Related Experiment Video

Updated: Aug 25, 2025

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

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An intrinsic temporal order of c-JUN N-terminal phosphorylation regulates its activity by orchestrating co-factor

Christopher A Waudby1,2, Saul Alvarez-Teijeiro3,4,5, E Josue Ruiz6

  • 1Institute of Structural and Molecular Biology, University College London, London, UK.

Nature Communications
|October 17, 2022
PubMed

Insights

c-JUN protein phosphorylation by JNK kinases has distinct temporal kinetics. This multisite phosphorylation controls c-JUN

Area of Science:

  • Cellular signaling
  • Molecular biology
  • Oncoprotein regulation

Background:

  • Protein phosphorylation is a key regulator of cellular signaling pathways.
  • The c-JUN proto-oncoprotein's transactivation domain (TAD) is phosphorylated by JNK kinases, but the functional impact of this multisite phosphorylation is not fully understood.

Purpose of the Study:

  • To investigate the temporal kinetics of c-JUN phosphorylation by JNK.
  • To elucidate the functional significance of distinct c-JUN phosphorylation states.

Main Methods:

  • Analysis of c-JUN phosphorylation kinetics by JNK family kinases.
  • Identification of factors influencing phosphorylation site kinetics.
  • Functional assays to determine the role of different c-JUN phosphorylation states.

Main Results:

  • c-JUN phosphorylation by JNK occurs with defined temporal kinetics: Serine63/73 are phosphorylated faster than Threonine91/93.
  • Phosphorylation site positioning and primary sequence dictate phosphorylation kinetics.
  • Three functional states of c-JUN were identified: unphosphorylated (recruits MBD3), doubly phosphorylated at S63/73 (binds TCF4), and fully phosphorylated (disfavors TCF4 binding, attenuates signaling).

Conclusions:

  • c-JUN multisite phosphorylation by JNK creates distinct functional states.
  • These phosphorylation states modulate interactions with repressors (MBD3) and co-activators (TCF4).
  • c-JUN phosphorylation acts as a complex signaling switch, translating a single JNK input into diverse cellular responses.

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