GRIK phosphorylates and activates KIN10 which also promotes its degradation

The sensor kinase Sucrose Non-fermenting-1-Related Kinase 1 (SnRK1) plays a central role in energy and metabolic homeostasis. KIN10 is a major catalytic (α) kinase subunit of SnRK1 regulated by transcription, posttranslational modification, targeted protein degradation, and its subcellular localization. Geminivirus Rep Interacting Kinase 1 and 2 (GRIK1 and 2) are immediate upstream kinases of KIN10. In the transient protein expression assays carried out in Nicotiana benthamiana (N. benthamiana) leaves, GRIK1 not only phosphorylates KIN10 but also simultaneously initiates its degradation. Posttranslational GRIK-mediated KIN10 degradation is dependent on both GRIK kinase activity and phosphorylation of the KIN10 T-loop. KIN10 proteins are significantly enriched in the grik1-1 grik2-1 double mutant, consistent with the transient assays in N. benthamiana. Interestingly. Among the enriched KIN10 proteins from grik1-1 grik2-1, is a longer isoform, putatively derived by alternative splicing which is barely detectable in wild-type plants. The reduced stability of KIN10 upon phosphorylation and activation by GRIK represents a mechanism that enables the KIN10 activity to be rapidly reduced when the levels of intracellular sugar/energy are restored to their set point, representing an important homeostatic control that prevents a metabolic overreaction to low-sugar conditions. Since GRIKs are activating kinases of KIN10, KIN10s in the grik1 grik2 double null mutant background remain un-phosphorylated, with only their basal level of activity, are more stable, and therefore increase in abundance, which also explains the longer isoform KIN10L which is a minor isoform in wild type is clearly detected in the grik1 grik2 double mutant.


Introduction
Plant Sucrose Non-fermenting-1-Related Kinase 1 (SnRK1) belongs to a family of Ca 2+ -independent serine/threonine protein kinases that are related to the Sucrose Non-Fermenting 1 (SNF1) kinase found in fungi, and the AMP-activated protein kinase (AMPK) in animals (Broeckx et al., 2016).In plants, SnRK1 functions as an important metabolic sensor kinase that is activated under low carbon/energy conditions.Activated SnRK1 phosphorylates a constellation of target proteins including key transcription factors and metabolic enzymes that results in a broad reprogramming of metabolism (Sugden et al., 1999;Baena-Gonzaĺez et al., 2007;Tsai and Gazzarrini, 2012;Mair et al., 2015;Zhai et al., 2017).SnRK1 is a heterotrimeric complex composed of a catalytic a subunit (encoded by KIN10 and KIN11, also known as SnRK1a1, and SnRK1a2, respectively) in Arabidopsis and regulatory subunits: b and bg (Ramon et al., 2013;Emanuelle et al., 2015;Peixoto and Baena-Gonzaĺez, 2022).KIN10 is also capable of activity independent of its regulatory subunits (Ramon et al., 2019).KIN10 is broadly expressed while KIN11 expression is restricted to specific tissues and developmental stages (Williams et al., 2014).Alternate splicing of KIN10 results in two KIN10 protein isoforms.The long KIN10 isoform (referred to as KIN10L herein) has a 23 residue N-terminal extension relative to the short KIN10 (referred to as KIN10).KIN10 appears to be the major KIN10 form in planta because the transcript levels of KIN10 in multiple tissues under laboratory growth conditions are reported to be much higher than those of KIN10L (Williams et al., 2014).In terms of physiological functions, overexpression of KIN10 leads to hypersensitivity to glucose and abscisic acid (ABA) (Jossier et al., 2009).It also increases leaf soluble sugar (i.e., glucose, Fructose, and sucrose) content (Jossier et al., 2009;Wang et al., 2019).Significant amounts of starch are detected in the kin10 kin11 double mutant at the end of the dark period suggesting that KIN10 and KIN11 are involved in mobilizing starch during darkness (Baena-Gonzaĺez et al., 2007).While the functions of KIN10 and KIN11 largely overlap, some differences have been noted, for instance, KIN10 overexpression delayed flowering while KIN11 overexpression promoted flowering (Baena-Gonzaĺez et al., 2007;Tsai and Gazzarrini, 2012;Williams et al., 2014;Wang et al., 2019).KIN10 was reported to positively regulate stomatal development under high sucrose conditions.Both kin10 and kin11 single mutants showed lower stomatal index relative to wild type (Han et al., 2020).For a broader description of the functions of KIN10 and KIN11, please refer to the following reviews (Crepin and Rolland, 2019;Margalha et al., 2019;Baena-Gonzaĺez and Lunn, 2020;Peixoto and Baena-Gonzaĺez, 2022).
Besides activation of KIN10/11 by its upstream kinases, selective degradation of KIN10/11 is a mechanism that attenuates SnRK1 signaling and prevents detrimental hyperactivation during responses to stresses.KIN10 can interact with Pleiotropic Regulatory Locus 1 (PRL1) (Bhalerao et al., 1999) and KIN10 degradation is mediated by the DDB1-CUL4-ROC1-PRL1 E3 ubiquitin ligase, via its interaction with the KIN10-PRL1 complex (Lee et al., 2008).Under low-nutrient conditions, myoinositol polyphosphate 5-phosphatase 13 (5PTase13) is required to stabilize KIN10 and slow its degradation by the 26S proteasomal pathway (Ananieva et al., 2008).It has been demonstrated that application of ABA to wheat roots can result in a dramatic reduction of KIN10 (Coello et al., 2012).KIN10 degradation is reported to be strictly dependent on its kinase activity because two KIN10 kinase mutants: T175A and K48M (impaired in their phosphotransferase activity) accumulate to higher levels than wild type KIN10 due to its reduced degradation (Baena-Gonzaĺez et al., 2007;Crozet et al., 2016).In other studies, KIN10/11 were also found to be SUMOylated by SIZ1 (E3 Small Ubiquitin-like Modifier (SUMO) ligase), marking them for proteasomal degradation (Crozet et al., 2016).
Based on the observations that GRIK is the major kinase that phosphorylates and activates KIN10 at its T-loop and that a KIN10 T-loop mutant [KIN10 (T175A)] shows increased stability relative to KIN10, we tested whether GRIK is directly involved in KIN10 degradation.
Here, we report that transient co-expression of KIN10 with GRIK1 in Nicotiana benthamiana (N.benthamiana) leaves results in significant degradation of KIN10 and that the GRIK1-dependent KIN10 degradation is contingent on the kinase activity of GRIK1 in phosphorylating the KIN10 T-loop.
Consistently, KIN10 protein levels are significantly elevated in the grik1-1 grik2-1 double mutant.Two isoforms of KIN10 are identified upon immunoprecipitations using KIN10 antibody from the grik1-1 grik2-1 double mutant, among them is a long alternative splicing isoform that is a minor isoform in wild-type plant.

GRIK1 phosphorylates KIN10 promoting its degradation
It was previously reported that KIN10 degradation is strictly dependent on its kinase activity (Baena-Gonzaĺez et al., 2007;Crozet et al., 2016).Since GRIK is the major kinase that phosphorylates and activates KIN10, we tested whether GRIK is involved in KIN10 degradation.GFP signal from GFP-tagged KIN10L was monitored upon transient co-expression of KIN10L with GRIK1 in Nicotiana benthamiana (N.benthamiana) leaves by fluorescence microscopy (Figure 1A) and western blotting with anti-GFP antibodies (Figure 1B).Previously, it was shown that Threonine-198 (T198) in the T-loop of KIN10L (equivalent to T175 in the T-loop of KIN10) is phosphorylated by GRIK and essential for KIN10 kinase activity (Shen et al., 2009).Consistent with Overexpression of GRIK1 results in KIN10 degradation in N. benthamiana Leaves.previous reports, expression of the KIN10L T198A phosphorylation mutant resulted in increased protein accumulation relative to KIN10L (Figures 1A-C).Co-expression of GRIK1 with KIN10L greatly reduced KIN10L accumulation relative to the expression of KIN10L alone (Figures 1A-C).Mn 2+ -Phos-tag gel electrophoresis was used to separate phosphorylated from non-phosphorylated proteins, revealed that most of the residual KIN10L upon its cotransformation with GRIK1 was present in phosphorylated form (Figure 1B).There was only a single detected band for GFP-KIN10L (T198A) visible in the Mn 2+ -Phos-tag gel blot upon cotransformation with GRIK1-HA (Figure 1B), suggesting that T198 in the T-loop is the only phosphorylation site for GRIK1.That the T-loop phosphorylation mutant of KIN10L was strongly stabilized relative to its parental wild-type sequence also suggests posttranslational regulation.To confirm this, we compared the levels of GFP-KIN10L mRNA upon its expression alone versus upon its co-expression with GRIK1-HA.The levels of GFP-KIN10L transcripts were equivalent for both treatments (Figure 1D), confirming that the observed reduction in GFP-KIN10 protein occurs at the posttranslational level.
To further understand GRIK-mediated KIN10 degradation we engineered a GRIK1 mutant, K137A.K137 is a key residue in the ATP binding domain of GRIK1 reported to be essential for its kinase activity (Shen et al., 2009).Co-transformation of KIN10 with GRIK1(K137A) did not result in substantial KIN10 degradation, confirming that GRIK1-mediated KIN10L degradation is dependent on the kinase activity of GRIK1 (Figures 1A, B, 2A, B).

The KIN10 long splicing isoform is enriched in a grik null mutant
To substantiate GRIK1-mediated KIN10 degradation we observed in transient N. benthamiana leaf assays, KIN10 protein levels were quantified in two Arabidopsis grik double mutants: grik1-2 grik2-1 containing the weaker grik1-2 allele and grik1-1 grik2-1 containing the stronger grik1-1 allele of GRIK1.Consistent with published results (Glab et al., 2017), phosphorylated KIN10 was observed in the grik1-2 grik2-1 line, but not in grik1-1 grik2-1, as evidenced by probing with the phosphorylated KIN10-specific antibody.This confirms that GRIK1 and GRIK2 are major activating kinases of KIN10 in vivo (Figure 3A).Immunoblot assays probed with the KIN10-specific antibody showed that two distinct forms of KIN10 are detected in WT and the grik mutants but no KIN10 immunoreactive species are visible in the kin10 mutant.The higher molecular mass KIN10 form was significantly more abundant in grik1-1 grik2-1 than in either WT or grik1-2 grik2-1 (Figure 3A).Since KIN10 has two alternative splicing Frontiers in Plant Science frontiersin.orgisoforms (i.e., KIN10L and KIN10) (Figure 3B), we hypothesize that the large and small KIN10s detected in grik1-1 grik2-1 are KIN10L and KIN10, respectively.The individual coding sequence (CDS) corresponding to KIN10L or KIN10 was transiently expressed in N. benthamiana leaves.Three days after agroinfiltration, protein samples were extracted and separated along with protein samples from the grik1-1 grik2-1 double mutant and subjected to immunoblotting.Transiently expressed KIN10L and KIN10 polypeptides in N. benthamiana showed similar SDS-PAGE mobilities to those of the putative KIN10L and KIN10 products in grik1-1 grik2-1 respectively (Supplementary Figure 1).To further confirm the identity of two sizes of KIN10 detected in the grik1-1 grik2-1 double mutant, KIN10s were immunoprecipitated with anti-KIN10 antibody from grik1-1 grik2-1 and separated by SDS-PAGE.The two protein bands were excised and analyzed with the use of tandem mass spectrometry.The faster migrating protein was confirmed to be KIN10.The slower migrating protein was identified as the KIN10L isoform (Supplementary Figure 2).These data show that KIN10L, a minor KIN10 isoform in WT, is enriched in the grik1-1 grik2-1 double mutant background due to the increased protein stability of its unphosphorylated form in grik1-1 grik2-1.

KIN10L accumulates to higher levels than KIN10 when transiently expressed in N. benthamiana leaves
To evaluate whether there are differences between KIN10L and KIN10, GFP-KIN10L or GFP-KIN10 were transiently co-expressed for 3 days in N. benthamiana leaves with either GRIK1 or an empty vector.As shown in Figure 4, compared with GFP-KIN10, more intense GFP fluorescence corresponding to GFP-KIN10L was observed as the puncta in the cytosol, consistent with reports by Williams (Williams et al., 2014) (Figure 4A).Immunoblot assays showed the levels of GFP-KIN10L were significantly higher than GFP-KIN10 (Figure 4B) upon co-expression with EV.Coexpression with GRIK1 dramatically reduced both KIN10L and KIN10 protein levels (Figure 4B).The accumulation of KIN10L seems related to its subcellular localization because a putative nuclear localization signal mutant of KIN10L (K250A, K251A, K253A) in the sequence LFKKIKG which is a match to monopartite nuclear localization signal K•(K/R) •X•(K/R) (Chelsky et al., 1989), accumulated to higher levels than native KIN10L.Conversely, fusing KIN10L with the SV40 NLS resulted in almost complete retention of KIN10L within the nucleus and promoted its degradation (Supplementary Figure 3).

Kinase activity of KIN10L is equivalent to that of KIN10
Next, we tested whether the kinase activity of KIN10L is equivalent to that of KIN10 i.e., whether the extra 23AA at the N-terminus of KIN10L has any effect on its in vitro kinase assay.A His-trigger factor (TF) followed by a factor Xa protease cleavage site domain was fused to the N-termini of KIN10L or KIN10.The constructs were expressed in E. coli and the resulting protein products were purified with the use of Ni-NTA chromatography.KIN10L or KIN10 were recovered after factor Xa protease digestion to remove the affinity tag, yielding proteins with equivalent, i.e., approximately 95% purity as assessed by SDS-PAGE and Coomassie Brilliant blue staining (Supplementary Figure 4).For KIN10 protein levels are significantly higher in the grik1-1grik2-1 double knockout mutant seedlings than that in wild type Arabidopsis.(A) immunoblot analysis of proteins extracted from 10-day-old of seedlings of WT, grik1-2grik2-1 (a weak grik1grik2 double mutant), grik1-1grik2-1 (a strong grik1grik2 double mutant) and kin10 respectively.KIN10 or P-AMPK a-1 (Thr-172) antibody was used to detect KIN10 or phosphorylated KIN10 respectively.Ponceau S staining of Rubisco is shown as a loading control.(B) Protein sequence alignment of long (KIN10L) and short (KIN10) alternative splicing isoforms of KIN10 shows 23 more amino acid residues on N terminus of KIN10L than KIN10.
the kinase assays, a recombinant KIN10 kinase domain (KIN10KD) and GRIK1 were used as a positive kinase control and activator, respectively.The expected low i.e., basal levels of phosphorylation activity were observed in for KIN10L or KIN10 in the absence of GRIK1 activation, although KIN10KD showed higher activity than either isoform.Upon GRIK1 activation, the kinase activities of both KIN10L and KIN10 were elevated dramatically during a 10-minute time course during which no significant differences in kinase activity were detected between KIN10L and KIN10 (Figure 5).

Discussion
SnRK1 is an evolutionarily conserved sensor kinase that plays critical roles in plant stress responses and development by regulating gene expression and enzyme activities.As a major kinase subunit of SnRK1, KIN10 is regulated by transcription, posttranscriptional modification, targeted protein degradation and subcellular localization.Among these regulatory mechanisms, targeted protein degradation of KIN10 is crucial for rapid SnRK1regulated plant responses to ever-changing energy stress conditions.Since KIN10 kinase mutants such KIN10 (T175A) and KIN10 (K48A) were previously shown to be more stable and GRIKs are major kinases that activate KIN10, in this research we focused on the regulatory role of GRIK1 on KIN10 stability.The results from both protein transient expression assays in N. benthamiana leaves and the characterization of grik mutants supports the hypothesis that GRIK not only phosphorylates and activates KIN10 but also promotes its degradation.For GRIK1-mediated KIN10 degradation, we reason that the reduced stability of KIN10 upon phosphorylation and activation by GRIK represents a mechanism that enables the KIN10 activity to be rapidly reduced when the levels of intracellular sugar/energy are restored to their set point, representing an important level of homeostatic control that prevents a metabolic overreaction to low sugar conditions.Since GRIKs are activating kinases of KIN10, KIN10s in the grik1 grik2 double null mutant background remain un-phosphorylated, with only their basal level of activity, are more stable, and therefore The long KIN10 isoform (KIN10L) accumulates to higher levels than KIN10 when transiently expressed in N. benthamiana leaves.increase in cellular abundance, which also explains why the longer isoform KIN10L which is a minor isoform in wild type plant can be clearly detected in the grik1 grik2 double mutant.
Several recent reports showed nuclear translocation of KIN10 plays an important regulatory contribution with respect to SnRK1 downstream regulation (Ramon et al., 2019;Belda-Palazoń et al., 2022;Shi et al., 2022).For example, abscisic acid (ABA) exposure triggers rapid subcellular re-localization of KIN10 from the nucleus to the cytoplasm and this is accompanied by the inhibition of the target of rapamycin (TOR) sensor kinase (Belda-Palazoń et al., 2022), implying that the subcellular re-localization of KIN10 likely exposes it to a different set of phosphorylation targets.In this study, characterizations of KIN10L and KIN10 show that although recombinant KIN10L and KIN10 demonstrate similar kinase activities with respect to in vitro kinase assay, KIN10L tends to accumulate to higher levels than KIN10 upon transient expression in N. benthamiana.Together with our observation that a putative subcellular localization mutant accumulates to different levels, implies that insufficient SnRK1 activity in vivo may activate a feedback mechanism to regulate the alternative splicing of KIN10.Future research on this feedback regulatory mechanism will likely provide additional insights into the complexity of SnRK1 signaling.
For expression in plants, the PCR products were cloned into the Invitrogen GATEWAY M pDONR/Zeo vector (Thermo Fisher Scientific, Waltham, MA; www.thermofisher.com) using the BP reaction and sub-cloned (LR reaction) into the plant GATEWAY ™ binary vector: pGWB414 (HA-tag at C terminal) or pMDC43 (GFP-tag at N terminal) (Nakagawa et al., 2007).

Nicotiana benthamiana agroinfiltration
Transient gene expression in N. benthamiana by agroinfiltration was carried out according to a previous described procedure (Schütze et al., 2009).Leaves were harvested 3 days after agroinfiltration for imaging with a Leica SP5 confocal laser scanning microscope or protein content analysis.In vitro protein kinase assays of purified recombinant full length of KIN10 or KIN10L show that two KIN10 splicing isoforms have similar kinase activity.KIN10 activity is quantified by the incorporation of 32 P from [g-32 P] ATP into the SPS peptide.Activity was measured in a 25µL-reaction containing the different KIN10 isoforms in the absence or presence of GRIK1 for the indicated times.KIN10 forms include KIN10(KD), the kinase domain of KIN10.KIN10, the short splicing isoform of KIN1.KIN10L, long splicing isoform of KIN10.The center line of the box and whisker plot denotes the mean, the box represents the interquartile range while the whiskers represent the 5th and 95th percentile (n = 3 or 4 independent biological replicates).One-way analysis of variance (ANOVA) and Tukey-Kramer Honestly Significant Difference (P <0.05) are used to compare means.Different letters above boxes indicate a significant difference.
(A) Representative fluorescence confocal images of N. benthamiana leaf samples 3 d after co-agroinfiltration with gene expression combinations as shown.EV, empty vector.KIN10L, long splicing protein isoform of KIN10.KIN10L(T198A), a KIN10L mutant.Bar = 250 mm.(B) immunoblot analysis of samples in (A) shows protein levels of total GFP-KIN10L or GRIK1-HA and respective phosphorylated (GFP-KIN10L-P) and non-phosphorylated GFP-KIN10L (Mn 2+ -Phos-tag is a 10% SDS-PAGE containing 50mM of Mn 2+ -Phos-tag™.Ponceau S staining of Rubisco is shown as a loading control.In all figures, multiple lanes for one gene combination or one genotype represent biological replicates.(C) Relative GFP-KIN10L protein levels in (B) quantified with GelAnalyzer2010 and normalized against corresponding protein loading.Data shown are mean ± SD, n=3 independent immunoblots; One-way analysis of variance (ANOVA) and Tukey-Kramer Honestly Significant Difference (P <0.05) are used to compare means.Different letters above boxes indicate a significant difference.(D) Reverse transcription quantitative PCR (RT-qPCR) results of KIN10L and GRIK1 in (A), values are means ± SD, n=5 independent experiments.Statistics is performed by using mean crossing point deviation analysis computed by the relative expression [REST] software algorithm.The blue bars represents gene transcript for GFP-KIN10L and the open bars represents for gene transcript for GRIK1-HA.
FIGURE 2 GRIK1 mediated KIN10 degradation is dependent on GRIK1 kinase activity.(A) Representative fluorescence confocal images of N. benthamiana leaf samples 3 d after co-agroinfiltration with gene expression combinations as shown.GRIK1(K137A) and GRIK1 (S261A) are GRIK1 mutants.Bar = 250 mm.(B) immunoblot analysis of samples in (A) shows protein levels of total GFP-KIN10 or GRIK1-HA and respective phosphorylated (GFP-KIN10-P) and non-phosphorylated GFP-KIN10 (Mn 2+ -Phos-tag).Ponceau S staining of Rubisco is shown as a loading control.
FIGURE 3 (A) Representative fluorescence confocal images of N. benthamiana leaf samples 3 d after co-agroinfiltration with gene expression combinations as shown.Bar = 250 mm.Fluorescence signal in nucleus is marked by white arrowhead.(B) immunoblot analysis of samples in (A) shows protein levels of GFP-KIN10L or GFP-KIN10 or GRIK1-HA.Ponceau S staining of Rubisco is shown as a loading control.