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Identification of domains responsible for ubiquitin-dependent degradation of dMyc by glycogen synthase kinase 3beta and casein kinase 1 kinases.

ABSTRACT: In the present study, we report that ubiquitin-mediated degradation of dMyc, the Drosophila homologue of the human c-myc proto-oncogene, is regulated in vitro and in vivo by members of the casein kinase 1 (CK1) family and by glycogen synthase kinase 3beta (GSK3beta). Using Drosophila S2 cells, we demonstrate that CK1alpha promotes dMyc ubiquitination and degradation with a mechanism similar to the one mediated by GSK3beta in vertebrates. Mutation of ck1alpha or -epsilon or sgg/gsk3beta in Drosophila wing imaginal discs results in the accumulation of dMyc protein, suggesting a physiological role for these kinases in vivo. Analysis of the dMyc amino acid sequence reveals the presence of conserved domains containing potential phosphorylation sites for mitogen kinases, GSK3beta, and members of the CK1 family. We demonstrate that mutations of specific residues within these phosphorylation domains regulate dMyc protein stability and confer resistance to degradation by CK1alpha and GSK3beta kinases. Expression of the dMyc mutants in the compound eye of the adult fly results in a visible defect that is attributed to the effect of dMyc on growth, cell death, and inhibition of ommatidial differentiation.

SUBMITTER: Galletti M

PROVIDER: S-EPMC2698736 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Identification of domains responsible for ubiquitin-dependent degradation of dMyc by glycogen synthase kinase 3beta and casein kinase 1 kinases.

Galletti Margherita M Riccardo Sara S Parisi Federica F Lora Carlina C Saqcena Mahesh Kumar MK Rivas Leinny L Wong Bonnie B Serra Alexis A Serras Florenci F Grifoni Daniela D Pelicci PierGiuseppe P Jiang Jin J Bellosta Paola P

Molecular and cellular biology 20090413 12

In the present study, we report that ubiquitin-mediated degradation of dMyc, the Drosophila homologue of the human c-myc proto-oncogene, is regulated in vitro and in vivo by members of the casein kinase 1 (CK1) family and by glycogen synthase kinase 3beta (GSK3beta). Using Drosophila S2 cells, we demonstrate that CK1alpha promotes dMyc ubiquitination and degradation with a mechanism similar to the one mediated by GSK3beta in vertebrates. Mutation of ck1alpha or -epsilon or sgg/gsk3beta in Drosop ...[more]

PMID: 19364825

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Project description:RationalePulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC) proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß) is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling.MethodsAll experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT)-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a), upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1) and canonical Wnt intracellular effectors (GSK3ß, Axin1) were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9) by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT) and constitutively active GSK3ß S9A by [(3)H]-thymidine incorporation assay.ResultsIncreased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation) were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3ß S9A, 9th serine replaced to alanine) inhibited MCT-PASMCs proliferation by decreasing ERK phosphorylation.ConclusionThis study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.

Dataset Information

Identification of domains responsible for ubiquitin-dependent degradation of dMyc by glycogen synthase kinase 3beta and casein kinase 1 kinases.

Publications

Identification of domains responsible for ubiquitin-dependent degradation of dMyc by glycogen synthase kinase 3beta and casein kinase 1 kinases.

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