Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes

Abstract

Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP) 1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP binding and hydrolysis are the driving processes in all living organisms. Hundreds of cellular proteins are able to bind and hydrolyze ATP to unfold proteins, transport molecules over membranes, or phosphorylate small molecules or proteins. Proteins with very different structures are able to bind ATP. A large and important class of ATP binding proteins is that of the kinases, which transfer the gamma phosphate from ATP to substrates. Kinases, and particularly protein kinases, play pivotal roles in signaling and protein regulation.The genome of the model plant Arabidopsis thaliana encodes for over 1099 protein kinases and hundreds of other ATP binding proteins (1, 2). Protein kinases are involved in nearly all signaling cascades and regulate processes ranging from cell cycle to flowering and from immunity to germination. Many protein kinases in plants are receptor-like kinases (RLKs), often carrying extracellular leucine-rich repeats (LRRs). The RLK class contains at least 610 members (3), including famous examples such as receptors involved in development (e.g. BRI1, ER, CLV1) and immunity (e.g. FLS2, EFR). Other important classes are mitogen-activated protein (MAP) kinases (MPKs) (20 different members), MPK kinase kinase kinases (MAP3Ks) (60 different members (4)), and calcium-dependent protein kinases (CPKs) (34 different members (5)). Because of their diverse and important roles, protein kinases have been intensively studied in plant science. The current approach is to study protein kinases individually-a daunting task, considering the remaining hundreds of uncharacterized p...

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