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Sequence‐ and Docking‐Site‐Dependent Contributions to Multi‐Site Phosphorylation of an Intrinsically Disordered MAPK Substrate

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Bibliographic Details
Title: Sequence‐ and Docking‐Site‐Dependent Contributions to Multi‐Site Phosphorylation of an Intrinsically Disordered MAPK Substrate
Authors: Thibault Orand, Elise Delaforge, Marion Chenal, Maud Tengo, Torsten Herrmann, Juan Cortés, Pau Bernadó, Malene Ringkjøbing Jensen
Source: Advanced Science, Vol 12, Iss 35, Pp n/a-n/a (2025)
Publisher Information: Wiley, 2025.
Publication Year: 2025
Collection: LCC:Science
Subject Terms: docking sites, intrinsically disordered protein, local effective concentration, mitogen‐activated protein kinases, NMR, scaffold protein, Science
Description: Abstract Protein kinases often rely on docking site motifs to enhance substrate interactions and facilitate phosphorylation. For example, mitogen‐activated protein kinases (MAPKs) utilize D‐ and F‐motifs, which frequently act in concert to enable bipartite substrate binding. While these motifs are known to modulate phosphorylation efficiency, their quantitative impact on target site phosphorylation within long intrinsically disordered substrates remains largely unexplored. Using NMR spectroscopy, JNK1‐dependent phosphorylation of JIP1, a 450‐amino acid disordered substrate, is investigated, identifying eleven phosphosites with distinct phosphorylation efficiencies. By selectively disrupting JNK1 binding to the D‐ and F‐motifs of JIP1, the determinants of phosphorylation efficiency are uncovered. Specifically, it is found that the D‐motif selectively enhances phosphorylation in the C‐terminal direction in a length‐dependent manner, impressively increasing the phosphorylation efficiencies of sites located at sequence distances exceeding 120 amino acids, while the F‐motif primarily promotes phosphorylation of a site located immediately N‐terminal to the F‐motif. Additionally, docking‐site‐independent phosphorylation is observed, whose efficiency is dictated by the intrinsic sequence preference of JNK1, as inferred from motif scores derived from positional scanning peptide arrays. The work highlights how docking site motifs and sequence context synergistically regulate phosphorylation efficiency, emphasizing the critical role of substrate architecture in determining MAPK‐mediated signaling outcomes.
Document Type: article
File Description: electronic resource
Language: English
ISSN: 2198-3844
Relation: https://doaj.org/toc/2198-3844
DOI: 10.1002/advs.202503987
Access URL: https://doaj.org/article/9ab639046d8a4b289595e112bbbc0f94
Accession Number: edsdoj.9ab639046d8a4b289595e112bbbc0f94
Database: Directory of Open Access Journals
Description
Abstract:Abstract Protein kinases often rely on docking site motifs to enhance substrate interactions and facilitate phosphorylation. For example, mitogen‐activated protein kinases (MAPKs) utilize D‐ and F‐motifs, which frequently act in concert to enable bipartite substrate binding. While these motifs are known to modulate phosphorylation efficiency, their quantitative impact on target site phosphorylation within long intrinsically disordered substrates remains largely unexplored. Using NMR spectroscopy, JNK1‐dependent phosphorylation of JIP1, a 450‐amino acid disordered substrate, is investigated, identifying eleven phosphosites with distinct phosphorylation efficiencies. By selectively disrupting JNK1 binding to the D‐ and F‐motifs of JIP1, the determinants of phosphorylation efficiency are uncovered. Specifically, it is found that the D‐motif selectively enhances phosphorylation in the C‐terminal direction in a length‐dependent manner, impressively increasing the phosphorylation efficiencies of sites located at sequence distances exceeding 120 amino acids, while the F‐motif primarily promotes phosphorylation of a site located immediately N‐terminal to the F‐motif. Additionally, docking‐site‐independent phosphorylation is observed, whose efficiency is dictated by the intrinsic sequence preference of JNK1, as inferred from motif scores derived from positional scanning peptide arrays. The work highlights how docking site motifs and sequence context synergistically regulate phosphorylation efficiency, emphasizing the critical role of substrate architecture in determining MAPK‐mediated signaling outcomes.
ISSN:21983844
DOI:10.1002/advs.202503987