A chemical genetic approach to engineer phototropin kinases for substrate labeling

The Journal of Biological Chemistry
Jonathan SchnabelJohn M Christie

Abstract

Protein kinases (PKs) control many aspects of plant physiology by regulating signaling networks through protein phosphorylation. Phototropins (phots) are plasma membrane-associated serine/threonine PKs that control a range of physiological processes that collectively serve to optimize photosynthetic efficiency in plants. These include phototropism, leaf positioning and flattening, chloroplast movement, and stomatal opening. Despite their identification over two decades ago, only a handful of substrates have been identified for these PKs. Progress in this area has been hampered by the lack of a convenient means to confirm the identity of potential substrate candidates. Here we demonstrate that the kinase domain of Arabidopsis phot1 and phot2 can be successfully engineered to accommodate non-natural ATP analogues by substituting the bulky gatekeeper residue threonine for glycine. This approach circumvents the need for radioactivity to track phot kinase activity and follow light-induced receptor autophosphorylation in vitro by incorporating thiophosphate from N6-benzyl-ATPγS. Consequently, thiophosphorylation of phot substrate candidates can be readily monitored when added or co-expressed with phots in vitro Furthermore, gatekeepe...Continue Reading

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Citations

Jan 8, 2020·Journal of Experimental Botany·Emmanuel LiscumKatelynn Koskie
Aug 12, 2020·The Plant Journal : for Cell and Molecular Biology·Shin-Ichiro InoueKen-Ichiro Shimazaki
Aug 28, 2020·Frontiers in Plant Science·Lixia Pan, Ive De Smet
Mar 31, 2021·The Journal of Biological Chemistry·Jaynee E Hart, Kevin H Gardner
Sep 7, 2021·Frontiers in Plant Science·Lixia PanIve De Smet

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