Abstract
p27 regulates the activity of Cdk complexes which are the principal governors of phase transitions during cell division. Members of the p27 family of proteins, which also includes p21 and p57, are called the Cip/Kip cyclin-dependent kinase regulators (CKRs). Interestingly, the Cip/Kip CKRs play critical roles in cell cycle regulation by being intrinsically unstructured, a characteristic contrary to the classical structure-function paradigm. They exhibit nascent helicity which has been localized to a segment referred to as sub-domain LH. The nascent helicity of this sub-domain is conserved and we hypothesize that it is an important determinant of their functional properties. To test this hypothesis, we successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/Cyclin A were less stable compared to the wild-type complex. Isothermal titration calorimetry experiments showed a decrease in the enthalpy of binding for all the mutants with respect to p27. The free energies of binding varied within a much narrower range. In vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Furthermore, when over-expressed in NIH 3T3 mouse fibroblast cells, the less helical p27 variants were less effective in causing cell cycle arrest relative to the wild-type p27. Our results indicate that the nascent helicity of sub-domain LH plays a key role mediating the biological function of p27.
Highlights
Strict temporal control of cell cycle phase transitions is governed primarily by a family of highly conserved serine/threonine kinases termed the Cyclin-dependent kinases (Cdks) [1,2]
We hypothesized that a p27-kinase inhibitory domain (KID) construct with enhanced helical character within the LH subdomain would exhibit a reduced entropic penalty and experience increased affinity for Cdk2/cyclin A
Our results indicated that factors in addition to entropy came into play when p27-KID and the p27-KID variants bound to Cdk2/cyclin A
Summary
Strict temporal control of cell cycle phase transitions is governed primarily by a family of highly conserved serine/threonine kinases termed the Cyclin-dependent kinases (Cdks) [1,2]. Given their cardinal role in controlling faithful duplication of cells, Cdk activity is tightly regulated through post-translational modifications and direct protein-protein interactions. Cdks require the binding of a regulatory subunit referred to as a cyclin (forming Cdk/cyclin complexes), [3,4,5] and phosphorylation on a conserved threonine by a Cdk activating kinase (CAK) [6,7,8] for complete activation. Recent studies from our lab have uncovered a mechanism involving tyrosine phosphorylation of p21 and p27 that explains the paradoxical outcome of their interaction with Cdk4/cyclin D and Cdk6/cyclin D [23,24,25]
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