A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The retinoblastoma protein (Rb) is regulated by phosphorylation during the cell cycle.
• Cyclin D:Cdk4/6 kinases play a role in early G1 phase, but their precise function in Rb inactivation and cell cycle entry remains unclear.
• Mono-phosphorylated Rb is generated by cyclin D:Cdk4/6, but its dispensability for early G1 progression raises questions about cyclin D:Cdk4/6's role.

Purpose of the Study:

• To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry.
• To identify novel substrates of cyclin D:Cdk4/6 beyond Rb.
• To understand the connection between early G1 phase events and subsequent cell cycle activation.

Main Methods:

• CRISPR/Cas9 gene editing to tag endogenous p27 with mCherry for a single-cell reporter.
• Single-cell tracing of cells with inhibited Cdk4/6 (Cdk4i).
• Whole proteomic and phospho-proteomic mass spectrometry to identify protein and phospho-peptide changes.

Main Results:

• A time-sensitive, early G1 phase-specific Cdk4/6-dependent phosphorylation gradient regulating cell cycle entry timing was identified.
• This gradient acts between serum sensing and cyclin E:Cdk2 activation.
• Proteomic analysis revealed 147 proteins and 82 phospho-peptides significantly altered by Cdk4 inhibition, including novel non-Rb substrates.

Conclusions:

• Cyclin D:Cdk4/6 establishes a phosphorylation gradient in early G1 phase that is critical for timely cell cycle entry.
• Novel non-Rb substrates of cyclin D:Cdk4/6 were identified, expanding our understanding of its regulatory network.
• These findings link early G1 phase functions of cyclin D:Cdk4/6 to cyclin E:Cdk2 activation and Rb hyper-phosphorylation.