Article

CVAK104 is a novel poly-L-lysine-stimulated kinase that targets the beta2-subunit of AP2.

Department of Genetics, Cell Biology, and Development, The University of Minnesota, Minneapolis, Minnesota 55455, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 07/2005; 280(22):21539-44. DOI: 10.1074/jbc.M502462200
Source: PubMed

ABSTRACT Isolated clathrin adaptor protein (AP) preparations are known to co-fractionate with endogenous kinase activities, including poly-L-lysine-stimulated kinases that target various constituents of the clathrin coat. We have identified CVAK104 (a coated vesicle-associated kinase of 104 kDa) using a mass spectroscopic analysis of adaptor protein preparations. CVAK104 is a novel serine/threonine kinase that belongs to the SCY1-like family of protein kinases, previously thought to be catalytically inactive. We found that CVAK104 co-fractionates with adaptor protein preparations extracted from clathrin-coated vesicles and directly binds to both clathrin and the plasma membrane adaptor, AP2. CVAK104 binds ATP, and kinase assays indicate that it functions in vitro as a poly-L-lysine-stimulated kinase that is capable of autophosphorylation and phosphorylating the beta2-adaptin subunit of AP2.

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    • "A possible alternate function of the kinase-like domain might be that of a protein–protein interaction module (Manning et al., 2002). This module might be regulated by nucleotides, because CVAK104 is an ATP-binding protein (Conner and Schmid, 2005). Using the gel chromatographic procedure of Hummel and Dreyer (1962), we also observed ATP binding to CVAK104 (our unpublished data). "
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    ABSTRACT: CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the beta2-subunit of the adaptor protein (AP) complex AP2 in vitro. Here, we demonstrate that a C-terminal segment of CVAK104 interacts with the N-terminal domain of clathrin and with the alpha-appendage of AP2. CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells, but it is also present on peripheral vesicular structures that are accessible to endocytosed transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor, and clathrin but not at all with its putative phosphorylation target AP2. RNA interference-mediated clathrin knockdown reduced the membrane association of CVAK104. Recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by guanosine 5'-O-(3-thio)triphosphate, and brefeldin A redistributes CVAK104 in cells. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of green fluorescent protein-CVAK104 with endocytosed transferrin and with red fluorescent protein-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D. Together, our data suggest a function for CVAK104 in clathrin-dependent pathways between the trans-Golgi network and the endosomal system.
    Molecular Biology of the Cell 11/2006; 17(10):4513-25. DOI:10.1091/mbc.E06-05-0390 · 4.55 Impact Factor
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    ABSTRACT: This thesis describes the use of a proteomics resource to define the spatial organization of the endoplasmic reticulum (ER) and to predict the function of poorly characterized proteins. By proteomics, resident proteins involved in translation and translocation (eIFs, Sec61, TRAP, and the OST complex) are enriched in rough ER. Resident chaperones and folding enzymes (PDIs, calnexin, and BiP) are in equal abundance in rough and smooth ER. The ER-associated degradation (ERAD) machinery (derlin-1, VCP/p97, sel-1L, ubiquitin conjugating enzymes, and the proteasome), and also cytosolic chaperones (Hsp90, TRiC) all colocalize with their highest enrichment in smooth ER. This suggests a model that the ER is segregated into functional subdomains, which is useful to predict functional features of poorly characterized proteins localized in the proteomics resource. Nudilin is a cytosolic-located (P body) protein affecting translation which colocalizes with translation constituents at the cytosolic face of the ER membrane. Nicalin clusters with translocon constituents and is found to associate with signal peptide peptidase as well as negatively regulate the model secretory protein tyrosinase at the translocon. Stexin also clusters with translocon constituents and associates transiently with an early N-glycosylated form of tyrosinase. Erlin-1 and TMX2 co-distribute with chaperones found throughout the rough and smooth ER and are found to associate transiently with later N-glycosylated forms of tyrosinase. The ER is spatially organized to coordinate the temporal processing of cargo. Spatial localization in the ER predicts the function of resident uncharacterized proteins. Cette thèse décrit l’usage d’une ressource protéomique afin de définir l’organisation spatiale du réticulum endoplasmique (RE) et de prévoir la fonction de protéines peu charactérisées à ce jour. Par la protéomique, des protéines résidentes impliquées dans la translocation et la traduction (eIFs, Sec61, TRAP, et le complexe OST) sont enrichies dans le RE granuleux. Des chaperones résidentes et des enzymes de pliage (PDIs, calnexin et BiP) sont d’abondance égale dans le RE granuleux et le RE lisse. Le système de dégradation associé au RE (ERAD) (derlin-1, VCP/p97, sel-1L, enzymes de conjugaison de l’ubiquitin, protéasome) ainsi que les chaperones cytosoliques (Hsp90, TriC) sont tous colocalisés dans le RE lisse, leur enrichissement y étant le plus important. Ceci suggère que le RE est ségrégué en sous-domaines fonctionnels, ce qui est utile pour prédire les charactéristiques fonctionelles de protéines peu décrites, localisées dans la ressource protéomique. Nudilin est une protéine cytosolique (P body) affectant la traduction qui est colocalisée avec des constituants de la traduction à la surface cytosolique de la membrane du RE. Nicalin se regroupe avec des constituants du translocon et s’associe avec le signal peptide peptidase, en plus de réguler négativement la protéine de sécrétion tyrosinase au niveau du translocon. Stexin se regroupe aussi avec des constituants du translocon et s’associe de manière transitoire avec une forme N-glycosylée de tyrosinase trouvée immédiatement après sa synthèse. Erlin-1 et TMX2 se co-distribuent avec des chaperones trouvées à travers le RE lisse et granuleux et s’associent de manière transitoire avec des configurations N-glycosylée plus tardives de tyrosinase. Le RE est organisé dans l’espace afin de coordoner le traitement temporel de sa cargaison. La localisation spatialle des protéines résidentes non-charactérisées dans le RE prédit leur fonction.
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