Review: How do SnRK1 protein kinases truly work?

Journal Pre-proof Review: How do SnRK1 protein kinases truly work? Eleazar Mart´ınez-Barajas, Patricia Coello

PII:

S0168-9452(19)31503-1

DOI:

https://doi.org/10.1016/j.plantsci.2019.110330

Reference:

PSL 110330

To appear in:

Plant Science

Received Date:

29 July 2019

Revised Date:

10 October 2019

Accepted Date:

1 November 2019

Please cite this article as: Mart´ınez-Barajas E, Coello P, Review: How do SnRK1 protein kinases truly work?, Plant Science (2019), doi: https://doi.org/10.1016/j.plantsci.2019.110330

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Review: How do SnRK1 protein kinases truly work?

Eleazar Martínez-Barajas and Patricia Coello*

Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Ciudad de México, 04510, México

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*Corresponding author: [email protected]

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Highlights  SnRK1 specific complexes might form in response to a particular stress.  SnRK1α subunit has kinase activity independently of the regulatory subunits.  Activation of the SnRK1α catalytic subunit might involve the phosphorylation of other residues than the Thr175.  Individual SnRK1 subunits could have other roles when they are not part of the complex

Abstract

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The AMPK/SNF1/SnRK1 family of protein kinases is involved in cellular responses to energy stress. They also interact with molecules of other signaling pathways to regulate many aspects of growth and development. The biochemical, genetic and molecular knowledge of SnRK1 in plants lags

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behind that of AMPK and SNF1 and is freely extrapolated such that, in many cases, it is assumed that plant enzymes behave in the same way as homologs in other organisms. In this review, we present data that support the evidence that the structural characteristics of the SnRK1 subunits determine the functional properties of the complex. We also discuss results suggesting that the

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SnRK1 subunits participate in the assembly of different complexes and that not all combinations are equally important. The activity of SnRK1 is dependent on the phosphorylation of SnRK1αThr175 found in the activation loop of the catalytic domain. However, we propose that the phosphorylation of sites close to SnRK1αThr175 might contribute to the fine-tuned regulation of SnRK1 activity and thus requires further evaluation. Finally, we also call attention to the interaction of the SnRK1α with regulatory proteins that are not typically identified as putative substrates. The additional functions of

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the SnRK1 subunits, in addition to those of the active complex, may be necessary for the cell to respond to the complicated conditions presented by energy stress.

Keywords: SnRK1 subunits; phosphorylation; activation; interactors.

1. Introduction Plants are sessile organisms living in a variable and frequently adverse environment. Some of the

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changes affecting plants are predictable (yearly seasonal changes and day/night transitions), while many others cannot be anticipated (intermittent drought, lack of nutrients, and extended darkness).

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These changes can affect the energy balance of plants and have adverse consequences on plant growth and development. Plants are very efficient in translating the challenges to energy

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homeostasis through complex responses to restore it. Some of the responses are rapid, while others require time for full realization. The posttranslational modification of enzymes and changes in

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gene expression are elements frequently used in adaptive plant responses. However, to have a proper response, a large number of changes need to be made. Some of these changes are

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general, while others are specific and restricted to organs and/or a particular stage of development. SNF1-related protein kinase 1 (SnRK1) is a plant ortholog of the evolutionarily conserved

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SNF1/AMPK/SnRK1 serine-threonine kinase family involved in controlling cellular energy homeostasis. The protein takes the form of a complex formed with a SnRK1α catalytic subunit and SnRK1β and SnRK1γ regulatory subunits. The role of SnRK1 in restoring energy homeostasis ranges from the regulation of metabolic processes through the phosphorylation of key enzymes to the regulation of significant changes in gene expression [1]. Arabidopsis has multiple isoforms of

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each of the canonical SnRK1 subunits that, with plant-specific subunits (SnRK1βγ and SnRK1β3), enable the formation of different complexes [2]. Assuming that each of these complexes has a specific function certainly helps to understand the multiple roles of SnRK1, but these functions remain to be demonstrated. In recent years, several papers in top journals have reported the effect of SnRK1 subunit overexpression on plant responses [3,4,5,6,7,8]. These cited studies raises the possibility that the SnRK1 subunits themselves have unique functions and that disrupted subunit

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balance affects critical aspects of SnRK1 complex formation. This short review is focused on showing how the versatility of SnRK1 (in complex and its subunits) is used to meet the need for a proper response to environmental and developmental demands. However, it also emphasizes the need to investigate the biochemical aspects of the SnRK1 complex that, in our opinion, are critical to understanding how it works and how it uses cross talk with other pathways to regulate cell growth and the stress response.

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2. Structure of SnRK1 complex

The composition and regulation of the SNF1/AMPK complex have been investigated in great detail

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in animal cells and yeast, and in both cases, the heterotrimeric nature of the complex comprising an α catalytic subunit and β and γ regulatory subunits has been illustrated by different approaches. In

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animal cells, the activation of AMPK depends on the binding of AMP to the γ subunit and on the phosphorylation of the AMPKαThr172 in the activation loop [9]. Mammals have multiple isoforms for

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each AMPK subunit (α1, α2, β1, β2, γ1, γ2, and γ3), and accordingly, as many as 12 heterotrimer combinations can be formed. However, although all the isoforms are expressed in the same tissue,

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it has been observed that not all of them are part of an AMPK complex, which suggests that an additional element regulates heterotrimer formation [10]. In yeast, snf1 encodes the α-catalytic

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subunit, there are three β isoforms (Sip1, Sip2, and Gal83), and snf4 encodes the γ subunit. The interaction between subunits has been studied both in vitro and in vivo, and it has been concluded that the complex assembly depends on the β subunit [11]. In addition, mutations in the regulatory subunits (snf4 and the triple mutant Gal83/Sip1/Sip2) lead to the inability to utilize other carbon sources, such as ethanol or glycerol, indicating that Snf1 by itself is not active [12]. Arabidopsis also

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has multiple isoforms of the subunits closely related to the AMPK/SNF1 kinases (SnRK1α1, SnRK1α2, SnRK1β1, SnRK1β2, and SnRK1γ), and two are plant-specific (SnRK1β3 and SnRK1βγ). It is assumed that SnRK1, similar to AMPK and SNF1, also forms heterotrimeric complexes. However, the composition of the SnRK1 complex has been carefully investigated in Arabidopsis, and six combinations represented by the formula αxβyβγ were observed: X=1 or 2 and y=1, 2 or 3 [13]. The available information clearly shows that the complexes might not be equally

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important. In fact, SnRK1β1 mutants did not show any defect in plant development or changes in SnRK1 activity [14], SnRK1γ does not behave as a functional γ-type subunit because it is unable to complement yeast snf4 mutants, and it has not been identified as part of the SnRK1 complex [13]. Accordingly, mutations to snrk1γ did not lead to changes in the SnRK1 responses [15]. Additionally, nonhomozygous snrk1βγ-knockout mutants have been isolated, suggesting that this subunit participates in the formation of complexes that have a critical role in plant development [16]. Heterozygous snrk1βγ+/- mutants show reduced numbers of mitochondria and peroxisomes and

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abnormal pollen morphology. Interestingly, snrk1α1/α2-knockdown mutants also showed the same phenotype as the snrk1βγ+/- mutants, supporting the idea that the SnRK1α and the plant-specific

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SnRK1βγ subunit are part of the same signaling pathway [16]. The available information suggests that, in plants, the function of the γ subunit is replaced by that of the βγ subunit. However, using a

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two-hybrid assay, the interaction between SnRK1γ and almost all SnRK1 subunits has been documented [17, 18]. On the other hand, essential differences in the expression of SnRK1γ and

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SnRK1βγ have been observed in response to abiotic stress [19]. Given the information described above, it is possible that SnRK1 complexes formed with SnRK1γ are restricted to specific conditions

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or particular tissues, which is why these complexes have not been identified. Another piece of new information indicates that the SnRK1γ subunit is phosphorylated in vivo [20], raising the question of

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whether this modification affects its assembly within the complexes. Therefore, it seems that, before reaching any conclusion regarding the participation of the SnRK1γ subunit in complex formation, it is necessary to perform additional experiments that better reflect the natural conditions in vivo. Nonetheless, the possibility that unique plant subunits could generate specific complexes with different functions is attractive. The SnRK1β1, β2, and β3 subunits interact with SnRK1βγ and form

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stable dimers when coexpressed in Escherichia coli [21]. It is noteworthy that, from a structural point of view, the exclusive plant heterotrimer SnRK1α1/SnRK1βγ/SnRK1β3 has only one carbohydrate-binding domain (CBM), which interacts directly with the catalytic subunit, instead of having two adjacent domains that would be found if SnRK1β3 were replaced by either the SnRK1β1 or SnRK1β2 subunit [21, 22]. The presence of two CBM motifs in recombinant complexes does not influence the kinase activity; however, when the CBM from the βγ subunit is eliminated and the

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truncated protein forms a dimer with the SnRK1β3 subunit, the activity decreases, suggesting that the CBM positively regulates kinase activity [23]. It will be essential to obtain crystal structures of the regulatory SnRK1β subunits interacting with the βγ and α subunits to learn the spatial arrangement of the two CBMs and understand how the regulatory subunits interact with the catalytic subunit. The kinase activity also depends on the structural characteristics of the catalytic subunit. Both the AMPKα and Snf1 have a self-inhibitory sequence (AIS) that interacts directly with the catalytic

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domain. This inhibitory interaction is terminated when the γ subunit interacts with the catalytic subunit (reviewed in [24). Only when the AIS domain and the C-terminal end are deleted is the

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catalytic domain active independently of the regulatory subunits [25]. In plants, a self-inhibiting domain has not been found; instead, the SnRK1α has an ubiquitinated protein-binding domain

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(UBA) that enhances T-loop phosphorylation by upstream kinases and maintains the catalytic activity for longer time assuming a regulatory role, but does not seem important for the interaction

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with the γ or βγ subunits [26]. Although in animal and yeast cells, the activity of the complex

have the same mechanism.

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depends on the interaction between subunits, there is not enough data to determine whether plants

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3. Phosphorylation of the catalytic subunit

The biochemical knowledge about the regulation of the SnRK1 complex is quite behind in relation to that of AMPK and SNF1. However, it is frequently assumed that SnRK1 will behave similarly to AMPK and SNF1. In a clear example, it is presumed that the phosphorylation of the AMPKαThr172 in the activation loop in AMPK indicates kinase activation. However, it has also been

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shown that phosphorylation does not always correlate with an increase in the activity of the kinase, suggesting that alternative mechanisms could also participate in its regulation [27]. In other systems, it has been observed that phosphorylation at different sites is necessary for the fine-tuned regulation of kinase activity, and it appears that AMPK/SNF1 behaves in this manner. It has been observed that, in addition to the phosphorylation of AMPKα1Thr172 (which is the canonical phosphorylation site in the activation loop), AMPKα1Ser173 and AMPKα1Ser176 are also relevant to the

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regulation of AMPK kinase activity. The phosphorylation of AMPKα1Ser173 by Protein Kinase A (PKA) prevents the phosphorylation of AMPKα1Thr172 by LKB and contributes to the modulation of AMPK activation [28,29]. In addition, AMPKα1Ser176 is phosphorylated when AMPK is inactive, suggesting it has an inhibitory role in AMPK activation [30]; in all these cases, different activating kinases are participating. In yeast, the catalytic subunit is phosphorylated at Snf1Thr210, but Snf1Ser211 is also phosphorylated; however, the change in Snf1S211A does not modulate SNF1 activity, indicating that phosphorylation of Snf1Ser211 has neither an activating nor inhibitory role. The

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catalytic subunit is also phosphorylated at Snf1Ser214, and this phosphorylation negatively modulates the activity of the kinase [31]. Both residues, Snf1Ser173 and Snf1Ser176, which are present in AMPK,

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are conserved in the catalytic subunits of SnRK1, and the phosphorylation of SnRK1α1Ser176 in Arabidopsis was detected in vivo [32]. The role of the phosphorylated SnRK1α1Ser176 has not been

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studied, but data from our lab indicate that the SnAK1-activating kinase can phosphorylate this residue in vitro and that the change of SnRK1α1S175 inhibits SnRK1 activity (unpublished data).

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Extensive biochemical studies are needed to supplement the current information on plant enzymes

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(Fig. 1).

Another important issue associated with the study of phosphoproteins arises from the fact that

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antibodies that have been designed to detect phosphorylation of Thr172 in AMPKα1 are commonly used to evaluate the phosphorylation status of the SnRK1 catalytic subunit. It is possible that these antibodies might not distinguish between the phosphorylation of contiguous residues, and this could lead to misinterpretations concerning kinase activation. In yeast, for example, a change in Snf1S211A/C abrogated the ability of the anti-pThr antibody to recognize the phosphorylation of

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Snf1Thr210, and this failed detection was interpreted as kinase inactivation [31]. In addition to more specific antibodies to determine the phosphorylation of the catalytic subunit, any evaluation of the SnRK1 activation status must include the measurement of SnRK1 activity as well as an assessment of the changes in the phosphorylation of a target protein.

4. Interactors

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The interaction of SnRK1 catalytic subunits with a large number of proteins unrelated to its kinase function has been described. This multitude of potential interactions dramatically expands the possibility that SnRK1 acts as a regulator of plant responses. A short list of examples includes its association with proteins containing a domain of unknown function (DUF) 581, which potentially facilitates the interaction of SnRK1 with other proteins to produce more specific responses [33]. The interaction with CYCLIN-DEPENDENT KINASE F;1 (CDKF;1) could provide a direct link to the signaling that modulates energy levels and controls the cell cycle [34]. The association with the

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WD40 repeated region of a myoinositol polyphosphate 5-phosphatase makes the SnRK1 catalytic subunits more stable for proteasome degradation [35]. However, the expression of both subunits

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increases the stability of the STOREKEEPER RELATED1/G-Element Binding Protein [36]. In rice, the N-terminal region of SnRK1A-interacting negative regulators (SKINs) interacts with the KD

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domain of SnRK1A to antagonize the function of SnRK1 and thus prevent its overactivation in response to the increase in ABA [37]. The interaction of the SnRK1 catalytic subunits with the

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trihelix transcription factor (ShCIGT) isolated from wild tomato Solanum habrochaites has also been described [38]. Finally, it has been reported that SnRK1β forms a complex with StInvInh2b, which

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inactivates the inhibitor of a vacuolar invertase (StvacINV1); however, the inhibitory effect of StInvInh2b can be restored by adding phosphorylating SnRK1αThr175 [39]. There is a consensus that

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the functional SnRK1 kinase is a complex formed by the catalytic subunit associated with the β and γ regulatory subunits. However, the information presented above clearly suggests that, by its interaction with other proteins, the free subunits could also have additional roles that complement SnRK1 functions. This hypothesis offers an attractive way to understand how SnRK1 participates in the regulation of multiple aspects of plant metabolism and development. It has been suggested that

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the SnRK1 catalytic subunits can be phosphorylated and activated as free subunits or as part of the SnRK1 complex [21,22]. Such functions would establish an essential difference between the system in plants and that in animals and yeast. According to this proposal, the catalytic subunit is constitutively activated and negatively regulated in response to specific physiological conditions, by sugar phosphates mainly by T6P [41, 42]. The idea that the kinase subunit can act independently of the regulatory subunits is exciting and could explain some of the changes that are observed when

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this subunit is expressed in a transient or stable form [36, 37, 38]. However, it is essential to emphasize that the catalytic subunit is not active if it is not phosphorylated and that autophosphorylation is low and not sufficient for good activation [21, 40]. It seems unlikely that the number of catalytic subunits limits SnRK1 activity in vivo (Fig. 1). We have observed that only a small proportion of the SnRK1α is phosphorylated and they are associated with fractions with high kinase activity [43]. The available information shows the need to develop sensitive procedures to assess the potential role of free phosphorylated catalytic subunits. Finally, moonlighting proteins are

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defined by their capacity to perform multiple functions depending on cell type, stage of development, intracellular distribution, and environmental changes. Moonlighting proteins are part

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to investigate whether the SnRK1 subunits fit in this category.

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of the cost-efficient and effective solution to solve complex problems [44]. It will be equally important

5. Future Perspectives

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SnRK1 kinases are part of a large interactive network of signaling in plants that respond to multiple external stimuli. Their activity is mostly dependent on the phosphorylation of the SnRK1α1Thr175 in

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the activation loop. However, since the phosphorylation of the catalytic subunit in other sites might affect SnRK1 activity, the implementation of new strategies to assess the activation state of the

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kinase should be performed. It is necessary to investigate the complex composition in vivo and to evaluate how it may contribute to the multiple processes in which SnRK1 is involved. It is possible that the catalytic subunit interactions with other proteins lead to additional functions that complement SnRK1 functions. It is necessary to investigate the molecular basis of the physiological

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responses observed when the catalytic subunits are overexpressed.

Acknowledgments We apologize to all the authors whose excellent works and reviews could not be cited due to space restrictions. Funding for PC lab and EMB lab was supported by PAPIIT IN227019 and PAPIIT IN203017, respectively.

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