AKIN10, a representative Arabidopsis SNF1‐related protein kinase 1 (SnRK1), phosphorylates and downregulates plant HMG‐CoA reductase

Abstract

HMG-CoA reductase (HMGR) is a key enzyme in the mevalonate pathway for sterols and cytosolic isoprenoid production. Although HMGR kinases from spinach, barley, and cauliflower tissues have been strongly suggested as members of SNF1-related protein kinases 1 (SnRK1), the phosphorylation and inactivation of HMGR by plant SnRK1s has not been demonstrated. In this study, we elucidated that AKIN10, an Arabidopsis SnRK1, acts as an HMGR kinase. The recombinant AKIN10 phosphorylates and inactivates AtHMGR1S using recombinant GRIK1 as the AKIN10 activator. In contrast, AKIN10-GRIK1 fails to inactivate AtHMGR1S-S577A, suggesting that this is achieved through Ser577 phosphorylation. Moreover, phosphorylation is detected not only in AtHMGR1S but also in AtHMGR1S-S577A, suggesting the presence of a novel regulatory mechanism of plant HMGR.

The SnRK1 Kinase as Central Mediator of Energy Signaling between Different Organelles

“…In fact, various examples for both possibilities have been reported as recently reviewed by Broeckx et al (2016). Key metabolic enzymes in the cytosol, such as Suc-P synthase, NR, TPS5, and HMG-CoA reductase, have been identified as direct targets of SnRK1 in vitro (Jossier et al, 2009;Robertlee et al, 2017) and in vivo (Nukarinen et al, 2016). Quantitative phosphoproteomics revealed altered phosphorylation levels of SnRK1 target sites in planta using an artificial miRNA knockdown approach to silence both AKIN10 and AKIN11 (Nukarinen et al, 2016).…”

Evidence that the <i>Arabidopsis thaliana</i> 3-hydroxy-3-methylglutaryl-CoA reductase 1 is phosphorylated at Ser577 <i>in planta</i>

“…The HMGR activity assay was started by adding 20 µl of the premix reaction buffer containing 1 µg BSA, 1 mM NADPH, 10 mM DTT, and 1 µl of [glutaryl-3-14 C] HMG-CoA (49.2 mCi mmol −1 , NEC642010UC, PerkinElmer, Inc., MA, USA) to the whole resulting products of phosphatase assay to reach a final assay volume of 25 µl at 30°C for 25 min. The reaction was stopped, and the resulting mevalonate was lactonized and then extracted with ethyl acetate to measure the radioactivity by a liquid scintillation counter (LSC-5100, Aloka, Tokyo, Japan) as previously described (Robertlee et al 2017). Twotailed students t-test was used to analyze all the data using the statistical software StatPlus:mac (AnalystSoft Inc.…”

“…The gel was treated using 10 mM EDTA (3×15 min) before transfer to polyvinylidene difluoride membranes (Milipore) using a semi-dry blotting apparatus at a constant voltage, 25 V, for 45 min (ATTO WSE-4020, Tokyo, Japan) with a three transfer buffers system (Kinoshita-Kikuta et al 2014). The presence of the AtHMGR1S band on the membrane was then analyzed by western blot using anti-AtHMG1 catalytic domain (AtHMG1cd) antibody, as previously described (Robertlee et al 2017). …”

“…We have previously shown that AtHMGR1S is inactivated through phosphorylation at S577 by the AKIN10-GRIK1 kinase cascade system in vitro (Robertlee et al 2017). …”

“…In the case of A. thaliana, three different HMGR isoenzymes are encoded by two genes, HMG1 for both AtHMGR1L and AtHMGR1S, and HMG2 for AtHMGR2 (Enjuto et al 1994;Lumbreras et al 1995), and it has been reported that HMG1 plays important physiological roles for plant growth and development Suzuki et al 2004Suzuki et al , 2009. We have also previously elucidated that AtHMGR1S is negatively regulated through phosphorylation by the AKIN10-GRIK1 kinase cascade system in vitro (Robertlee et al 2017). Because AKIN10 is the ortholog of AMPK and the phosphorylated residue (S577) corresponds to the phosphorylated serine of mammalian HMGR by AMPK, this system resembles the regulatory system of mammal HMGR by the AMPK-AMPKK kinase cascade.…”