Identification of kinases phosphorylating 13 sites in the nuclear, DNA-binding protein NUCKS

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Nuclear casein kinase substrate (NUCKS) is a nuclear, DNA-binding phosphoprotein.
• NUCKS is highly expressed in rapidly dividing cells and overexpressed in various cancers.
• NUCKS phosphorylation is regulated by the cell cycle and DNA damage, but responsible kinases are largely unknown.

Purpose of the Study:

• To identify the kinases responsible for NUCKS phosphorylation.
• To elucidate the role of NUCKS in cell cycle control and DNA repair.

Main Methods:

• In vitro and in vivo phosphorylation assays using isolated NUCKS and synthetic peptides.
• Mass spectrometry, phosphopeptide mapping, and phosphoamino acid analyses.
• Phosphospecific antibodies and kinase inhibitors were employed.

Main Results:

• NUCKS is phosphorylated on 11 sites by CK2, with at least 7 sites phosphorylated in vivo.
• ATM kinase and DNA-PK phosphorylate NUCKS on two sites in vitro.
• ATM kinase phosphorylates NUCKS in vivo in gamma-irradiated cells.

Conclusions:

• CK2 and ATM kinase phosphorylate NUCKS in vivo, identifying key kinases involved in its regulation.
• These findings support the involvement of NUCKS in cell cycle control and DNA repair pathways.