A chemical genetic approach to engineer phototropin kinases for substrate labeling

Schnabel, J., Hombach, P., Waksman, T., Guiriani, G., Petersen, J. and Christie, J. M. (2018) A chemical genetic approach to engineer phototropin kinases for substrate labeling. Journal of Biological Chemistry, 293(15), pp. 5613-5623. (doi:10.1074/jbc.RA118.001834) (PMID:29475950) (PMCID:PMC5900772)

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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 serve 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, gatekeeper-modified phot1 retained its functionality and its ability to accommodate N6-benzyl-ATPγS as a phosphodonor when expressed in Arabidopsis. We therefore anticipate that this chemical genetic approach will provide new opportunities for labeling and identifying substrates for phots and other related AGC kinases under in vitro and near-native in vivo conditions.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Waksman, Thomas and Hombach, Mr Peter and Schnabel, Mr Jonathan and Christie, Professor John and Petersen, Dr Jan
Authors: Schnabel, J., Hombach, P., Waksman, T., Guiriani, G., Petersen, J., and Christie, J. M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
ISSN (Online):1083-351X
Published Online:23 February 2018
Copyright Holders:Copyright © 2018 Schnabel et al.
First Published:First published in Journal of Biological Chemistry 293(15):5613-5623
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
659801Photoreceptor Engineering to Modulate Plant GrowthJohn ChristieBiotechnology and Biological Sciences Research Council (BBSRC)BB/M002128/1RI MOLECULAR CELL & SYSTEMS BIOLOGY
581491Regulation of Auxin Fluxes Required For Phototropic GrowthJohn ChristieBiotechnology and Biological Sciences Research Council (BBSRC)BB/J016047/1RI MOLECULAR CELL & SYSTEMS BIOLOGY