Myc phosphorylation in its basic helix-loop-helix region destabilizes transient α-helical structures, disrupting Max...

Area of Science:

• Molecular Biology
• Protein Phosphorylation
• Transcription Regulation

Background:

• Myc is a key transcription factor regulating cellular homeostasis and cancer.
• Myc activity is tightly controlled by phosphorylation, particularly in its transactivation domain and C-terminal region.
• P21 (RAC1)-activated kinase 2 (PAK2) phosphorylates Myc, influencing its transcriptional function.

Purpose of the Study:

• To investigate the specific sites and effects of PAK2-mediated phosphorylation on Myc.
• To elucidate how Myc phosphorylation impacts its interaction with Myc-associated factor X (Max) and DNA binding.
• To understand the structural and functional consequences of Myc phosphorylation by PAK2.

Main Methods:

• Isothermal titration calorimetry (ITC) to measure binding affinities.
• Site-directed mutagenesis and phosphomimetic substitutions.
• Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy for structural analysis.

Main Results:

• PAK2 preferentially phosphorylates Myc at Thr-358 and Ser-373, with minor modification at Thr-400.
• Myc phosphorylation by PAK2 significantly decreases Myc's affinity for Max (by 2 orders of magnitude).
• Ser-373 phosphorylation is the primary driver of reduced Myc-Max heterodimerization, while T400D substitution also impairs affinity.
• Phosphorylation-induced secondary structure disruption during heterodimerization, not disordered state changes or electrostatic repulsion, underlies the weakened Myc-Max interaction.

Conclusions:

• PAK2-mediated phosphorylation at specific C-terminal sites (Thr-358, Ser-373) critically regulates Myc transcriptional activity.
• Phosphorylation destabilizes the Myc-Max heterodimer by altering secondary structure, thereby affecting DNA binding.
• These findings offer crucial insights into the regulatory mechanisms of Myc function through post-translational modification.