Expression and regulation of the AMP-activated protein kinase-SNF1 (sucrose non-fermenting 1) kinase complexes in yeast and mammalian cells: studies using chimaeric catalytic subunits.

Abstract

Mammalian AMP-activated protein kinase (AMPK) and yeast SNF1 (sucrose non-fermenting 1) kinase are members of a highly conserved protein kinase family that plays an important role in energy homoeostasis. AMPK and SNF1 kinase are heterotrimeric complexes consisting of a catalytic subunit and two regulatory subunits. We swapped the C-terminal regulatory domains of the catalytic subunits of AMPK (alpha) and SNF1 kinase (Snf1) and compared the expression and regulation of these chimaeric proteins with the native catalytic subunits in both mammalian and yeast cells. In mammalian cells, alpha1-Snf1 yielded a functional kinase complex following co-expression with the yeast regulatory subunits Sip2 and Snf4. Unlike native AMPK, the alpha 1-Snf1 complex was not activated by the stresses that deplete intracellular AMP. Significantly, hyperosmotic stress led to the marked activation of both the alpha 1-Snf1 complex and AMPK, without a detectable change in adenine nucleotide levels, indicating that an alternative, non-AMP-dependent, pathway was responsible for activation. alpha1-Snf1 was able to restore growth of snf1 mutant yeast on raffinose and phosphorylated the transcriptional repressor protein Mig1. Co-expression of the AMPK trimeric complex in yeast yielded an activity, increased by low glucose, that was similar to native SNF1 kinase. Importantly, expression of AMPK restored growth of a snf1 mutant on raffinose. Our results provide clues to the regulation of AMPK and SNF1 kinase and demonstrate that, in mammalian cells, there are at least two pathways that can activate AMPK, namely one that involves an increase in the AMP/ATP ratio and one that is independent of this ratio. In yeast, the glucose signalling pathway is able to activate AMPK, suggesting that the mammalian and yeast kinase pathways are conserved.