Recombinant Xenopus laevis Wee1-like protein kinase 2-A (wee2-a), partial
Description
Functional Role in Cell Cycle Regulation
The recombinant Wee1-like kinase delays mitotic initiation by phosphorylating Cdc2 on Tyr-15, thereby inhibiting cyclin B-Cdc2 complex activation . Key findings include:
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Cyclin-dependent activity: Phosphorylation of Cdc2 is strictly dependent on cyclin binding .
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Cell cycle phase-specific regulation:
Table 2: Functional Assays of Recombinant Wee1 Activity
Phosphorylation
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Autophosphorylation: Occurs at Tyr-90, Tyr-103, and Tyr-110 in the NRD, enhancing Wee1 activity in vivo .
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Mitotic inactivation: Mediated by phosphorylation at multiple sites (e.g., Ser-38, Thr-329) by Cdc2 and MPM-2 kinase .
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Acetylation: K177 acetylation by GCN5 activates Wee1, reversed by SIRT1 deacetylase .
Isoform-Specific Differences
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Wee1A (maternal isoform): Expressed in oocytes and early embryos; less stable during meiosis II .
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Wee1B (zygotic isoform): Post-gastrula expression; stronger kinase activity due to a truncated CRD .
Research Implications
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Cell cycle synchronization: Recombinant Wee1 is used to study G2/M checkpoint control .
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Cancer therapeutic targeting: Structural insights into Wee1’s kinase domain inform inhibitor design .
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Developmental biology: Isoform switching (Wee1A to Wee1B) correlates with embryonic cell cycle remodeling .
Unresolved Questions
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The exact correspondence between "Wee2-A" and the Wee1A/Wee1B isoforms requires further clarification.
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Functional differences between full-length and partial recombinant constructs remain understudied.
Product Specs
Target Background
KEGG: xla:398049
UniGene: Xl.1694
Q&A
FAQs for Researchers on Recombinant Xenopus laevis Wee1-like Protein Kinase 2-A (Wee2-A)
What expression systems are optimal for producing functional recombinant Wee2-A, and how do purification methods affect kinase activity?
Recombinant Wee2-A (or Wee1B, based on nomenclature overlaps in Xenopus studies) is typically expressed in E. coli or baculovirus systems. Critical steps include:
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Tag selection: Use His-tags for affinity chromatography, but ensure tags do not interfere with the kinase domain (residues 200–595) .
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Activity preservation: Add phosphatase inhibitors (e.g., okadaic acid) during purification to maintain phosphorylation-dependent activity .
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Validation: Confirm activity via in vitro kinase assays using Cdc2–cyclin B complexes as substrates, monitoring Tyr-15 phosphorylation .
How is Wee2-A activity assayed in cell-free systems, and what controls are essential?
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Cycling Xenopus egg extracts: Add recombinant Wee2-A (10–50 nM) and monitor mitotic timing via cyclin B degradation or histone H1 kinase activity .
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Key controls:
What structural features distinguish Wee2-A from other Wee1 isoforms?
Wee2-A (likely analogous to Wee1B) has:
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N-terminal PEST sequences (residues 37–82) linked to rapid degradation during meiosis II .
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Truncated C-terminal regulatory domain (CRD), enhancing its kinase activity compared to Wee1A .
Table 1: Key Differences Between Wee1A and Wee2-A (Wee1B)
| Feature | Wee1A (Maternal) | Wee2-A/Wee1B (Zygotic) |
|---|---|---|
| Expression Timing | Pregastrula embryos | Postgastrula embryos |
| C-terminal Domain | Long CRD | Short CRD |
| Stability in M-phase | Stable | Labile (PEST sequences) |
| Kinase Activity | Moderate | Strong |
| Developmental Role | Rapid embryonic cycles | Tissue-specific regulation |
How does Wee2-A’s regulatory phosphorylation impact its role in developmental cell cycle transitions?
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Interphase activity: Wee2-A is hypophosphorylated and active, phosphorylating Cdc2 on Tyr-15 to delay mitosis .
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Mitotic inactivation: Hyperphosphorylation by Cdc2 and kinase X (e.g., Plk1) reduces activity. Dephosphorylation via PP2A restores function .
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Methodological note: Use phospho-specific antibodies (e.g., anti-pSer549) to track cell cycle-dependent phosphorylation .
What experimental approaches resolve contradictions in Wee2-A’s dose-dependent effects on mitotic delay?
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Dose-response in extracts: In Xenopus egg extracts, 10 nM Wee2-A restores endogenous activity, while >20 nM causes prolonged G2 (Figure 5 in ).
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Contradictions: Some studies report linear delays (e.g., ), while others note threshold effects. Address via:
How do Wee2-A’s interactions with 14-3-3 proteins and Chk1 regulate its subcellular localization and activity?
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14-3-3 binding: Phosphorylation at Ser-549 (via Chk1) promotes 14-3-3 association, enhancing kinase activity and nuclear retention .
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Functional assay: Compare wild-type Wee2-A vs. S549A mutant in nuclear-cytoplasmic fractionation experiments .
Table 2: Regulatory Partners of Wee2-A
Methodological Best Practices
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Handling instability: Add proteasome inhibitors (e.g., MG132) when expressing Wee2-A in oocytes or embryos .
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Kinase assays: Use γ-³²P-ATP and autoradiography to quantify Cdc2 phosphorylation, normalizing to total Cdc2 levels .
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Developmental studies: Combine microinjection of Wee2-A mRNA with time-lapse microscopy to track cell viability in early embryos .
