Docking-Dependent Regulation of the Rb Tumor Suppressor Protein by Cdk4

Abstract

Phosphorylation of target proteins by cyclin D1-Cdk4 requires both substrate docking and kinase activity. In addition to the ability of cyclin D1-Cdk4 to catalyze the phosphorylation of consensus sites within the primary amino acid sequence of a substrate, maximum catalytic activity requires the enzyme complex to anchor at a site remote from the phospho-acceptor site. A novel Cdk4 docking motif has been defined within a stretch of 19 amino acids from the C-terminal domain of the Rb protein that are essential for Cdk4 binding. Mutation or deletion of the docking motif prevents Cdk4-dependent phosphorylation of full-length Rb protein or C-terminal Rb fragments in vitro and in cells, while a peptide encompassing the Cdk4 docking motif specifically inhibits Cdk4-dependent phosphorylation of Rb. Cyclin D1-Cdk4 can overcome the growth-suppressive activity of Rb in both cell cycle progression and colony formation assays; however, while mutants of Rb in which the Cdk4 docking site has been either deleted or mutated retain growth suppressor activity, they are resistant to inactivation by cyclin D1-Cdk4. Finally, binding of Cdk4 to its docking site can inhibit cleavage of exogenous and endogenous Rb in response to distinct apoptotic signals. The Cdk4 docking motif in Rb gives insight into the mechanism by which enzyme specificity is ensured and highlights a role for Cdk4 docking in maintaining the Rb protein in a form that favors cell survival rather than apoptosis.The mechanisms employed by the protein kinase family of enzymes to ensure substrate and phospho-acceptor site specificity are beginning to receive increased interest as it becomes apparent that, in general, the information contained within the consensus phosphorylation motif is not sufficient to explain the fidelity of a given kinase in vivo. The broad consensus motif at the phosphorylation site targeted by the cyclin-cyclin-dependent kinases (cyclin-CDKs) is T/SPXR/K (23, 52); however, in order to gain specificity, these enzymes participate in a direct interaction with their substrate at a site distinct from the phospho-acceptor site (1, 2, 35, 48). In addition, at least some cyclin-CDKs are able to phosphorylate a given substrate through an interaction with an associated binding protein. For example, E2F1 can function as a targeting factor for cyclin A-Cdk2-dependent phosphorylation of its heterodimeric partner DP1 (14,30,58). Thus, the cyclin-CDKs belong to a growing class of kinases that require a specific docking interaction for efficient site-specific phosphorylation of a substrate (5).To date, the G 1 -associated kinase cyclin D-Cdk4 has been shown to phosphorylate only a limited subset of possible CDK target proteins (20, 28), suggesting that it has a strict requirement for determinants outside the phosphorylation motif and that these determinants are distinct from those required by other family members. There is good evidence that as well as providing increased specificity, cyclin D-Cdk4 substrate docking is required for the enzyme's maximal catalytic ...

p27Kip1 Inhibits Cyclin D-Cyclin-Dependent Kinase 4 by Two Independent Modes

“…This may be more applicable to cdk2 complexes, because cyclins A and E have better-defined p27 and substrate binding domains. Cyclin D-cdk4 might associate with Rb by a different mode, independent of the cyclin-binding domain (39), but p27 binding to cyclin D-cdk4 has not been fully defined due to the lack of structural information about the ternary complex. Yphosphorylated p27, which "opens" the active site, might permit phosphorylation of a cdk2 substrate that does not depend on the cyclin targeting domain or of a prebound substrate, such as p27 itself.…”

Crystal structure of human CDK4 in complex with a D-type cyclin

“…Unlike CDK2/cyclin A (34), CDK4 does not appear to spontaneously activate when bound to cyclin D and exhibits ordered addition kinetics (35,36). Phosphorylation studies using peptidic or Rb-protein substrates reveal a low specific activity of CDK4/cyclin D1 versus peptides and a modulation of the enzymatic K m for ATP (36)(37)(38)(39). On the basis of these data and our structure it can be speculated that protein substrate and/or cofactor binding may be necessary to promote remodeling of the CDK4/cyclin D1 interface and ␣C-helix into active conformations.…”

Suppression of Activation of Signal Transducer and Activator of Transcription-5B Signaling in the Vessel Wall Reduces Balloon Injury-Induced Neointima Formation

“…CDK4 phosphorylates and inactivates Rb protein. 37 The phosphorylated Rb protein, in turn, dissociates from E2Fs and thereby facilitates the activation of the latter transcriptional factors, which in their turn, influence the transcription of genes required for DNA synthesis. 38,39 Therefore, to test whether PDGF-BB-induced CDK4 activity correlates with increased phosphorylation of its substrate(s), we measured Rb protein phosphorylation.…”

“…35 Cyclin D1 association is required for CDK4 activity, 37,38 and CDK4, in turn, phosphorylates Rb protein. 39 The phosphorylated Rb protein dissociates from and thereby facilitates activation of transcriptional factors, E2Fs, which in their turn influence the expression of genes required for cell-cycle progression.…”