Site-specific monophosphorylation of RB fine-tunes its regulation of multiple cellular activities.

  • Major finding: Site-specific monophosphorylation of RB fine-tunes its regulation of multiple cellular activities.

  • Approach: Mass spectrometry and RNA-seq of 14 monophospho-RB mutants defined the regulatory network of RB.

  • Impact: This work expands the role of RB in governing cell proliferation beyond its interaction with E2F.

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The interaction between the tumor suppressor RB and the E2F transcription factor represents an important checkpoint on cell proliferation and is disrupted at the G1/S transition via hyperphosphorylation of RB. RB has been shown to physically associate with hundreds of proteins beyond E2F family members, and monophosphorylation of RB at any of 14 sites targeted by cyclin-dependent kinases (CDK) is thought to confer specific changes to RB activity in G1 phase. Sanidas and colleagues employed quantitative proteomic and transcriptomic analysis across a panel of isogenic cell lines containing a doxycycline-inducible knockdown/replacement system in which endogenous RB was replaced with a mutant version containing only one of each of the 14 CDK phosphorylation sites (mP-RB). Although mass spectrometry profiles identified shared interactors for each mP-RB (including E2F proteins), unique interactions were identified, and hierarchical clustering analysis revealed that mutants containing phosphorylation sites in related domains grouped together: mP-RB proteins with phosphorylation sites in the C-terminal domain grouped together, whereas mutants with phosphorylation sites in the N-terminal domain grouped together with a phosphorylation-deficient RBΔcdk mutant. Specifically, RBΔcdk bound Cyclin D1 more than wild-type RB, but monophosphorylation of several residues decreased the affinity of RB for Cyclin D1–CDK6. Conversely, monophosphorylation of RB affected its association with the nuclear remodeling and deacetylase (NuRD) complex and affected NuRD-dependent transcriptional repression by RB. Gene set enrichment analysis also revealed that individual mP-RBs regulate transcriptional programs for many cellular functions such as purine and pyrimidine metabolism, nucleotide excision, WNT signaling, proliferation, differentiation, proinflammatory functions, and oxidative phosphorylation. The latter of these resulted in increased rates of cellular oxygen consumption due to greater numbers of mitochondria in the mP-RB mutants compared with both wild-type RB and RBΔcdk. Taken together, these results reveal a phosphorylation code that can activate RB in G1 phase and dictates its ability to regulate a diverse series of E2F-dependent and E2F-independent transcriptional programs.

Sanidas I, Morris R, Fella KA, Rumde PH, Boukhali M, Tai EC, et al. A code of mono-phosphorylation modulates the function of RB. Mol Cell 2019 Jan 30 [Epub ahead of print].

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