Protein Kinase A-Mediated Septin7 Phosphorylation Disrupts Septin Filaments and Ciliogenesis

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Septins are essential GTP-binding proteins forming heteromeric filaments crucial for cell growth and migration.
• Septin 7 (SEPT7) is a core component of all septin filament structures.
• The regulation of septin filament dynamics is critical for various cellular processes.

Purpose of the Study:

• To investigate the role of protein kinase A (PKA) in regulating septin filament dynamics.
• To identify specific phosphorylation sites on SEPT7 and their functional consequences.
• To elucidate the impact of SEPT7 phosphorylation on ciliogenesis.

Main Methods:

• Site-directed mutagenesis to identify the PKA phosphorylation site on SEPT7.
• Treatment with cAMP and overexpression of PKA catalytic subunit (PKACA2) to induce phosphorylation.
• Co-immunoprecipitation assays to analyze protein interactions.
• Immunofluorescence microscopy to assess septin filament formation and primary cilia.

Main Results:

• PKA phosphorylates SEPT7 at a conserved residue, Thr197.
• SEPT7 phosphorylation by PKA disrupts septin filament polymerization and dynamics.
• Phosphorylation at Thr197 specifically reduces SEPT7-SEPT7 interaction, but not interactions with SEPT6, SEPT2, or SEPT4.
• SEPT7 interacts with PKACA2 via its GTP-binding domain.
• PKA-mediated SEPT7 phosphorylation inhibits primary cilia formation.

Conclusions:

• SEPT7 phosphorylation by PKA is a novel regulatory mechanism for septin filament assembly.
• PKA-mediated disruption of SEPT7 function impacts ciliogenesis.
• This study reveals a new role for PKA signaling in controlling cell structure and function through SEPT7 modification.