Posttranslational modifications of the retinoblastoma tumor suppressor protein as determinants of function

Abstract

The retinoblastoma tumor suppressor protein (pRB) plays an integral role in G1-S checkpoint control and consequently is a frequent target for inactivation in cancer. The RB protein can function as an adaptor, nucleating components such as E2Fs and chromatin regulating enzymes into the same complex. For this reason, pRB's regulation by posttranslational modifications is thought to be critical. pRB is phosphorylated by a number of different kinases such as cyclin dependent kinases (Cdks), p38 MAP kinase, Chk1/2, Abl, and Aurora b. Although phosphorylation of pRB by Cdks has been extensively studied, activities regulated through phosphorylation by other kinases are just starting to be understood. As well as being phosphorylated, pRB is acetylated, methylated, ubiquitylated, and SUMOylated. Acetylation, methylation, and SUMOylation play roles in pRB mediated gene silencing. Ubiquitinylation of pRB promotes its degradation and may be used to regulate apoptosis. Recent proteomic data have revealed that pRB is posttranslationally modified to a much greater extent than previously thought. This new information suggests that many unknown pathways affect pRB regulation. This review focuses on posttranslational modifications of pRB and how they influence its function. The final part of the review summarizes new phosphorylation sites from accumulated proteomic data and discusses the possibilities that might arise from this data.

Keywords: acetylation; cell cycle; methylation; phosphorylation; proteomics.

Figure 1.

A model for cell cycle regulation by pRB. In G₁, pRB is mostly dephosphorylated (hypophosphorylated) and binds to E2F/DP1 heterodimers to repress transcription. As part of the repressor complex, pRB also recruits chromatin modifying proteins that contribute in the repression of E2F dependent transcription. As the cell progresses through G₁, hyperphosphorylation of pRB (ppRB) by cyclin D/Cdk4/6 and cyclin E/Cdk2 compromises the integrity of the repressor complex, resulting in the dissociation of E2F and allowing for the transcription of S-phase genes.

Figure 2.

Schematic representation of the Cdk phosphorylation sites in pRB. Position of the consensus Cdk phosphorylation sites in relation to the pRB protein is indicated. Phosphorylation of S230 has not been confirmed by either site directed methods or mass spectrometry in human pRB, but phosphorylation of the corresponding serine in mouse pRb (S224) has been observed by site directed methods (see text). Unless specified, amino acid numbering throughout the text will refer to human pRB. The A and B domains of the small pocket and large pocket and the carboxyl-terminus are indicated.

Figure 3.

Identified tyrosine phosphorylation sites in pRB. Sites of tyrosine phosphorylation in pRB identified by site specific methods or mass spectrometry are indicated. Tyrosine phosphorylation at Y651/Y659 has yet to be observed in human pRB, but phosphorylation of the corresponding tyrosines (Y644/Y652) in murine pRb has been identified by mass spectrometry (see text). The site at Y805 in pRB is a target of the c-Abl tyrosine kinase.

Figure 4.

Nonphosphorylation posttranslational modifications of pRB. Sites of acetylation, methylation, ubiquitinylation, and SUMOylation in pRB identified by a combination of site specific methods and mass spectrometry are indicated. *Ubiquitinylation of the corresponding lysine in mouse pRb (K57) has been identified by mass spectrometry (see text). Ubiquitinylation of this site in human pRB has yet to be observed.

Figure 5.

Non-Cdk targeted serine/threonine phosphorylation sites on pRB identified in proteomic studies. Information regarding the sites of phosphorylation was obtained from the literature and from the PhosphoSite database (www.phosphosite.org).