The UBA domain of SnRK1 promotes activation and maintains catalytic activity

https://doi.org/10.1016/j.bbrc.2018.02.039Get rights and content

Highlights

  • SnRK1 α-subunits possess a UBA domain following its kinase domain (KD).

  • The UBA enhances activating phosphorylation by SnRK1 upstream kinase.

  • The UBA maintains catalytic activity over time, in contrast to KD-only.

  • SnRK1 regulation by UBA is distinct from AMPK regulation by the orthologous AID.

Abstract

Sucrose non-fermenting 1-related protein kinase 1 (SnRK1) is a central metabolic regulator and the plant orthologue of the mammalian AMP-activated protein kinase (AMPK); both are energy-sensing heterotrimeric enzymes comprising a catalytic α- and regulatory β- and γ-subunits. α-Subunits contain a serine/threonine kinase domain (KD) at their N-terminus that is immediately followed by a small regulatory domain termed the auto-inhibitory domain (AID) in AMPK and the ubiquitin-associated domain (UBA) in SnRK1. Association of the AID with the AMPK KD inhibits activating phosphorylation of the KD by upstream kinases and promotes dephosphorylation, as well as inhibiting AMPK catalytic activity. Despite these mechanistic insights regarding the AMPK AID, the SnRK1 UBA regulatory implications have not been investigated. Using recombinant protein comprising either the KD-only or KD-AID/KD-UBA, we found that the UBA of SnRK1 acts in a distinct regulatory manner to its orthologous AID of AMPK. Firstly, the plant upstream kinase GRIK2 preferentially phosphorylates the SnRK1 KD-UBA. Secondly, the SnRK1 KD in the absence of the UBA shows near identical initial catalytic activity to the KD-UBA, but in comparison a rapid loss of catalytic activity is observed. Our findings indicate that the role of the UBA in SnRK1 regulation may be more akin to that of the UBA in the mammalian AMPK-related kinases rather than its immediate functional orthologue, AMPK. This study adds to a growing body of work demonstrating the divergent regulatory mechanisms of the orthologous plant SnRK1 and mammalian AMPK.

Introduction

SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of an evolutionarily-conserved family of energy-sensing, central metabolic regulators that include yeast sucrose non-fermenting 1 (SNF1) and mammalian AMP-activated protein kinase (AMPK). Members of the SnRK1/SNF1/AMPK family sense changes in cellular energy status and act at both cellular and systemic levels to restore energy balance [1,2]. These heterotrimeric enzymes comprise a catalytic α- and regulatory β- and γ-subunits, for each of which multiple isoforms exist [3]; for example, there are two α-isoforms for both AMPK and SnRK1. The catalytic subunits comprise an N-terminal serine/threonine kinase domain (KD) that is immediately followed by a three-helix bundle; this is termed the auto-inhibitory domain (AID) for AMPK and the ubiquitin-associated domain (UBA) for SnRK1 (Fig. 1A). The remainder of the α-subunits comprise regions responsible for interaction with β- and γ-subunits and other regulatory elements. Whilst the AMPK and SnRK1 KDs share 63% sequence identity, the respective AID and UBA share only 33–37% sequence identity (Table S1), suggesting their functions in relation to the kinase may differ.

Mammalian AMPK is primarily regulated by reversible phosphorylation of the α-subunit T-loop threonine – Thr172 in the α2 isoform – whose phosphorylation is critical for significant activity [4]. Decreasing adenylate charge – i.e. increasing ADP and AMP concentrations relative to ATP – enhances phosphorylation by upstream kinases [5] and protects against dephosphorylation [6]. In addition, AMP further allosterically activates the kinase [6]. The mechanism(s) by which ADP and AMP, which bind to the γ-subunit, causes activation involves conformational change in the heterotrimer whereby the AID dissociates from the hinge region of the KD. This results in a closed, active conformation that renders the KD less susceptible to phosphatases [7] and, as yet inexplicably, a better substrate for its upstream kinases [5,8]. T-loop phosphorylation – at Thr175 in the Arabidopsis thaliana SnRK1 α1 isoform, Thr176 in α2 – is likewise critical for SnRK1 activity [9], and two upstream kinases, Geminivirus Rep-interacting kinase 1 (GRIK1) and GRIK2 [10], have been described, with others likely yet to be identified. Adenylate nucleotides do not, however, play a significant role in the regulation of SnRK1 [2,3,11,12]. Rather, SnRK1 appears to sense energy charge primarily through high-energy sugar-phosphates, most importantly trehalose-6-phosphate, which leads to inhibition of the kinase (through an unresolved mechanism most likely involving a proteinaceous co-factor) [13,14]. The role of reversible phosphorylation in planta is also somewhat unclear, with multiple studies demonstrating that SnRK1 T-loop phosphorylation state does not predictably correlate with SnRK1 activity [9,[15], [16], [17]]. SnRK1 belongs to a larger family of related kinases that include the plant-specific SnRK2 and SnRK3 sub-groups. Whilst these contain regulatory regions C-terminal to their kinase domains that regulate T-loop phosphorylation and catalytic activity, they are unrelated to the UBA of SnRK1 [18]. This then begs the question as to what role the UBA plays in the regulation of SnRK1.

UBAs are present in ten of the twelve mammalian AMPK-related kinases (AMPK-RKs), the only known kinases to possess UBAs in the human genome. Though UBAs in proteins commonly bind ubiquitin, this is not the case for the mammalian AMPK-RKs. Instead, the UBA in these kinases allows liver kinase B1 (LKB1) to phosphorylate their T-loop and thus render them active, though the domain itself is not involved in the LKB1 interaction [19]. LKB1 is one of two AMPK upstream kinases, the other being Ca2+/Calmodulin-dependent kinase kinase β (CAMKKβ) [8], though the AID, in contrast to the UBA of AMPK-RKs, inhibits AMPK T-loop phosphorylation. Despite the observed role in enhancing activating T-loop phosphorylation, investigation of the UBAs of the AMPK-RK microtubule-associated protein/microtubule affinity regulating kinase 1 (MARK1) and MARK2 suggests that they act to reduce catalytic activity [20]. To resolve a seemingly contradictory mechanism of concurrent activation and inhibition, the AMPK-RK UBA has been proposed as a bistable switch element. This hypothesis postulates that the UBA stabilises both the open, inactive and the closed, active conformation depending on phosphorylation state or interaction with co-factors [21].

We sought to define a role for the UBA of the plant SnRK1 α-subunit using recombinantly-expressed A. thaliana (Atα1/2) KD or KD-UBA constructs in comparison to Homo sapiens AMPK α2 isoform (Hsα2) KD and KD-AID equivalents. These constructs comprise only the KD or the KD extended to include the UBA or AID but truncated thereafter (Fig. 1B). We show that the SnRK1 UBA shares sequence similarities with the related but distinct AMPK AID and AMPK-RK UBAs. In an opposite manner to AMPK, the SnRK1 UBA promotes phosphorylation by its upstream kinase GRIK2. Finally, we show that the UBA contributes to the preservation of catalytic activity over time but does not influence initial catalytic activity. The data suggest that the role of the SnRK1 UBA in regulating kinase activity is more akin to that of UBAs from mammalian AMPK-RKs and distinct from that of the AID for AMPK itself. These findings build on a growing body of work showing distinct regulatory mechanisms of SnRK1 in contrast to its opisthokont orthologues.

Section snippets

Sequence alignment and statistical analyses

Multiple sequence alignments were performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Sequences were obtained from UniProt (http://www.uniprot.org). Graphing and statistical analyses were performed using Prism7 (GraphPad).

Constructs

AMPK and SnRK1 (Fig. 1B) and GRIK2 constructs were cloned into and expressed from pGEX-6P-3 plasmid (GE Healthcare Life Sciences) to contain an N-terminal glutathione-S-transferase (GST) tag for purification. pGEX-6P-3 was digested with BamHI and SalI

Analysis of AID/UBA from AMPK and related kinases

The alignment of the UBA sequences of SnRK1 α-subunits with the AID of AMPK and SNF1 and the UBA from AMPK-RKs reveals several interesting aspects (Fig. 2). Firstly, the residues comprising the hydrophobic core of the AID of AMPK [24] and the UBA of MARKs [20] are conserved in SnRK1, suggesting a conserved three-dimensional fold. Secondly, SnRK1 UBAs share identity or similarity in residues contacting the kinase domain of both AMPK and MARKs. Thirdly, the conserved glycine residue reported by

Discussion

SnRK1 is the plant orthologue of mammalian AMPK and fungal SNF1, sharing their conserved role as an energy sensor to mediate central metabolism as well as the heterotrimeric subunit composition. Despite this, it is clear that the mechanism(s) by which SnRK1 senses changes in cellular energy charge and by which these changes regulate catalytic activity are fundamentally distinct from those of its opisthokont orthologues [2,3,11]. The manner by which adenylate nucleotides transmit their effect in

Acknowledgements

This work was supported by Kentucky Science and Energy Foundation [grants KSEF-2268RDE-014, KSEF-2971-RDE-017], National Science Foundation [grants IIA-1355438, MCB-1252345], Australian Research Council Centre of Excellence in Plant Cell Walls [grant CE1101007] and an Australia Research Council Discovery [grant DP110103161].

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    Present address: La Trobe Institute for Agriculture and Food (LIAF), Department of Animal, Plant and Soil Sciences, AgriBio, La Trobe University, Bundoora, Victoria 3086, Australia.

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