| 1. |
- Alsterfjord, Magnus, et al.
(författare)
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Plasma membrane H+-ATPase and 14-3-3 Isoforms of Arabidopsis leaves: Evidence for isoform specificity in the 14-3-3/H+-ATPase interaction
- 2004
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Ingår i: Plant and Cell Physiology. - : Oxford University Press (OUP). - 1471-9053 .- 0032-0781. ; 45:9, s. 1202-1210
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Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane H+-ATPase is activated by binding of 14-3-3 protein to the phosphorylated C terminus. Considering the large number of 14-3-3 and H+-ATPase isoforms in Arabidopsis (13 and 11 expressed genes, respectively), specificity in binding may exist between 14-3-3 and H+-ATPase isoforms. We now show that the H'-ATPase is the main target for 14-3-3 binding at the plasma membrane, and that all twelve 14-3-3 istiforms tested bind to the H+-ATPase in vitro. Using specific antibodies for nine of the 14-3-3 isoforms, we show that GF14epsilon, mu, lambda, omega, chi, phi, nu, and upsilon are present in leaves, but that isolated plasma membranes lack GF14chi, phi and upsilon. Northern blots using isoform-specific probes for all 14-3-3 and H+-ATPase isoforms showed that transcripts were present for most of the isoforms. Based on mRNA levels, GF14epsilon, mu, lambda and chi are highly expressed 14-3-3 isoforms, and AHA1, 3, and 11 highly expressed H+-ATPase isoforms in leaves. However, mass peptide fingerprinting identified AHA1 and 2 with the highest score, and their presence could be confirmed by MS/MS. It may be calculated that under 'unstressed' conditions less than one percent of total 14-3-3 is attached to the H+-ATPase. However, during a condition requiring full activation of H+ pumping, as induced here by the presence of the fungal toxin fusicoccin, several percent of total 14-3-3 may be engaged in activation of the H+-ATPase.
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| 2. |
- Choi, Young Hun, et al.
(författare)
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Alterations in regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B-null mice
- 2006
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Ingår i: Journal of Clinical Investigation. - 0021-9738. ; 116:12, s. 3240-3251
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Tidskriftsartikel (refereegranskat)abstract
- Cyclic nucleotide phosphodiesterase 3B (PDE3B) has been suggested to be critical for mediating insulin/IGF-1 inhibition of cAMP signaling in adipocytes, liver, and pancreatic beta cells. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated hpogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The beta(3)-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance.
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| 3. |
- Jones, Helena, et al.
(författare)
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Early and rapid development of insulin resistance, islet dysfunction and glucose intolerance after high-fat feeding in mice overexpressing phosphodiesterase 3B.
- 2006
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Ingår i: Journal of Endocrinology. - : Bioscientifica. - 1479-6805 .- 0022-0795. ; 189:3, s. 629-641
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Tidskriftsartikel (refereegranskat)abstract
- Inadequate islet adaptation to insulin resistance leads to glucose intolerance and type 2 diabetes. Here we investigate whether β-cell cAMP is crucial for islet adaptation and prevention of glucose intolerance in mice. Mice with a β-cell-specific, 2-fold overexpression of the cAMP-degrading enzyme phosphodiesterase 3B (RIP-PDE3B/2 mice) were metabolically challenged with a high-fat diet. We found that RIP-PDE3B/2 mice early and rapidly develop glucose intolerance and insulin resistance, as compared with wild-type littermates, after 2 months of high-fat feeding. This was evident from advanced fasting hyperinsulinemia and early development of hyper-glycemia, in spite of hyperinsulinemia, as well as impaired capacity of insulin to suppress plasma glucose in an insulin tolerance test. In vitro analyses of insulin-stimulated lipogenesis in adipocytes and glucose uptake in skeletal muscle did not reveal reduced insulin sensitivity in these tissues. Significant steatosis was noted in livers from high-fat-fed wild-type and RIP-PDE3B/2 mice and liver triacyl-glycerol content was 3-fold higher than in wild-type mice fed a control diet. Histochemical analysis revealed severe islet perturbations, such as centrally located α-cells and reduced immunostaining for insulin and GLUT2 in islets from RIP-PDE3B/2 mice. Additionally, in vitro experiments revealed that the insulin secretory response to glucagon-like peptide-1 stimulation was markedly reduced in islets from high-fat-fed RIP-PDE3B/2 mice. We conclude that accurate regulation of β-cell cAMP is necessary for adequate islet adaptation to a perturbed metabolic environment and protective for the development of glucose intolerance and insulin resistance.
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| 4. |
- Svennelid, Fredrik
(författare)
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The Plasma Membrane H+-ATPase - Identification of a 14-3-3 binding motif
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The P-type plasma membrane H+-ATPases form a group of proteins only found in plants and fungi. The pumping of protons across the plasma membrane, energized by ATP hydrolysis, creates an electrochemical gradient that is essential for solute transport and internal pH regulation. The H+-ATPase genes are present as multigene families in the genomes of higher plants and all cell types investigated express some H+-ATPase gene. The fundamental importance of the electrochemical gradient makes precise regulation of the H+-ATPase important. Several internal and external factors, such as hormones, light, pH, and fungal toxins, are involved in regulating the plant plasma membrane H+-ATPase activity. Besides the direct regulation by pH and ATP availability, the activity is controlled by an autoinhibitory C-terminal domain in the H+-ATPase. Removing this C-terminal domain by proteolysis or by fusicoccin-induced 14-3-3 binding irreversibly activates the enzyme. Normally, 14-3-3 binds to phosphorylated motifs and by incubating spinach leaves with 32P-orthophosphate and the fungal toxin fusicoccin in vivo it was possible to radiolabel the H+-ATPase. The radiolabeling could be removed by proteolysis and sequencing the released radiolabeled peptides identified the phosphorylated amino acid as the penultimate threonine in the C terminus, in the relatively conserved motif QQXYTV. This phosphorylated threonine is essential for 14-3-3 binding in the absence of fusicoccin, whereas fusicoccin-induced 14-3-3 binding occurs regardless of phosphorylation but still requires the YTV residues. The physiological importance of this motif was shown by heterologous expression of a plant H+-ATPase in yeast. Mutations in the motif abolished or heavily reduced 14-3-3 binding and activation of the plant H+-ATPase. In vitro phosphorylation of isolated plasma membranes with [ g -32P]ATP radiolabels the H+-ATPase in a calcium-dependent way and creates a 14-3-3 binding site in the H+-ATPase, containing a phosphothreonine. The H+-ATPase isoforms AHA1 and AHA2 are present in Arabidopsis leaf plasma membranes under normal conditions; fusicoccin treatment induces expression of three additional isoforms, AHA3, AHA8, and AHA11. Five 14-3-3 isoforms, epsilon, mu, nu, omega, and upsilon, are associated with the plasma membrane, where the H+-ATPase is the main target for 14-3-3 binding; after infiltration with fusicoccin there is a change in isoforms, omega disappears and the chi isoform appears. In summary, the data show that in vivo phosphorylation of the penultimate threonine in the motif QQXYTV regulates the H+-ATPase activity by 14-3-3 binding and that a calcium-dependent protein kinase activity phosphorylating this threonine in vitro is present in the plasma membrane. The appearance of additional H+-ATPase isoforms and the shift in 14-3-3 isoforms after fusicoccin-treatment is interesting and further research might answer questions regarding 14-3-3 isoform specificity and the function of the different H+-ATPase isoforms, in e.g. adaptation to stress.
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