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Updated: Jun 8, 2026
Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
Published on: March 3, 2016
Acetylation of Rb by PCAF is required for nuclear localization and keratinocyte differentiation
Adam Pickard1, Ping-Pui Wong, Dennis J McCance
1Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, BT9 7BL, UK.
Abstract:
Although the retinoblastoma protein (Rb) functions as a checkpoint in the cell cycle, it also regulates differentiation. It has recently been shown that Rb is acetylated during differentiation; however, the role of this modification has not been identified. Depletion of Rb levels with short hairpin RNA resulted in inhibition of human keratinocyte differentiation, delayed cell cycle exit and allowed cell cycle re-entry. Restoration of Rb levels rescued defects in differentiation and cell cycle exit and re-entry; however, re-expression of Rb with the major acetylation sites mutated did not. During keratinocyte differentiation, acetylation of Rb is mediated by PCAF and it is further shown that PCAF acetyltransferase activity is also required for normal differentiation. The major acetylation sites in Rb are located within the nuclear localization sequence and, although mutation did not alter Rb localization in cycling cells, the mutant is mislocalized to the cytoplasm during differentiation. Studies indicate that acetylation is a mechanism for controlling Rb localization in human keratinocytes, with either reduction of the PCAF or exogenous expression of the deacetylase SIRT1, resulting in mislocalization of Rb. These findings identify PCAF-mediated acetylation of Rb as an event required to retain Rb within the nucleus during keratinocyte differentiation.
Insights
Retinoblastoma protein (Rb) acetylation by PCAF is crucial for human keratinocyte differentiation. This modification ensures Rb nuclear retention, regulating cell cycle exit and differentiation processes.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The retinoblastoma protein (Rb) is a key regulator of cell cycle checkpoints and cellular differentiation.
- Recent studies indicate Rb undergoes acetylation during differentiation, but the functional significance of this modification remains unclear.
Purpose of the Study:
- To investigate the role of retinoblastoma protein (Rb) acetylation in human keratinocyte differentiation.
- To identify the enzymes involved in Rb acetylation and its impact on Rb localization and function.
Main Methods:
- Short hairpin RNA (shRNA) was used to deplete Rb levels.
- Rb acetylation sites were mutated to assess their functional importance.
- PCAF (p300/CBP-associated factor) and SIRT1 (Sirtuin 1) were manipulated to study their roles in Rb acetylation and localization.
Main Results:
- Rb depletion inhibited keratinocyte differentiation and delayed cell cycle exit.
- Mutating major Rb acetylation sites prevented proper differentiation and caused cytoplasmic mislocalization of Rb during differentiation.
- PCAF-mediated acetylation was essential for maintaining Rb in the nucleus during differentiation, and PCAF activity was required for normal differentiation.
Conclusions:
- PCAF-mediated acetylation of Rb is a critical event for retaining Rb in the nucleus during human keratinocyte differentiation.
- Acetylation controls Rb localization, impacting its function in regulating both cell cycle exit and differentiation.
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