Maria Adelaida Garcia-Gimeno

The PP1-R6 protein phosphatase holoenzyme is involved in the glucose-induced dephosphorylation and inactivation of AMP-activated protein kinase, a key regulator of insulin secretion, in MIN6 β cells

Mammalian AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status. It is activated by phosphorylation of the catalytic subunit on Thr172. The main objective of this study was the identification of a phosphatase involved in the regulation of AMPK activity. Mouse MIN6 β cells were used to study the glucose-induced regulation of the phosphorylation of AMPK. Small interfering RNA (siRNA) technology was used to deplete putative phosphatase candidate genes that could affect AMPK regulation. The effect of the siRNAs used in the study was compared with the effect observed using a negative control siRNA. A protein phosphatase complex composed of the catalytic subunit of protein phosphatase-1 (PP1) and the regulatory subunit R6 participates in the glucose-induced dephosphorylation of AMPK. R6 interacts physically with the β-subunit of the AMPK complex and recruits PP1 to dephosphorylate the catalytic α-subunit on Thr172. siRNA depletion of R6 decreases glucose-induced insulin secretion due to the presence of a constitutively active AMPK complex. The characterization of the PP1-R6 complex identifies this holoenzyme as a possible target for therapeutic intervention with the aim of regulating the activity of AMPK in pancreatic β cells.

Molecular Characterization of Ypi1, a Novel Saccharomyces cerevisiae Type 1 Protein Phosphatase Inhibitor

The Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1). Ypi1 interacts physically in vitro with both Glc7 and Ppz1 phosphatase catalytic subunits, as shown by pull-down assays. Ypi1 inhibits Glc7 but appears to be less effective toward Ppz1 phosphatase activity under the conditions tested. Ypi1 contains a 48RHNVRW53 sequence, which resembles the characteristic consensus PP1 phosphatase binding motif. A W53A mutation within this motif abolishes both binding to and inhibition of Glc7 and Ppz1 phosphatases. Deletion of YPI1 is lethal, suggesting a relevant role of the inhibitor in yeast physiology. Cells overexpressing Ypi1 display a number of phenotypes consistent with an inhibitory role of this protein on Glc7, such as decreased glycogen content and an increased growth defect in a slt2/mpk1 mitogen-activated protein kinase-deficient background. Taking together, these results define Ypi1 as the first inhibitory subunit of Glc7 identified in budding yeast.

YPI1 and SDS22 Proteins Regulate the Nuclear Localization and Function of Yeast Type 1 Phosphatase Glc7

We have recently characterized Ypi1 as an inhibitory subunit of yeast Glc7 PP1 protein phosphatase. In this work we demonstrate that Ypi1 forms a complex with Glc7 and Sds22, another Glc7 regulatory subunit that targets the phosphatase to substrates involved in cell cycle control. Interestingly, the combination of equimolar amounts of Ypi1 and Sds22 leads to an almost full inhibition of Glc7 activity. Because YPI1 is an essential gene, we have constructed conditional mutants that demonstrate that depletion of Ypi1 leads to alteration of nuclear localization of Glc7 and cell growth arrest in mid-mitosis with aberrant mitotic spindle. These phenotypes mimic those produced upon inactivation of Sds22. The fact that progressive depletion of either Ypi1 or Sds22 resulted in similar physiological phenotypes and that both proteins inhibit the phosphatase activity of Glc7 strongly suggest a common role of these two proteins in regulating Glc7 nuclear localization and function.

Clinical Heterogeneity in Monogenic Diabetes Caused by Mutations in the Glucokinase Gene (GCK-MODY)

OBJECTIVE To evaluate the heterogeneity in the clinical expression in a family with glucokinase mature-onset diabetes of the young (GCK-MODY). RESEARCH DESIGN AND METHODS Members (three generations) of the same family presented either with overt neonatal hyperglycemia, marked postprandial hyperglycemia, or glucosuria. Homeostasis model assessment of insulin resistance (HOMAIR) and insulinogenic and disposition indexes were calculated. Oral glucose tolerance test (OGTT) results in the GCK mutation carriers from this family were compared with those from other subjects with GCK mutations in the same codon (GCK261), with other missense and other types of GCK mutations in different codons from the European MODY Consortium database (GCKm). RESULTS Mutation G261R was found in the GCK gene. During the OGTT, glucose (P = 0.02) and insulin (P = 0.009) response at 2 h as well as at the 2-h glucose increment (GCK261 versus other missense GCK mutations, P = 0.003) were significantly higher in GCK261 than in GCKm carriers. CONCLUSIONS Differing from other GCKm carriers, the glucose and insulin response to oral glucose was significantly higher in GCK261 carriers, indicating clinical heterogeneity in GCK-MODY.

Glycogenic activity of R6, a protein phosphatase 1 regulatory subunit, is modulated by the laforin-malin complex

Protein phosphatase type 1 (PP1) plays a major role in the regulation of glycogen biosynthesis. PP1 is recruited to sites of glycogen formation by its binding to specific targeting subunits. There, it dephosphorylates different enzymes involved in glycogen homeostasis leading to an activation of glycogen biosynthesis. Regulation of these targeting subunits is crucial, as excess of them leads to an enhancement of the action of PP1, which results in glycogen accumulation. In this work we present evidence that PPP1R3D (R6), one of the PP1 glycogenic targeting subunits, interacts physically with laforin, a glucan phosphatase involved in Lafora disease, a fatal type of progressive myoclonus epilepsy. Binding of R6 to laforin allows the ubiquitination of R6 by the E3-ubiquitin ligase malin, what targets R6 for autophagic degradation. As a result of the action of the laforin-malin complex on R6, its glycogenic activity is downregulated. Since R6 is expressed in brain, our results suggest that the laforin-malin complex downregulates the glycogenic activity of R6 present in neuron cells to prevent glycogen accumulation.

Opposite clinical phenotypes of glucokinase disease: Description of a novel activating mutation and contiguous inactivating mutations in human glucokinase (GCK) gene

Glucokinase is essential for glucose-stimulated insulin release from the pancreatic beta-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.

AMPKbeta subunits, more than just a scaffold in the formation of AMPK complex

AMP‐activated protein kinase (AMPK) is a sensor of energy status composed of a catalytic subunit (AMPKα), a scaffolding subunit (AMPKβ) and a regulatory subunit involved in nucleotide binding (AMPKγ). Activation of AMPK results in enhancement of catabolic processes and downregulation of anabolic pathways with the aim to equilibrate the energy status of the cell. The study of the regulation of the activity of the AMPK complex has been traditionally focused on modifications of AMPKα and AMPKγ subunits by post‐translational changes (i.e. phosphorylation of the catalytic subunit) and allosteric activation by AMP. In this review, we summarize recent reports that indicate that AMPKβ subunits are also critical players in AMPK function, because they can regulate the phosphorylation status and activity of the AMPK complex. AMPKβ1‐ and AMPKβ2‐containing complexes differ in their capacity to be activated by specific drugs (i.e. A769622, salicylate) and also by the ability to undergo post‐translational modifications. This selective behavior opens the possibility to design specific drugs that activate AMPK complexes containing specific β‐isoforms.

Structure—function analysis of the α5 and the α13 helices of human glucokinase: Description of two novel activating mutations

It was recently described that the α5 and the α13 helices of human pancreatic glucokinase play a major role in the allosteric regulation of the enzyme. In order to understand the structural importance of these helices, we have performed site‐directed mutagenesis to generate glucokinase derivatives with altered residues. We have analyzed the kinetic parameters of these mutated forms and compared them with wild‐type and previously defined activating mutations in these helices (A456V and Y214C). We found two new activating mutations, A460R and Y215A, which increase the affinity of the enzyme for glucose. Our results suggest that substitutions in the α5 or the α13 helices that favor the closed, active conformation of the enzyme, either by improving the interaction with surrounding residues or by improving the flexibility of the region defined by these two helices, enhance the affinity of the enzyme for glucose, and therefore its performance as a glucose phosphorylating enzyme.

Glucose-dependent regulation of AMP-activated protein kinase in MIN6 beta cells is not affected by the protein kinase A pathway

AMP‐activated protein kinase (AMPK) is a sensor of cellular energy status. In pancreatic beta cells, glucose induces the dephosphorylation of Thr172 within the catalytic subunit and the inactivation of the AMPK complex. Here we demonstrate that glucose also activates protein kinase A (PKA), leading to the phosphorylation of AMPKα at Ser485 and Ser497. However, these modifications do not impair the phosphorylation of Thr172 by upstream kinases, and phosphorylation of Thr172 does not affect the phosphorylation of AMPKα by PKA either. Thus, although phosphorylation of Thr172 and Ser485/Ser497 are inversely correlated in response to glucose, they follow an independent regulation.

Biochemical characterization of novel glucokinase mutations isolated from Spanish maturity-onset diabetes of the young (MODY2) patients

AbstractMature-onset diabetes of the young, type 2 (MODY2) is associated with mutations in the glucokinase (GCK) gene that result in impaired glucokinase activity. Although more than 200 inactivating GCK mutations have been reported, only less than 20% of these mutations have been functionally characterized. In this work, we describe the biochemical characterization of six missense glucokinase mutations associated with MODY2 from Spanish patients, namely, Y61S, V182L, C233R, E265K, A379V, and K420E. All these mutations produced enzymes that presented reduced enzymatic activity in various degrees, from a mild affectation (K420E) to a more severe effect (C233R). Mutation severity correlated with the importance of the structural changes introduced by the mutations. For example, C233R affected a critical residue of the active center of the enzyme and rendered a protein with undetectable enzymatic activity. These data add new information on the structure–function relationship of human glucokinase.