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1.
J Cell Biol ; 219(6)2020 06 01.
Article in English | MEDLINE | ID: mdl-32422653

ABSTRACT

The TGN is a key compartment for the sorting and secretion of newly synthesized proteins. At the TGN, soluble proteins are sorted based on the instructions carried in their oligosaccharide backbones or by a Ca2+-mediated process that involves the cargo-sorting protein Cab45. Here, we show that Cab45 is phosphorylated by the Golgi-specific protein kinase Fam20C. Mimicking of phosphorylation translocates Cab45 into TGN-derived vesicles, which goes along with an increased export of LyzC, a Cab45 client. Our findings demonstrate that Fam20C plays a key role in the export of Cab45 clients by fine-tuning Cab45 oligomerization and thus impacts Cab45 retention in the TGN.


Subject(s)
Calcium-Binding Proteins/metabolism , Casein Kinase I/metabolism , Extracellular Matrix Proteins/metabolism , Glycoproteins/metabolism , Protein Transport/genetics , trans-Golgi Network/metabolism , CRISPR-Cas Systems , Calcium-Binding Proteins/genetics , Casein Kinase I/deficiency , Casein Kinase I/genetics , Cell Line, Tumor , Extracellular Matrix Proteins/deficiency , Extracellular Matrix Proteins/genetics , Gene Knockout Techniques , Glycoproteins/genetics , Humans , Isoantigens/metabolism , Mutation , Phosphorylation , Protein Transport/physiology , RNA, Small Interfering , Recombinant Proteins , Seminal Plasma Proteins/metabolism
2.
Elife ; 52016 12 20.
Article in English | MEDLINE | ID: mdl-27996937

ABSTRACT

The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling ß-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the ß-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems.


Subject(s)
Gene Expression Regulation , Gene Regulatory Networks , Genetic Testing/methods , Wnt Signaling Pathway , Casein Kinase I/deficiency , Cytoskeletal Proteins/deficiency , Genes, Reporter , Haploidy , Humans , Wnt Proteins/genetics , Wnt Proteins/metabolism
3.
J Proteome Res ; 9(11): 6025-6032, 2010 Nov 05.
Article in English | MEDLINE | ID: mdl-20883044

ABSTRACT

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown, we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos.


Subject(s)
Chromosome Segregation/drug effects , Cloning, Organism , Oocytes/ultrastructure , Proteomics/methods , Spindle Apparatus/chemistry , Animals , Aurora Kinase B , Aurora Kinases , Casein Kinase I/deficiency , Chromosome Segregation/genetics , Clathrin Heavy Chains/analysis , Clathrin Heavy Chains/drug effects , Dynactin Complex , Embryo, Mammalian , Mice , Mice, Inbred Strains , Microtubule-Associated Proteins/deficiency , Oocytes/chemistry , Protein Serine-Threonine Kinases/deficiency , Proteins/analysis , RNA, Messenger/administration & dosage , RNA, Messenger/therapeutic use
4.
Neuron ; 67(5): 810-20, 2010 Sep 09.
Article in English | MEDLINE | ID: mdl-20826312

ABSTRACT

Cyclic AMP signaling in Drosophila mushroom body neurons, anchored by the adenylyl cyclase encoded by the rutabaga gene, is indispensable for olfactory memory formation. From a screen for new memory mutants, we identified alleles of the gilgamesh (gish) gene, which encodes a casein kinase Iγ homolog that is preferentially expressed in the mushroom body neurons. The gish-encoded kinase participates in the physiology of these neurons underlying memory formation since the mutant memory deficit was rescued with expression of a gish cDNA in these neurons only during adulthood. A cellular memory trace, detected as increased calcium influx into the α'/ß' neuron processes in response to the odor used for conditioning, was disrupted in gish mutants. Epistasis experiments indicated a lack of genetic interactions between gish and rutabaga. Therefore, gish participates in a rutabaga-independent pathway for memory formation and accounts for some of the residual learning that occurs in rutabaga mutants.


Subject(s)
Adenylyl Cyclases/metabolism , Avoidance Learning/physiology , Casein Kinase I/physiology , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , Smell/physiology , Adenylyl Cyclases/genetics , Animals , Animals, Genetically Modified , Behavior, Animal , Casein Kinase I/deficiency , Casein Kinase I/genetics , Conditioning, Classical/physiology , Drosophila , Drosophila Proteins/deficiency , Drosophila Proteins/genetics , Gene Expression Regulation/genetics , Memory Disorders/genetics , Mushroom Bodies/growth & development , Mushroom Bodies/metabolism , Mutation/genetics , Odorants
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