Direct phosphorylation of proliferative and survival pathway proteins by RET

Area of Science:

• Oncology
• Molecular Biology
• Biochemistry

Background:

• Gain-of-function mutations in the RET tyrosine kinase receptor are linked to Multiple Endocrine Neoplasia types 2A/2B and medullary thyroid cancer.
• Previous studies indicated RET signaling through focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) in medullary thyroid cancer cells.
• The role of RET in directly phosphorylating FAK and ERK was investigated.

Purpose of the Study:

• To determine if RET directly phosphorylates FAK and ERK.
• To elucidate the direct interaction of RET with downstream signaling molecules.
• To understand the molecular mechanisms contributing to RET-driven tumor pathogenesis.

Main Methods:

• In vitro kinase assays were used to measure RET and ERK kinase activity.
• Cells were treated with MEK inhibitor (PD98059) and RET inhibitor (PP2) to assess RET's contribution to ERK phosphorylation.
• Immunoprecipitation and recombinant RET kinase domain were employed to identify direct phosphorylation targets.

Main Results:

• Mutant RET directly phosphorylated specific tyrosine residues on FAK (Y576/577, Y861, Y925) and ERK2 (Y187).
• Inhibition of both MEK and RET demonstrated a synergistic effect on reducing ERK activity.
• RET was also shown to directly phosphorylate tyrosine residues on DOK1, PI3K p85 subunit, JNK 1/2, P-38, and phospholipase-gamma.

Conclusions:

• RET directly phosphorylates a wide array of effector molecules, including FAK, ERK, DOK1, PI3K p85, JNK, P-38, and phospholipase-gamma.
• These findings reveal a direct interaction between RET and its downstream targets.
• This direct interaction likely plays a significant role in the pathogenesis of RET-driven tumors.