Specificity Determinants of Recruitment Peptides Bound to Phospho-CDK2/Cyclin A^,

Abstract

Progression through S phase of the eukaryotic cell cycle is regulated by the action of the cyclin dependent protein kinase 2 (CDK2) in association with cyclin A. CDK2/cyclin A phosphorylates numerous substrates. Substrate specificity often employs a dual recognition strategy in which the sequence flanking the phospho-acceptor site (Ser.Pro.X.Arg/Lys) is recognized by CDK2, while the cyclin A component of the complex contains a hydrophobic site that binds Arg/Lys.X.Leu ("RXL" or "KXL") substrate recruitment motifs. To determine additional sequence specificity motifs around the RXL sequence, we have performed X-ray crystallographic studies at 2.3 A resolution and isothermal calorimetry measurements on complexes of phospho-CDK2/cyclin A with a recruitment peptide derived from E2F1 and with shorter 11-mer peptides from p53, pRb, p27, E2F1, and p107. The results show that the cyclin recruitment site accommodates a second hydrophobic residue either immediately C-terminal or next adjacent to the leucine of the "RXL" motif and that this site makes important contributions to the recruitment peptide recognition. The arginine of the RXL motif contacts a glutamate, Glu220, on the cyclin. In those substrates that contain a KXL motif, no ionic interactions are observed with the lysine. The sequences N-terminal to the "RXL" motif of the individual peptides show no conservation, but nevertheless make common contacts to the cyclin through main chain interactions. Thus, the recruitment site is able to recognize diverse but conformationally constrained target sequences. The observations have implications for the further identification of physiological substrates of CDK2/cyclin A and the design of specific inhibitors.

Discovery of a distinct domain in cyclin A sufficient for centrosomal localization independently of Cdk binding

“…Several conserved residues are on the surface of the α1 helix in a position accessible for interactions with substrates and regulators of the cyclin A-Cdk complexes (29)(30)(31). Therefore, point mutations were generated to determine whether these residues are important for cyclin A CLS functions.…”

Diversity within the pRb pathway: is there a code of conduct?

“…The p300 transcriptional co-activator acetylates pRb on the adjacent lysine residues K873/K874 (Chan et al, 2001a), and acetylation is enhanced by the viral oncoprotein E1A, which recruits p300 and pRb into a multimeric protein complex. Acetylation of pRb impedes the phosphorylation of pRb by cyclin/Cdk complexes, which is perhaps not entirely unexpected as the site of acetylation falls within the motif believed to be required for binding Cdks (residues 868-878) (Adams et al, 1999;Lowe et al, 2002). Acetylation of pRb at K873/K874 is under cell cycle control and increased levels of acetylation occur during S phase (Chan et al, 2001a).…”

“…A structural study examining the association between pRb and PP1 revealed an enzyme docking site in the Cterminal domain of pRb that is essential for PP1 activity towards pRb (Hirschi et al, 2010). The phosphatase binding site maps to amino acid residues 870-882, which overlaps with a well-documented Cdk binding site (amino acid 868-878) (Schulman et al, 1998;Adams et al, 1999;Lowe et al, 2002). PP1 competes with Cdkcyclins for pRb binding, maintains pRb in an active growth-suppressing form and blocks cell cycle progression (Hirschi et al, 2010).…”

Modeling the Relationship between the p53 C-Terminal Domain and Its Binding Partners Using Molecular Dynamics

“…This crystal structure solved at 2.24 Å resolution contains several important elements. 30 The foundation is formed by a complex of two proteins, cyclin A and phosphorylated cyclin-dependent protein kinase 2 (pCDK2), of which there are two copies in the crystallographic asymmetric unit. A p53 CTD 9-mer fragment (residues 378-386) is bound to one of the cyclin A chains.…”