AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy
homeostasis which achieves this through the phosphorylation of a myriad of downstream
targets. One target is TBC1D1 a Rab-GTPase-activating protein that regulates glucose
uptake in muscle cells by integrating insulin signalling with that promoted by muscle contraction. Ser237 in TBC1D1 is a target for phosphorylation by AMPK, an event which may
be important in regulating glucose uptake. Here, we show AMPK heterotrimers containing
the α1, but not the α2, isoform of the catalytic subunit form an unusual and stable association with TBC1D1, but not its paralogue AS160. The interaction between the two proteins is direct, involves a dual interaction mechanism employing both phosphotyrosinebinding (PTB) domains of TBC1D1 and is increased by two different pharmacological
activators of AMPK (AICAR and A769962). The interaction enhances the efficiency by
which AMPK phosphorylates TBC1D1 on its key regulatory site, Ser237. Furthermore, the
interaction is reduced by a naturally occurring R125W mutation in the PTB1 domain of
TBC1D1, previously found to be associated with severe familial obesity in females, with a
concomitant reduction in Ser237 phosphorylation. Our observations provide evidence for
a functional difference between AMPK α-subunits and extend the repertoire of protein
kinases that interact with substrates via stabilisation mechanisms that modify the efficacy
of substrate phosphorylation.