SnRK1 from <i>Arabidopsis thaliana</i> is an atypical <scp>AMPK</scp>

<jats:title>Summary</jats:title><jats:p>SNF1‐related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily‐conserved <jats:styled-content style="fixed-case">SNF</jats:styled-content>1/<jats:styled-content style="fixed-case">AMPK</jats:styled-content>/Sn<jats:styled-content style="fixed-case">RK</jats:styled-content>1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non‐catalytic β and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The <jats:italic>Arabidopsis thaliana</jats:italic> genome contains homologues of each subunit type, and, in addition, two atypical subunits, β<jats:sub>3</jats:sub> and βγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The <jats:italic>At</jats:italic>Sn<jats:styled-content style="fixed-case">RK</jats:styled-content>1 subunit structure was determined using recombinant protein expression and endogenous co‐immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique βγ subunit and one of three non‐catalytic β subunits: β<jats:sub>1</jats:sub>, β<jats:sub>2</jats:sub> or the plant‐specific β<jats:sub>3</jats:sub> isoform. Thus, the <jats:italic>At</jats:italic>Sn<jats:styled-content style="fixed-case">RK</jats:styled-content>1 heterotrimers contain the atypical βγ subunit rather than a conventional γ subunit. Mammalian <jats:styled-content style="fixed-case">AMPK</jats:styled-content> heterotrimers are phosphorylated on the T–loop (<jats:styled-content style="fixed-case">pT</jats:styled-content>hr175/176) within both catalytic a subunits. However, <jats:italic>At</jats:italic>Sn<jats:styled-content style="fixed-case">RK</jats:styled-content>1 is insensitive to <jats:styled-content style="fixed-case">AMP</jats:styled-content> and <jats:styled-content style="fixed-case">ADP</jats:styled-content>, and is resistant to T–loop dephosphorylation by protein phosphatases, a process that inactivates other <jats:styled-content style="fixed-case">SNF</jats:styled-content>1/<jats:styled-content style="fixed-case">AMPK</jats:styled-content> family members. In addition, we show that Sn<jats:styled-content style="fixed-case">RK</jats:styled-content>1 is inhibited by a heat‐labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three Sn<jats:styled-content style="fixed-case">RK</jats:styled-content>1 carbohydrate‐binding modules, located in the β<jats:sub>1</jats:sub>, β<jats:sub>2</jats:sub> and βγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which <jats:styled-content style="fixed-case">AMPK</jats:styled-content> binds <jats:italic>in vitro</jats:italic>. These data clearly demonstrate that <jats:italic>At</jats:italic>Sn<jats:styled-content style="fixed-case">RK</jats:styled-content>1 is an atypical member of the <jats:styled-content style="fixed-case">SNF</jats:styled-content>1/<jats:styled-content style="fixed-case">AMPK</jats:styled-content>/Sn<jats:styled-content style="fixed-case">RK</jats:styled-content>1 family.</jats:p>