Figure 1. C elegans AAKG isoforms contribute differentially to AMPK's phospho-activation, and its role in lifespan and reproductive health:(A) AMP-binding sites within AAKG-2, AAKG-4, and AAKG-5 modelled using PyMOL. AAKG-1-3 show high conservation of predicted key AMP-binding residues with human PRKAG1, and AAKG-2 is shown as an example. AAKG-4 and AAKG-5 do not show conservation of AMP-binding residues with PRKAG1. For each isoform, the predicted binding residues are shown as sticks: residues in green are conserved at the functional level with PRKAG1, residues in red are not. AMP molecules are shown in magenta. A multiple sequence alignment is shown below: deeper colouring indicates higher conservation between sequences, and key predicted AMP binding residues are outlined in red. (B) C. elegans has five AMPKγ isoforms which can be grouped by the presence or absence of conserved residues predicted to influence its ability to bind AMP (as in (A)). Mutant alleles of each γ isoform were combined genetically to create strains that we predict act AMP-dependently or AMP-independently. (C) Phosphorylation of the AAK-2 protein (α subunit) at Thr172. Western blot analysis with and without 4.5mM phenformin. Graph shows quantification from three biological replicates, representative blot shown below the graph. Data shown is normalised to untreated WT/N2 phosphorylation. (D) Effect of aakg mutation on WT lifespan. Representative plot of three biological replicates shown. n=>100 for each group. (E) Effect of aakg mutation on fecundity. Data pooled from three biological replicates. n=>27 for each group. (F) Effect of aakg mutation on the lifespan of animals with reduced insulin signalling (
daf-2 mutants). Representative plot of four biological replicates shown. n=>100 for each group. In C and E, *= p<0.05, **= p<0.01, ***= p<0.001, using Student's two-tailed t-test. Error bars indicate SEM. For D and F, log-rank test was used - see text for p values.