Abstract
The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.
Highlights
SKP2 (FBXL1) and SKP1 (S-phase kinaseassociated proteins 1 and 2) were identified as components of a pentameric cyclin-dependent kinase 2 (CDK2)-cyclin A-cyclin-dependent kinases regulatory subunit 1 (CKS1)-containing complex whose levels were found to be elevated in transformed cell lines compared to their nontumorigenic counterparts.[1]
Using purified proteins we show that a stable CDK2-cyclin ASKP1-SKP2-CKS1 complex can be formed either through CKS1 bridging CDK2 and SKP216 or through a direct interaction between the SKP2 N
As we were not able to detect an interaction between the cyclin A interacting motif (CAIM)-containing peptides and cyclin A, we cannot distinguish between models in which the interaction is dependent on the context in which the CAIM is presented or is stabilized to bind by additional interactions within the assembly thereby rigidifying the resulting SKP1-SKP2N-CDK2-cyclin A complex
Summary
SKP2 (FBXL1) and SKP1 (S-phase kinaseassociated proteins 1 and 2) were identified as components of a pentameric cyclin-dependent kinase 2 (CDK2)-cyclin A-cyclin-dependent kinases regulatory subunit 1 (CKS1)-containing complex whose levels were found to be elevated in transformed cell lines compared to their nontumorigenic counterparts.[1]. To verify the ITC results we used SEC to confirm that full-length SKP2-SKP1 does not form a complex with CDK2-cyclin Amut[5] (Figure 1 (a)).
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