Deciphering the retinoblastoma protein phosphorylation code

Abstract

Multisite phosphorylation modulates the function of regulatory proteins with complex signaling properties and outputs. The retinoblastoma tumor suppressor protein (Rb) is inactivated by cyclin-dependent kinase (Cdk) phosphorylation in normal and cancer cell cycles, so understanding the molecular mechanisms and effects of Rb phosphorylation is imperative. Rb functions in diverse processes regulating proliferation, and it has been speculated that multisite phosphorylation might act as a code in which discrete phosphorylations control specific activities. The idea of an Rb phosphorylation code is evaluated here in light of recent studies of Rb structure and function. Rb inactivation is discussed with an emphasis on how multisite phosphorylation changes Rb structure and associations with protein partners.

Figure 1

Retinoblastoma protein (Rb) structure, phosphorylation sites, and protein interactions. (a) Domain organization and location of Cdk phosphorylation sites. The structured domains, which are colored, include the N-terminal domain (RbN) and the pocket domain. In contrast, several regions are intrinsically disordered including two large loops in RbN (RbNL) and the pocket (RbPL), an interdomain linker (RbIDL), and parts of the C-terminal domain (RbC). (b) Model of unphosphorylated Rb from crystal structures of individual domains and complexes with E2F and an LxCxE peptide (PDB codes: 2QDJ, 1GUX, 1N4M, and 2ACE). Unstructured loops and linkers connecting the structured domains are represented as broken lines. Approximate locations of phosphorylation sites are indicated. The Rb domains are colored as in (a); E2F is in pink, and the LxCxE peptide is in yellow. The C-terminal helix of RbN, which becomes disordered upon T356 phosphorylation is colored brown. (c) Model of phosphorylated Rb from crystal structures (PDB codes: 4ELJ and 4ELL). Only phosphates that are known to promote intramolecular interactions (T373, S608/S612, and T821/T826) are shown. T821/T826 induces binding of RbC to the pocket domain at the LxCxE site, although there is no high-resolution structure detailing this interaction. The N-terminal helix of the pocket domain that is nucleated by T373 phosphorylation is shown in green.

Figure 2

Specific phosphorylation events can inhibit protein interactions relevant to different Rb tumor suppressor functions. (a) S249/T252 phosphorylation inhibits EID1 binding through electrostatic repulsion. (b) T373 phosphorylation induces RbN-pocket docking and inhibits E2F^TD and LxCxE protein binding. (c) S608/S612 phosphorylation inhibits E2F^TD association by promoting RbPL binding to the pocket. (d) S788/S795 and T821/T826 phosphorylation inhibit E2F1^MB-DP^MB binding. T821/T826 promotes binding of RbC to the pocket and inhibits LxCxE protein binding.