Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B <i>in vitro</i> by protein phosphatase type 2A

Resjö, Svante; Oknianska, Alina; Zołnierowicz, Stanisław; Manganiello, Vincent C.; Degerman, Eva

doi:10.1042/bj3410839

Cited by 50 publications

(5 citation statements)

References 51 publications

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“…We used 12B2 to examine the regulation of GSK3β by protein phosphatases in cells. We treated HEK cells for 30 min with 10 nM calyculin A, a potent protein phosphatase inhibitor (Ishihara et al, 1989; Resjo et al, 1999), which increases S9 phosphorylation in GSK3β (Morfini et al, 2004; Kim et al, 2009; Xiao et al, 2010). The cell lysates were analyzed in sandwich ELISAs, GSK3β kinase activity assays, immunofluorescence, and western blot.…”

Section: Resultsmentioning

confidence: 99%

Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

Grabinski

Kanaan

2016

Front. Mol. Neurosci.

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Glycogen synthase kinase 3 (GSK3) β and α are serine/threonine kinases involved in many biological processes. A primary mechanism of GSK3 activity regulation is phosphorylation of N-terminal serine (S) residues (S9 in GSK3β, S21 in GSK3α). Phosphorylation is inhibitory to GSK3 kinase activity because the phosphorylated N-terminus acts as a competitive inhibitor for primed substrates. Despite widespread interest in GSK3 across most fields of biology, the research community does not have reagents that specifically react with nonphosphoS9/21 GSK3β/α (the so-called “active” form). Here, we describe two novel monoclonal antibodies that specifically react with nonphosphoS9/21 GSK3β/α in multiple species (human, mouse, and rat). One of the antibodies is specific for nonphospho-S9 GSK3β (clone 12B2) and one for nonphospho-S9/21 GSK3β/α (clone 15C2). These reagents were validated for specificity and reactivity in several biochemical and immunochemical assays, and they show linear detection of nonphosphoS GSK3. Finally, these reagents provide significant advantages in studying GSK3β regulation. We used both antibodies to study the regulation of S9 phosphorylation by Akt and protein phosphatases. We used 12B2 (due to its specificity for GSK3β) and to demonstrate that protein phosphatase inhibition reduces nonphospho-S9 GSK3β levels and lowers kinase activity within cells. The ability to use the same reagent across biochemical, immunohistological and kinase activity assays provides a powerful approach for studying serine-dependent regulation of GSK3β/α.

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Section: Resultsmentioning

confidence: 99%

Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

Grabinski

Kanaan

2016

Front. Mol. Neurosci.

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“…Consistent with a feedback regulatory mechanism, this is believed to contribute to fine-tuning of cAMP levels and PKA activity and thereby control of lipolysis. Regarding dephosphorylation and deactivation of PDE3B, PP2A was shown to act as a PDE3B phosphatase ( Resjö et al, 1999 ).…”

Section: Intracellular Brakes On Adipocyte Lipolysismentioning

confidence: 99%

The Molecular Brakes of Adipose Tissue Lipolysis

Ngandiri

et al. 2022

Front. Physiol.

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Adaptation to changes in energy availability is pivotal for the survival of animals. Adipose tissue, the body’s largest reservoir of energy and a major source of metabolic fuel, exerts a buffering function for fluctuations in nutrient availability. This functional plasticity ranges from energy storage in the form of triglycerides during periods of excess energy intake to energy mobilization via lipolysis in the form of free fatty acids for other organs during states of energy demands. The subtle balance between energy storage and mobilization is important for whole-body energy homeostasis; its disruption has been implicated as contributing to the development of insulin resistance, type 2 diabetes and cancer cachexia. As a result, adipocyte lipolysis is tightly regulated by complex regulatory mechanisms involving lipases and hormonal and biochemical signals that have opposing effects. In thermogenic brown and brite adipocytes, lipolysis stimulation is the canonical way for the activation of non-shivering thermogenesis. Lipolysis proceeds in an orderly and delicately regulated manner, with stimulation through cell-surface receptors via neurotransmitters, hormones, and autocrine/paracrine factors that activate various intracellular signal transduction pathways and increase kinase activity. The subsequent phosphorylation of perilipins, lipases, and cofactors initiates the translocation of key lipases from the cytoplasm to lipid droplets and enables protein-protein interactions to assemble the lipolytic machinery on the scaffolding perilipins at the surface of lipid droplets. Although activation of lipolysis has been well studied, the feedback fine-tuning is less well appreciated. This review focuses on the molecular brakes of lipolysis and discusses some of the divergent fine-tuning strategies in the negative feedback regulation of lipolysis, including delicate negative feedback loops, intermediary lipid metabolites-mediated allosteric regulation and dynamic protein–protein interactions. As aberrant adipocyte lipolysis is involved in various metabolic diseases and releasing the brakes on lipolysis in thermogenic adipocytes may activate thermogenesis, targeting adipocyte lipolysis is thus of therapeutic interest.

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“…We also observed that immunoprecipitation with a phospho-Serine (pSer) 14-3-3 motif antibody pulled down USP43, and importantly, this interaction was hypoxia dependent ( Fig S7B ). Moreover, the interaction between 14-3-3 and USP43 could be replicated using Culyculin A as a phosphatase inhibitor (Resjö et al , 1999), and conversely, prevented by Lambda PP ( Fig S7C ).…”

Section: Resultsmentioning

confidence: 81%

Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response

Pauzaite,

Wit,

Seear

et al. 2024

Preprint

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The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results unveil the DUB landscape in HIF signalling, and highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.

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Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A

Cited by 50 publications

References 51 publications

Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

The Molecular Brakes of Adipose Tissue Lipolysis

Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response

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