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Laboratory of Molecular Biophysics
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Previous: Tyr15pThr160pCDK2-cyclinA complex, Next: p13suc1 and CDK2/cyclin A/HsCks1 complexes, Up: Cell Cycle Proteins., Return to: Contents.
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Human SKP1 and SKP2 were identified as components of a stable SKP1/SKP2/CDK2/cyclin A/Cks (cyclin-dependent kinase subunit) complex that was present in elevated levels in human transformed cell lines compared to their non-transformed counterparts [7]. To generate the quarternary human SKP1/SKP2/CDK2/cyclin A complex, we have developed an E. coli-based co-expression strategy to yield a stable complex between SKP2, residues 1-153 (GST-SKP2(1-153)) and full-length SKP1 (Yield 30mg/L cells). This complex includes the SKP2 sequence N-terminal to the F-box (residues 112-153) that is required for association with CDK2/cyclin A. A complex of CDK2 phosphorylated on Thr160 with cyclin A3 (a construct that encodes residues 173-432) (pCDK2/cyclin A3) suitable for crystallisation trials is purified from E. coli cells as previously described [5]. Purified SKP1/SKP2(1-153) and pCDK2/cyclin A3 are mixed in equimolar amounts on ice and then the complex is purified by gel filtration (GF) (Figure 3a, run A). Conditions have been identified under which the SKP1/SKP2(1-153) complex forms quasicrystals. We are currently refining these conditions to grow crystals suitable for X-ray analysis. Crystallisation trials on the SKP1/SKP2(1-153)/CDK2/cyclin A complex have also identified promising leads.
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An initial goal is to characterise the interaction between SKP2 and CDK2/cyclin A. Certain CDK substrates and cyclin-dependent kinase inhibitors (CKIs) of the Cip/Kip1 class stably associate with CDK2/cyclin A through a cyclin-binding RXL motif (single-letter amino acid code) [5]. pRB (the product of the retinoblastoma protein) is an RXL-dependent CDK2/cyclin A substrate and can be phosphorylated by SKP1/SKP2/CDK2/cyclin A. This result suggests that the interaction of cyclin A and/or CDK2 with SKP2 may not involve RXL binding, and may be a novel interaction the nature of which might be illuminated by structural analysis. Short peptides containing an RXL motif efficiently compete with RXL-dependent pCDK2/cyclin A3 substrates in kinase assays ([5], Figure 4). A complex of SKP1/SKP2(1-153)/CDK2/cyclin A3 phosphorylates SKP2 (Figure 4). Unlike CDK2/cyclin A3 phosphorylation of GST-pRB, the activity is not inhibited by excess p107c peptide (2mM) containing an RXL motif ([5], Figure 4). In the absence of ATP, GST-pRB forms a quinternary complex with SKP1/SKP2(1-153)CDK2/cyclin A. Addition of ATP results in GST-pRB and SKP2 phosphorylation and release of GST-pRB but not SKP2 from the complex.
Previous: Tyr15pThr160pCDK2-cyclinA complex, Next: p13suc1 and CDK2/cyclin A/HsCks1 complexes, Up: Cell Cycle Proteins., Return to: Contents.