Abstract
Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi.
Highlights
Among the most ancient signalling routes are the mitogenactivated protein kinase (MAPK) pathways that transduce extracellular stimuli into appropriate cellular responses
Whereas D-motifs interact with acidic residues in the common docking (CD) domain of MAPKs, DEF motifs bind to a hydrophobic pocket that is only exposed upon MAPK activation [8]
The IYT motif of S. cerevisiae strain YPH499 transformed with plasmids YCplac22Sdp1m (Sdp1) mediates binding to the cell wall integrity (CWI) MAPK Slt2
Summary
Among the most ancient signalling routes are the mitogenactivated protein kinase (MAPK) pathways that transduce extracellular stimuli into appropriate cellular responses. MAPKs contain a highly conserved TXY motif in the kinase activation loop in which both Thr and Tyr residues must be phosphorylated to achieve kinase activation [1]. Their main negative regulators are protein phosphatases. These enzymes are designated as dual-specificity MAPK phosphatases (MKPs) and play an essential role in the regulation of MAPK signalling [2,3] This MKP-mediated dephosphorylation event requires a transient enzyme-substrate interaction involving the active site of these enzymes, and the formation of a complex through specific docking interactions between conserved regions within both MKPs and MAPKs [4]. Whereas D-motifs interact with acidic residues in the common docking (CD) domain of MAPKs, DEF motifs bind to a hydrophobic pocket that is only exposed upon MAPK activation [8]
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