The UBA domain of SnRK1 promotes activation and maintains catalytic activity.

Sucrose non-fermenting 1-related protein kinase 1 (SnRK1) is a central metabolic regulator and the plant orthologue of the mammalian AMP-activated protein kinase (AMPK); both are energy-sensing heterotrimeric enzymes comprising a catalytic α- and regulatory β- and γ-subunits. α-Subunits contain a serine/threonine kinase domain (KD) at their N-terminus that is immediately followed by a small regulatory domain termed the auto-inhibitory domain (AID) in AMPK and the ubiquitin-associated domain (UBA) in SnRK1. Association of the AID with the AMPK KD inhibits activating phosphorylation of the KD by upstream kinases and promotes dephosphorylation, as well as inhibiting AMPK catalytic activity. Despite these mechanistic insights regarding the AMPK AID, the SnRK1 UBA regulatory implications have not been investigated. Using recombinant protein comprising either the KD-only or KD-AID/KD-UBA, we found that the UBA of SnRK1 acts in a distinct regulatory manner to its orthologous AID of AMPK. Firstly, the plant upstream kinase GRIK2 preferentially phosphorylates the SnRK1 KD-UBA. Secondly, the SnRK1 KD in the absence of the UBA shows near identical initial catalytic activity to the KD-UBA, but in comparison a rapid loss of catalytic activity is observed. Our findings indicate that the role of the UBA in SnRK1 regulation may be more akin to that of the UBA in the mammalian AMPK-related kinases rather than its immediate functional orthologue, AMPK. This study adds to a growing body of work demonstrating the divergent regulatory mechanisms of the orthologous plant SnRK1 and mammalian AMPK.