Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Mitogen-activated protein kinases (MAPKs) regulate cellular processes through substrate phosphorylation.
• MAPKs utilize docking interactions with short linear motifs for substrate specificity.
• Characterizing these weak, transient interactions and their motifs is challenging.

Purpose of the Study:

• To develop and implement a yeast-based genetic screening pipeline for evaluating MAPK docking sequences.
• To identify novel MAPK interactors and define critical binding features for stress-activated MAPKs JNK1 and p38α.
• To investigate the role of specific MAPK docking groove residues in selective binding.

Main Methods:

• A yeast-based genetic screening platform was established to assess large libraries of MAPK docking sequences.
• A combinatorial library based on MKK6 and MKK7 docking sequences was analyzed against JNK1 and p38α.
• Analysis involved creating p38α/JNK1 exchange mutants to pinpoint residues mediating selective binding.
• Identified docking sequences were validated for substrate recruitment in vitro and in cell culture.

Main Results:

• The screening platform successfully evaluated ~12,000 human proteome sequences.
• Multiple selective interactors for p38α and JNK1 were identified, including novel motifs.
• Specific residues within the MAPK docking groove were found to mediate selective binding.
• Validated docking sequences demonstrated functional substrate recruitment.

Conclusions:

• A robust yeast-based screening approach for characterizing MAPK docking sequences has been established.
• This study provides a valuable resource for understanding p38 and JNK signaling pathways.
• New insights into MAPK-substrate interactions and selectivity have been gained.