Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens...

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• The N-terminal domain of c-Myc proteins is crucial for their transactivation and biological functions.
• Understanding the post-translational modifications of c-Myc, particularly phosphorylation, is key to elucidating its regulatory mechanisms.

Purpose of the Study:

• To investigate the detailed phosphorylation patterns on the N-terminal domain of c-Myc proteins.
• To identify specific phosphorylation sites and the kinases involved.
• To determine the functional consequences of these phosphorylation events on c-Myc activity and cellular transformation.

Main Methods:

• Detailed phosphopeptide mapping analyses were employed.
• In vitro kinase assays using various glycogen synthase kinase 3 alpha/beta, p42 mitogen-activated protein kinase, and cdc2 kinase preparations were performed.
• In vivo experiments were conducted to assess kinase roles and phosphorylation site localization.

Main Results:

• A cluster of four regulated phosphorylation events was identified on the N-terminal domain, including Thr-58, Ser-62, and Ser-71.
• Phosphorylation of Thr-58 is dependent on prior Ser-62 phosphorylation, while Ser-62 phosphorylation is enhanced in Thr-58 mutated v-Myc proteins.
• Hierarchical phosphorylation was observed in vitro with a specific glycogen synthase kinase 3 alpha.
• Thr-58 and Ser-62 phosphorylation can occur in the cytoplasm, independent of nuclear translocation.
• A block in Ser-62 phosphorylation during mitosis was noted.
• Enhanced transforming properties correlated with reduced Thr-58 and increased Ser-62 phosphorylation, but transactivation ability remained unaffected.

Conclusions:

• The N-terminal phosphorylation of c-Myc proteins is complex and hierarchical, with specific sites like Thr-58 and Ser-62 playing critical roles.
• Cytoplasmic phosphorylation of c-Myc is possible, challenging previous assumptions about nuclear dependence.
• While these phosphorylation events influence cellular transformation, they do not alter the core transactivation function of c-Myc via the Myc/Max binding site.