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Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors
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DOI：10.1126/scisignal.abm5518.png)
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Abstract
En 中文
Essential functions of mitogen-activated protein kinases (MAPKs) depend on their capacity to selectively phos-phorylate a limited repertoire of substrates. MAPKs harbor a conserved groove located outside of the catalytic cleft that binds to short linear sequence motifs found in substrates and regulators. However, the weak and tran-sient nature of these docking interactions poses a challenge to defining MAPK interactomes and associated sequence motifs. Here, we describe a yeast-based genetic screening pipeline to evaluate large collections of MAPK docking sequences in parallel. Using this platform, we analyzed a combinatorial library based on the docking sequences from the MAPK kinases MKK6 and MKK7, defining features critical for binding to the stress-activated MAPKs JNK1 and p38 alpha. Our screen of a library consisting of similar to 12,000 sequences from the human proteome revealed multiple MAPK-selective interactors, including many that did not conform to previ-ously defined docking motifs. Analysis of p38 alpha/JNK1 exchange mutants identified specific docking groove res-idues that mediate selective binding. Last, we verified that docking sequences identified in the screen functioned in substrate recruitment in vitro and in cultured cells. Together, these studies establish an approach to characterize MAPK docking sequences and provide a resource for future investigation of signaling down-stream of p38 and JNK.
Keywords:
MAP KINASE
STRUCTURAL BASIS
SUBSTRATE SELECTIVITY
CRYSTAL-STRUCTURE
PHAGE DISPLAY
SPECIFICITY
SITES
BINDING
PHOSPHATASE
P38-ALPHA