The non-canonical CDK2 activator SPY1 uses separable mechanisms to activate phosphorylated and unphosphorylated CDK2.

The cyclin-dependent kinases (CDKs) regulate cell cycle progression subject to strict control by activating cyclin subunits and by site-specific phosphorylation on the activation loop. These mechanisms are highly conserved across eukaryotic organisms. The non-canonical CDK activator Speedy A (SPY1) is unrelated in sequence to the cyclins and activates CDK2 in G1/S phase in somatic cells and promotes meiosis in germ cells without requiring activation-loop phosphorylation of CDK2 on T160. This has been attributed to a single aspartic acid residue of SPY1 (D136) that mimics T160 phosphorylation by forming similar salt-bridging interactions that stabilize the active conformation of the SPY1-CDK2 complex. Here, we show that SPY1 activates both phosphorylated and unphosphorylated CDK2 with similar efficacy to its canonical activator CyclinA. FRET experiments show that SPY1 binding causes a similar magnitude of CDK2 conformational shift to that triggered by CyclinA binding to phosphorylated CDK2, but regardless of the kinase phosphorylation state. SPY1 binding allosterically enhances binding of ATP-competitive CDK2 inhibitors to a similar degree as CyclinA, resulting in similarly high-affinity inhibitor binding and inhibition. Remarkably, we found that the D136A mutant of SPY1, while inactive towards unphosphorylated CDK2, can still activate phosphorylated CDK2 as effectively as WT SPY1, demonstrating functional non-equivalence of its two activation mechanisms.