The interaction between AMPK beta 2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

11 páginas, 7 figuras.AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at threonine-148 (Thr-148), which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. Here, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid analyses and co-immunoprecipitation of the overexpressed proteins in HEK293T cells revealed that both AMPKβ1 and β2 wild-type (WT) isoforms bind to R6. The AMPKβ/R6 interaction was stronger with the muscle-specific β2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced AMPKβ2/R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous to increased AMPKβ2 Thr-148 autophosphorylation. Such model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.DN is recipient of a VIDI-Innovational Research Grant from the Netherlands Organization of Scientific Research (NWO-ALW Grant no. 864.10.007). This work has further been supported by grants from the Spanish Ministry of Education and Science SAF2014-54604-C3-1-R and a grant from Generalitat Valenciana (PrometeoII/2014/029) to PS.Peer reviewe

HLA and Genomewide Allele Sharing in Dizygotic Twins

Gametic selection during fertilization or the effects of specific genotypes on the viability of embryos may cause a skewed transmission of chromosomes to surviving offspring. A recent analysis of transmission distortion in humans reported significant excess sharing among full siblings. Dizygotic (DZ) twin pairs are a special case of the simultaneous survival of two genotypes, and there have been reports of DZ pairs with excess allele sharing around the HLA locus, a candidate locus for embryo survival. We performed an allele-sharing study of 1,592 DZ twin pairs from two independent Australian cohorts, of which 1,561 pairs were informative for linkage on chromosome 6. We also analyzed allele sharing in 336 DZ twin pairs from The Netherlands. We found no evidence of excess allele sharing, either at the HLA locus or in the rest of the genome. In contrast, we found evidence of a small but significant (P=.003 for the Australian sample) genomewide deficit in the proportion of two alleles shared identical by descent among DZ twin pairs. We reconciled conflicting evidence in the literature for excess genomewide allele sharing by performing a simulation study that shows how undetected genotyping errors can lead to an apparent deficit or excess of allele sharing among sibling pairs, dependent on whether parental genotypes are known. Our results imply that gene-mapping studies based on affected sibling pairs that include DZ pairs will not suffer from false-positive results due to loci involved in embryo survival

Is there a Mendelian transmission ratio distortion of the c.429_452dup(24bp) polyalanine tract ARX mutation?

Advance online publication 11 April 2012Intellectual disability is common. Aristaless-related homeobox (ARX) gene is one of the most frequently mutated and pleiotropic genes, implicated in 10 different phenotypes. More than half of ~100 reported cases with ARX mutations are due to a recurrent duplication of 24 bp, c.429_452dup, which leads to polyalanine tract expansion. The excess of affected males among the offspring of the obligate carrier females raised the possibility of transmission ratio distortion for the c.429_452dup mutation. We found a significant deviation from the expected Mendelian 1:1 ratio of transmission in favour of the c.429_452dup ARX mutation. We hypothesise that the preferential transmission of the c.429_452dup mutation may be due to asymmetry of meiosis in the oocyte. Our findings may have implications for genetic counselling of families segregating the c.429_452dup mutation and allude to putative role of ARX in oocyte biology.Cheryl Shoubridge, Alison Gardner, Charles E. Schwartz, Anna Hackett, Michael Field and Jozef Gec