Analysis of E1A domains involved in the enhancement of CDK2 activity.

E1A is an adenoviral protein which is expressed at the early phase after viral infection and contains four conserved regions (CR1, CR2, CR3 and CR4). Our previous work suggests that E1A facilitates the formation of cyclin A-CDK2 complex and thereby enhances CDK2 activity. However, the molecular function of E1A in CDK2 activation has been unclear. Here, we studied the mechanism of enhancement of CDK2 activity by E1A, using the E1A variant forms which selectively contain CR domains. We isolated four E1A variant forms, i.e. 13S (containing CR1, CR2, CR3, CR4), 12S (CR1, CR2, CR4), 10S (CR2, CR4) and 9S (CR4), derived from HEK293 cells which express E1A. 13S promoted G2/M-phase arrest, upon CDK2 hyper-activation by co-expressing a stabilized cyclin A mutant, most strongly among those E1A variant forms. Concomitantly, the specific activity of the 13S-associated CDK2 was highest among them. 10S exhibited lower affinity for CDK2 than the 13S while the affinity for CDK2 was comparable between 13S and 12S. Nonetheless, 12S did not enhance the CDK2 specific activity. On the other hand, a mutation in CR2 domain, which is essential for binding to p107, suppressed both the binding and activation of CDK2. These results suggest that CR1 domain, in addition to CR2 domain via p107 interaction, is important for binding to CycA-CDK2 complex while CR3 domain facilitates CDK2 activation. Since the function of CR3 in cell cycle regulation has been relatively unknown, we propose the enhancement of CDK2 activity as a novel function of CR3 domain.

The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.