HEY1 functions are regulated by its phosphorylation at Ser-68

Area of Science:

• Cellular Biology
• Molecular Biology
• Signal Transduction

Background:

• HEY1 (hairy/enhancer-of-split related with YRPW motif 1) is a transcription repressor involved in Notch signaling.
• HEY1 positively regulates the tumor suppressor p53 through mechanisms not fully understood.
• A novel regulatory phosphorylation site on HEY1 was investigated.

Purpose of the Study:

• To identify and characterize a novel regulatory phosphorylation event on HEY1.
• To elucidate the role of HEY1 phosphorylation in regulating its stability, function, and interaction with p53.
• To explore the connection between HEY1, ribosomal stress, and p53 activation.

Main Methods:

• Mass spectrometry (MALDI-TOF/TOF MS) to identify phosphorylation sites.
• Site-directed mutagenesis to create phosphomimetic mutants (HEY1-S68D).
• Co-immunoprecipitation and Western blotting to study protein interactions and stability.
• Cell cycle analysis and drug sensitivity assays in U2OS cells.

Main Results:

• A novel regulatory phosphorylation event was identified at Ser-68 of HEY1.
• Phosphorylation at Ser-68 enhances HEY1 protein stability but inhibits its ability to enhance p53 transcriptional activity.
• STK38 and STK38L were identified as kinases that phosphorylate HEY1 at Ser-68.
• Phosphorylated HEY1 failed to induce p53-dependent cell cycle arrest and did not sensitize cells to chemotherapeutic drugs.
• HEY1 interacts with ribosomal protein RPL11 and cooperates in inhibiting MDM2-mediated p53 degradation.
• Ribosomal stress induces HEY1 relocalization to nucleolar caps, an event prevented by Ser-68 phosphorylation.
• Ser-68 phosphorylation prevents HEY1 interaction with p53, RPL11, and MDM2.

Conclusions:

• Phosphorylation at Ser-68 by STK38/STK38L is a critical regulatory mechanism for HEY1 stability and function.
• This phosphorylation event modulates HEY1's role in p53 regulation and cellular responses to stress.
• Findings reveal a novel cross-talk mechanism between Notch signaling and nucleolar stress pathways involving HEY1.