# Deciphering the code of RB phosphorylation

> **NIH NIH R01** · MASSACHUSETTS GENERAL HOSPITAL · 2020 · $503,198

## Abstract

Project summary
For simplicity we tend to imagine that most proteins have a single mechanism of action. The retinoblastoma
tumor suppressor is an example of a protein that defies this simple classification. RB1 is functionally
inactivated in most human cancers and the molecular properties of its protein product (RB) have been studied
intensively. Despite this research, RB's mechanism of action has remained an enigma. RB has been reported
to physically associate with hundreds of proteins and many different interactions have been proposed to
contribute to its tumor suppressive properties. The RB research community is faced with a conundrum: which
of these interactions are real, which are not, and how could one protein co-ordinate its effects on so many
potential targets? Recent studies from several laboratories have suggested that the answers to these
questions lie in a code of RB phosphorylation. The concept is that normal cells do not contain a single form of
RB, but that differential phosphorylation generates multiple isoforms of RB that have different binding
properties and, presumably, perform different roles. In essence, the action of RB is tailored by phosphorylation.
RB is known to have 14 sites of CDK phosphorylation. We have recently developed methods that allow us to
use mass spectrometry-based proteomics to profile RB complexes. We have also generated panels of isogenic
cell cultures in which we can replace the endogenous RB protein with mutant RB proteins that contain just a
single cdk phosphorylation site, or a single phospho-mimicking mutation. In this application we propose to use
these tools to decipher this code of RB phosphorylation. In Aim 1 we will use state-of-the-art proteomics to
define the binding properties of each of the mono-phosphorylated isoforms of RB. In Aim 2 we will identify the
functional consequences of these interactions by identifying the transcriptional programs that they control and
by genomic loci that they target. Using this binding information and transcription profiles we will identify the
molecular interactions that allow specific mono-phosphorylated isoforms of RB to control distinct programs of
transcription. Together these experiments will generate a framework of molecular information that is critical to
be able to understand RB's mechanism of action.

## Key facts

- **NIH application ID:** 9957058
- **Project number:** 5R01CA236538-03
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** NICHOLAS J DYSON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $503,198
- **Award type:** 5
- **Project period:** 2018-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9957058

## Citation

> US National Institutes of Health, RePORTER application 9957058, Deciphering the code of RB phosphorylation (5R01CA236538-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9957058. Licensed CC0.

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