A Complex Containing SNF1-Related Kinase (SnRK1) and Adenosine Kinase in Arabidopsis

<div><p>SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5′-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5′-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK <i>in vitro</i> by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for <i>in vivo</i> linkage but also revealing that <i>in vivo</i> regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.</p></div

ADK is phosphorylated by SnRK1 <i>in vitro</i>.

<p>Kinase assays were conducted using γ³²P-ATP with SnRK1-KD or SnRK1-KD-K49R either alone or with ADK. With the exception of autophosphorylation assays, SnRK1-KD and SnRK1-KD-K49R were pre-incubated with unlabeled ATP to obscure autophosphorylation. Samples were fractioned by SDS-PAGE and signals detected using a phosphor-imager. Immunoblots were probed with anti-ADK to detect ADK, or with anti-HA to detect SnRK1-KD or SnRK1-KD-K49R. (A) Kinase assays with SnRK1-KD (10 ng) and SnRK1-KD-K49R (30 ng) were performed without pre-incubation with unlabeled ATP. (B) ADK protein (3 µg) was incubated with SnRK1-KD (10 ng) or SnRK1-KD-K49R (30 ng). Note that SnRK1-KD autophosphorylation (in the lane lacking ADK) was nearly undetectable due to pre-incubation of SnRK1 with unlabeled ATP. The same pre-incubated SnRK1-KD and SnRK1-KD-K49R preparations were used to perform the GST-SAMS phosphorylation experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087592#pone-0087592-g001" target="_blank">Figure 1C</a>. Activity data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087592#pone.0087592.s006" target="_blank">Table S3</a>.</p

Cellular SnRK1 and ADK activities are linked.

<p>In all cases, γ³²P-ATP and appropriate substrates were added to total soluble protein extracts to measure ADK (200 ng extract) and SnRK1 (20 µg extract) activities. (A) Arabidopsis plants containing a dexamethasone (dex)-inducible ADK transgene (lines ADK-L5 and ADK-L6) were dex- or mock-treated prior to extract preparation. (B) As before, except that ADK activity was reduced by RNA interference in Arabidopsis plants containing a dex-inducible dsADK transgene (lines dsADK-L4 and dsADK-L6). (C) Transgenic <i>N. benthamiana</i> plants (lines S-3 and S-5) expressed full-length, sense SnRK1 mRNA from the constitutive 35S promoter. Non-transgenic, wild-type (WT) plants served as control. (D) As before, except that SnRK1 activity was reduced by RNA interference in transgenic <i>N. benthamiana</i> plants (lines AS-12 and AS-4) that expressed antisense SnRK1 RNA. In (A) and (B), three independent experiments using pooled tissue from three plants, with two replicates each, were performed. In (C) and (D), two independent experiments were performed, each using two individual plants from each line and two replicate samples. Values indicate the mean +/− SE. Asterisks indicate significant differences at the 95% (*) or 99% (<b>**</b>) confidence level, as determined by Student's <i>t</i> test. ADK and SnRK1 activity data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087592#pone.0087592.s009" target="_blank">Tables S6A</a>-D.</p

SnRK1-KD and SnRK1-KD-K49R stimulate ADK activity <i>in vitro</i>.

<p>(A) ADK (10 ng) was incubated alone, or with two-fold molar excess of HA₂His₆-tagged SnRK1-KD, SnRK1-KD-K49R, GFP, or APT1 purified from <i>N. benthamiana</i>. ADK activity was measured and normalized to ADK+GFP. (B) ADK (10 ng) was incubated alone, or with a two-fold molar excess of GST, GST-fused SnRK1-KD, or SnRK1-KD-K49R purified from <i>E. coli</i>. ADK activity was normalized to ADK+GST. In each case, data were obtained from three independent experiments, with two replicates each. Values indicate the mean +/− SE. Asterisks (<b>**</b>) indicate significant differences in ADK activity at 99% confidence, as determined by Student's <i>t</i> test. (C) The graph shows relative ADK activity plotted against increasing SnRK1-KD:ADK ratio. HA₂His₆-SnRK1-KD (diamonds); GST-SnRK1-KD (squares). The data are representative of three experiments. ADK activity data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087592#pone.0087592.s007" target="_blank">Table S4A</a>-C.</p

The <i>in vitro</i> SnRK1 assay.

<p>(A) Diagrams of GST-SAMS and GST-SAMA substrates. The target serine in GST-SAMS, containing a SNF1/AMPK/SnRK1 consensus site, is replaced by alanine in GST-SAMA (residues underlined). The line connecting the SAMS/SAMA sequence to GST indicates a flexible polyglycine linker. These substrates were expressed and purified from <i>E. coli</i> cells. (B) Autophosphorylation assay confirmed that SnRK1-KD, but not SnRK1-KD-K49R, expressed in <i>N. benthamiana</i> is active. HA₂His₆-tagged SnRK1-KD and SnRK1-KD-K49R (inactive mutant, negative control) expressed and purified from <i>N. benthamiana</i> were incubated with γ³²P-ATP alone or with GST-SAMS or GST-SAMA. Aliquots were fractionated on polyacrylamide gels containing SDS (SDS-PAGE) and exposed to a phosphor-imager to detect labeled proteins. For loading controls, GST-SAMS and GST-SAMA were monitored by Coomassie staining, while immunoblots were probed with anti-HA (α-HA) to detect the kinases. (C) Kinase assays in which GST-SAMS or GST-SAMA (3 µg) were incubated with varying amounts of SnRK1-KD or SnRK1-KD-K49R, as indicated. (D) A linear correlation (R² = 0.9664) was observed between the intensity of labeled GST-SAMS signal and the amount of SnRK1-KD added in the range tested. The data shown are representative of three experiments, and were normalized to activity observed with 5 ng SnRK1-KD. (E) Extracts from <i>N. benthamiana</i> cells expressing full-length SnRK1, SnRK1-KD, or GUS (control for endogenous activity) from a plasmid vector were assayed following the addition of γ²P-ATP and GST-SAMS. SnRK1-KD activity data (autophosphorylation and phosphorylation of GST-SAMS) is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087592#pone.0087592.s004" target="_blank">Table S1</a>.</p

SnRK1 co-immunoprecipitates with ADK.

<p>(A) Immunoprecipitation (IP) and immunodetection were performed with ADK antibody (α-ADK) and TMD1 antibody (α-TMD1). Activated SnRK1 was detected using an antibody raised against phosphorylated AMPK (α-pT172). (B) Immunoprecipitates obtained with α-ADK or α-TMD1 were incubated with 1 µM adenosine and γ³²P-ATP. Controls contained purified ADK or no added protein. Reaction mixtures were resolved by thin layer chromatography and labeled AMP was detected using a phosphor-imager. (C) Immunoprecipitates obtained with α-ADK or α-TMD1 were incubated with 3 µg of GST-SAMS or GST-SAMA and γ³²P-ATP. Assays with purified SnRK1-KD were included as controls. Samples were fractionated on SDS-PAGE gels and exposed to a phosphor-imager for 2 h (SnRK1-KD controls) or 2 days (immunoprecipitate samples). GST-SAMS and GST-SAMA were monitored by Coomassie stain.</p

Endogenous <i>N. benthamiana</i> SnRK1 activity co-purifies with expressed ADK.

<p>(A) HA₂His₆-tagged Arabidopsis ADK and HA₂His₆-GFP (control) were expressed in <i>N. benthamiana</i> and enriched using nickel-NTA resin. Proteins were fractionated by SDS-PAGE, and Coomassie stained samples are shown. (B) GST-SAMS and γ³²P-ATP were added to ADK or GFP preparations (3 µg protein). Samples were separated by SDS-PAGE and exposed to a phosphor-imager for 2 days. The Coomassie stained panel is a loading control for GST-SAMS and GST-SAMA, while the immunoblot (bottom panel, probed with anti-HA) is a loading control for ADK and GFP.</p

ADK interacts with SnRK1 and SnRK1-KD in yeast cells.

<p>The indicated bait proteins were expressed as GAL4 DNA binding domain fusions, and the prey proteins as GAL4 activation domain fusions, in yeast Y190 cells. SnRK1 indicates the full-length protein and SnRK1-KD denotes the kinase domain. Chloramphenicol acetyl transferase (CAT) and p53 were negative controls. Interaction was indicated by cell growth on medium lacking histidine and containing 50 mM aminotriazole. As an additional indicator of interaction, colonies were monitored for LacZ activity (blue color) using a filter-lift assay.</p

SnRK1 and ADK interact in the cytoplasm.

<p>Constructs expressing full-length SnRK1, ADK, DCL4, or DRB4 proteins fused to the N- or C-terminal portion of YFP were delivered by agroinfiltration to <i>N. benthamiana</i> leaves. Images were captured at 40x magnification 48 h post-infiltration using a confocal microscope. Representative images of epidermal cells, which have a characteristic irregular shape and a large vacuole that restricts the cytoplasm to the cell periphery, are shown. Histone H2B fused to RFP (RFP-H2B) is a marker for the nucleus.</p