP2A/B55 Inhibition Mechanism: Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than by directly affecting Cdk1 kinase activity .

• Experimental Evidence:

  • Mastl knockout (Mastl NULL) MEFs show premature disappearance of the essential SAC protein Mad1 at kinetochores

  • The duration of mitotic arrest caused by microtubule poisons in Mastl NULL MEFs is shortened

  • Treatment with the phosphatase inhibitor okadaic acid (OKA) rescues defects in MPS1 kinase activity, mislocalization of phospho-MPS1 and Mad1 at the kinetochore, and premature SAC silencing

This suggests a model wherein Greatwall→Arpp19/ENSA→PP2A/B55 inhibition pathway is essential for maintaining SAC through regulation of MPS1 phosphorylation status.

What is the relationship between Greatwall kinase and protein phosphatase regulation during mitosis?

Greatwall kinase establishes a regulatory network with protein phosphatases that is essential for proper mitotic progression:

• PP2A/B55 Regulation: In G2 phase, Greatwall binds active PP2A/B55 but dissociates upon entry into M phase . This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase.

• Mechanism of Inhibition: Greatwall phosphorylates two small proteins, Arpp19 and ENSA, which then bind to and inhibit PP2A/B55 . This inhibition creates a dominant kinase environment required for mitotic entry and maintenance.

• Feedback Loop: The relationship forms a regulatory circuit:

  • Cdk1 activation leads to Greatwall activation

  • Activated Greatwall phosphorylates Arpp19/ENSA

  • Phosphorylated Arpp19/ENSA inhibit PP2A/B55

  • Inhibited PP2A/B55 cannot dephosphorylate Cdk1 substrates

  • This maintains high Cdk1 activity and keeps Greatwall active

• Temporal Regulation: This system ensures switch-like transitions between interphase and mitosis, preventing inappropriate partial activation of mitotic processes.

How can I study the effects of Greatwall mutations in Xenopus tropicalis?

To investigate the effects of Greatwall mutations in Xenopus tropicalis, researchers can employ several complementary approaches:

• CRISPR/Cas9 Genome Editing:

  • Design guide RNAs targeting specific regions of the mastl gene

  • Introduce donor templates for precise mutations or gene replacements

  • Screen F0 founders for germline transmission of the mutation

• Morpholino Knockdown Combined with Rescue:

  • Design morpholinos targeting endogenous mastl mRNA

  • Co-inject morpholinos with mRNA encoding mutant versions of Greatwall

  • This approach allows analysis of specific mutations in a knockdown background

• Dominant-Negative Approaches:

  • Express kinase-dead mutants (e.g., K71M, known as Scant in Drosophila)

  • Study phenotypic effects on cell cycle progression and mitotic fidelity

  • This K71M mutation is particularly interesting as it induces M phase in the absence of progesterone when expressed in oocytes, despite reduced stability

• Xenopus Egg Extract System:

  • Immunodeplete endogenous Greatwall from egg extracts

  • Add back recombinant wild-type or mutant Greatwall protein

  • Measure effects on downstream pathways and mitotic progression

For analyzing the K71M (Scant) mutation specifically, note that despite having reduced stability and elevated degradation by the proteasome, this mutant induces M phase in the absence of progesterone when expressed in oocytes .

What are the key phosphorylation sites on Xenopus tropicalis Greatwall kinase and their functions?

The activity and localization of Xenopus tropicalis Greatwall kinase is regulated by multiple phosphorylation events:

Key findings regarding phosphorylation:

• Greatwall dissociation from PP2A/B55 when progesterone-treated oocytes reach M phase does not require phosphorylation at T748 in the presumptive T loop of the kinase

• Greatwall is activated during mitosis by phosphorylation, with evidence indicating that maturation promoting factor (MPF) is an upstream kinase

• The phosphorylation status affects Greatwall's ability to phosphorylate downstream targets including endosulfine and Arpp19

How does Xenopus tropicalis Greatwall kinase compare to its orthologs in mammals and other model organisms?

Greatwall kinase shows evolutionary conservation with functional specialization across species:

The Greatwall–Arpp19–ENSA–PP2A-B55 pathway plays an essential role in the control of M and S phases from yeast to human, though with species-specific adaptations . The mammalian ortholog MASTL is required for embryonic development and proper cell division, as Mastl knockout (MastlNULL/NULL) mice die within 7–8 days while heterozygous Mastl+/NULL mice remain viable with no visible phenotypical abnormalities .

What are common issues in expressing and purifying active recombinant Xenopus tropicalis Greatwall kinase?

When working with recombinant Xenopus tropicalis Greatwall kinase, researchers commonly encounter several technical challenges:

• Protein Solubility Issues:

  • Problem: Insoluble protein expression or aggregation during purification

  • Solution: Express with solubility tags (MBP, SUMO); optimize buffer conditions with increased salt (150-300mM NaCl) and mild detergents (0.01% NP-40)

• Low Kinase Activity:

  • Problem: Purified protein shows minimal catalytic activity

  • Solution: Ensure proper phosphorylation status by co-expressing with Cdk1/Cyclin B; add phosphatase inhibitors throughout purification; confirm protein folding by circular dichroism

• Protein Degradation:

  • Problem: Rapid degradation during expression or storage

  • Solution: Include protease inhibitor cocktails; express at lower temperatures (16-18°C); store with 15% glycerol at -80°C in small aliquots

• Expression Level Variations:

  • Problem: Inconsistent yield between expression batches

  • Solution: Standardize induction conditions; optimize codon usage for expression system; consider stable cell line development for consistent expression

• Preserving Post-translational Modifications:

  • Problem: Loss of critical phosphorylation during purification

  • Solution: Include phosphatase inhibitors (PhosSTOP); perform purification steps rapidly at 4°C; validate phosphorylation status by phospho-specific antibodies or mass spectrometry

How can I design effective experiments to study Greatwall kinase interactions with PP2A/B55?

To effectively study the interactions between Greatwall kinase and PP2A/B55, consider these methodological approaches:

• Co-immunoprecipitation Studies:

  • Use antibodies against Greatwall to precipitate protein complexes from Xenopus egg extracts in different cell cycle states

  • Analyze by Western blotting for PP2A subunits (catalytic C, scaffolding A, and regulatory B55)

  • Compare interacting proteins in interphase versus mitotic extracts

• Proximity Ligation Assays in Xenopus Cells:

  • Detect protein-protein interactions in situ

  • Visualize spatial and temporal dynamics of interactions

  • Quantify interaction frequency under different conditions

• Fluorescence Resonance Energy Transfer (FRET):

  • Generate fluorescently tagged Greatwall and PP2A/B55 constructs

  • Express in Xenopus cells or inject mRNA into embryos

  • Measure interaction dynamics in real-time during cell cycle progression

• In vitro Reconstitution Assays:

  • Purify recombinant Greatwall (wild-type and mutants)

  • Isolate PP2A/B55 from Xenopus egg extracts or use recombinant components

  • Assess direct binding through pull-down assays

  • Measure effects of Arpp19/ENSA on complex formation

• Analytical Approaches:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics

  • Isothermal Titration Calorimetry (ITC) to determine binding thermodynamics

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to map binding interfaces

Evidence shows that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates when progesterone-treated oocytes reach M phase, and this dissociation does not require Greatwall kinase activity or phosphorylation at T748 .

What controls should be included when studying Greatwall kinase activity in Xenopus egg extracts?

When studying Greatwall kinase activity in Xenopus egg extracts, include these essential controls:

• Depletion Controls:

  • Mock-depleted extracts (using non-specific IgG)

  • Extracts depleted of Greatwall and rescued with recombinant wild-type protein

  • Extracts depleted of Greatwall and rescued with kinase-dead mutant

• Cell Cycle State Verification:

  • Histone H1 kinase assay to confirm MPF activity

  • Western blotting for cyclin B levels

  • Microscopic examination of chromatin morphology in extracts supplemented with sperm nuclei

• Phosphatase Inhibition Controls:

  • Addition of okadaic acid at specific concentrations to inhibit PP2A

  • Comparison of phenotypes between Greatwall depletion and phosphatase inhibition

  • Combined treatments to determine epistatic relationships

• Substrate Specificity Controls:

  • Parallel kinase assays with known Greatwall substrates (Arpp19, ENSA)

  • Assays with mutated substrate proteins lacking key phosphorylation sites

  • Competition assays with phosphomimetic substrate variants

• Temporal Controls:

  • Time-course experiments to track kinase activity through cell cycle progression

  • Synchronization controls using cell cycle inhibitors (e.g., roscovitine for Cdk inhibition)

Research has demonstrated that the addition of neutralizing antibody into M-phase extracts results in loss of mitotic phosphorylation of Greatwall, Plk1, and Cdk1 substrates , providing a useful tool for functional inhibition.