Physiology - Research Publications
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ItemThe Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2015-05-01)The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.
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ItemNo Preview AvailableAMP-Activated Protein Kinase β-Subunit Requires Internal Motion for Optimal Carbohydrate Binding(CELL PRESS, 2012-01-18)AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β(1) and β(2)). Muscle-specific β(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β(2)-CBM is concurrent with an increase in flexibility in β(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to β(1)-CBM, unbound β(2)-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β(2)-CBM mutant. Insertion of a threonine into the background of β(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.
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ItemNo Preview AvailableSingle fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle(AMER PHYSIOLOGICAL SOC, 2012-12)To understand how glycogen affects skeletal muscle physiology, we examined enzymes essential for muscle glycogen synthesis and degradation using single fibers from quiescent and stimulated rat skeletal muscle. Presenting a shift in paradigm, we show these proteins are differentially associated with glycogen granules. Protein diffusibility and/or abundance of glycogenin, glycogen branching enzyme (GBE), debranching enzyme (GDE), phosphorylase (GP), and synthase (GS) were examined in fibers isolated from rat fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscle. GDE and GP proteins were more abundant (~10- to 100-fold) in fibers from EDL compared with SOL muscle. GS and glycogenin proteins were similar between muscles while GBE had an approximately fourfold greater abundance in SOL muscle. Mechanically skinned fibers exposed to physiological buffer for 10 min showed ~70% total pools of GBE and GP were diffusible (nonbound), whereas GDE and GS were considerably less diffusible. Intense in vitro stimulation, sufficient to elicit a ~50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (~15-60%) and decreased GBE diffusibility (~20%). Amylase treatment, which breaks α-1,4 linkages of glycogen, indicated differential diffusibilities and hence glycogen associations of GDE and GS. Membrane solubilization (1% Triton-X-100) allowed a small additional amount of GDE and GS to diffuse from fibers, suggesting the majority of nonglycogen-associated GDE/GS is associated with myofibrillar/contractile network of muscle rather than membranes. Given differences in enzymes required for glycogen metabolism, the current findings suggest glycogen particles have fiber-type-dependent structures. The greater catabolic potential of glycogen breakdown in fast-twitch fibers may account for different contraction induced rates of glycogen utilization.