A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun

Area of Science:

• Cellular Biology
• Molecular Biology
• Signal Transduction

Background:

• c-Jun is a transcription factor involved in cellular responses.
• Multisite phosphorylation regulates c-Jun activity.
• Specific kinases mediating c-Jun phosphorylation have not been fully elucidated.

Purpose of the Study:

• To investigate the specific kinases responsible for multisite phosphorylation of c-Jun.
• To differentiate the roles of JNK and ERK pathways in c-Jun regulation.
• To understand the mechanisms of c-Jun dephosphorylation.

Main Methods:

• Use of phospho-specific antibodies to detect phosphorylated c-Jun.
• Experiments in murine RAW macrophages and embryonic fibroblasts.
• Stimulation with various agents like LPS, anisomycin, TNF-alpha, TPA, and EGF.
• Studies in wild-type and JNK-deficient fibroblasts.

Main Results:

• JNK isoforms are essential for Thr91, Thr93, Ser63, and Ser73 phosphorylation in response to LPS/anisomycin and TNF-alpha/anisomycin.
• ERK1/ERK2, along with JNK1/JNK2, mediate TPA/EGF-induced Ser63/Ser73 phosphorylation in wild-type fibroblasts.
• ERK1/ERK2 alone mediates TPA/EGF-induced Ser63/Ser73 phosphorylation in JNK-deficient fibroblasts.
• GSK3 catalyzes Thr239 phosphorylation, and an unknown kinase phosphorylates Ser243.
• GSK3 inhibition is not required for Thr239 dephosphorylation after LPS stimulation.

Conclusions:

• JNK and ERK pathways exhibit distinct roles in c-Jun phosphorylation depending on the stimulus and cell type.
• Agonist-induced dephosphorylation of Thr239 may involve conformational changes or phosphatase activation.
• Further research is needed to identify the kinase responsible for Ser243 phosphorylation.