c-Abl phosphorylates E6AP and regulates its E3 ubiquitin ligase activity

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• The p53 tumor suppressor is degraded in human papillomavirus (HPV)-infected cells via the HPV-E6-E6AP complex.
• c-Abl tyrosine kinase previously showed protective effects on p53 under stress, but the mechanism was unclear.

Purpose of the Study:

• To investigate if c-Abl targets E6AP to protect p53 from degradation.
• To elucidate the molecular mechanism of c-Abl-mediated protection of p53.

Main Methods:

• Co-immunoprecipitation to detect protein interactions.
• In vitro kinase assays to assess phosphorylation.
• Site-directed mutagenesis to identify phosphorylation sites.
• Ubiquitination assays to measure E3 ligase activity.
• Structural modeling of E6AP.

Main Results:

• c-Abl directly interacts with and phosphorylates E6AP.
• Phosphorylation by c-Abl impairs E6AP's E3 ligase activity.
• Tyrosine 636 in E6AP's HECT domain is the critical phosphorylation site.
• This phosphorylation regulates E6AP oligomerization and substrate specificity.

Conclusions:

• c-Abl protects p53 from HPV-E6-E6AP-mediated degradation by inhibiting E6AP's ligase activity through phosphorylation.
• This provides a molecular basis for stress-induced p53 protection in HPV-infected cells.