Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades.

Nagy, Szilvia Krisztina and Darula, Zsuzsanna and Kállai, Brigitta M. and Bögre, László and Bánhegyi, Gábor and Medzihradszky F., Katalin and Horváth V., Gábor and Mészáros, Tamás (2015) Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades. BIOCHEMICAL JOURNAL, 467 (1). pp. 167-175. ISSN 0264-6021

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Abstract

Mitogen activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organised into three-tiered kinase cascades. The activation of MAPKs in these pathways is fully dependent on the bisphosphorylation of the TXY motif in the T-loop by the pertinent dual-specificity MAPK kinases (MAPKKs). The plant AtMPK9 is a member of an atypical class of MAPKs. Representatives of this MAPK family have TDY phosphoacceptor site, a long C-terminal extension, and lack the common MAPKK binding docking motif. Here, we present multiple in vitro and in vivo data that AtMPK9 is activated independently of any upstream MAPKKs but it is activated through autophosphorylation. We mapped the autophosphorylation sites by mass spectrometry to the TDY motif and to the C-terminal regulatory extension. We mutated the phosphoacceptor sites on the TDY, which confirmed the requirement for bisphorylation at this site for full kinase activity. Next, we demonstrated that the kinase inactive mutant form of AtMPK9 is not transphosphorylated on the TDY site when mixed with an active AtMPK9, implying that the mechanism of the autocatalytic phosphorylation is intramolecular. Furthermore, we show that in vivo AtMPK9 is activated by salt and is regulated by okadaic acid-sensitive phosphatases. We conclude that the plant AtMPK9 shows similarities to the mammalian atypical MAPKs, ERK7/8 in terms of MAPKK-independent activation mechanism.

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