Phosphorylated Human Keratinocyte Ornithine Decarboxylase Is Preferentially Associated with Insoluble Cellular Proteins
| Autor: | Olli A. Jänne, Rocky S. Tuan, Noreen J. Hickok, Irene M. Leigh, Mary M. Pomidor, Ping Zheng, James A. Gurr, Rebecca Cimildoro, Bien Lazatin |
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| Rok vydání: | 1999 |
| Předmět: |
Keratinocytes
genetic structures Fluorescent Antibody Technique macromolecular substances Cell Fractionation Ornithine Decarboxylase Article Ornithine decarboxylase 03 medical and health sciences chemistry.chemical_compound Tubulin Keratin Humans Phosphorylation Cytoskeleton Molecular Biology Cells Cultured Actin 030304 developmental biology Cytochalasin D chemistry.chemical_classification 0303 health sciences biology fungi 030302 biochemistry & molecular biology Cell Biology Actin cytoskeleton Precipitin Tests Molecular biology chemistry biology.protein Keratins Cell fractionation |
| Zdroj: | Molecular Biology of the Cell. 10:4299-4310 |
| ISSN: | 1939-4586 1059-1524 |
| DOI: | 10.1091/mbc.10.12.4299 |
| Popis: | Ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, is highly regulated by many trophic stimuli, and changes in its levels and organization correlate with cytoskeletal changes in normal human epidermal keratinocytes (NHEK). NHEK ODC exhibits a filamentous perinuclear/nuclear localization that becomes more diffuse under conditions that alter actin architecture. We have thus asked whether ODC colocalizes with a component of the NHEK cytoskeleton. Confocal immunofluorescence showed that ODC distribution in NHEK was primarily perinuclear; upon disruption of the actin cytoskeleton with cytochalasin D, ODC distribution was diffuse. The ODC distribution in untreated NHEK overlapped with that of keratin in the perinuclear but not cytoplasmic area; after treatment with cytochalasin D, overlap between staining for ODC and for keratin was extensive. No significant overlap with actin and minimal overlap with tubulin filament systems were observed. Subcellular fractionation by sequential homogenizations and centrifugations of NHEK lysates or detergent and salt extractions of NHEK in situ revealed that ODC protein and activity were detectable in both soluble and insoluble fractions, with mechanical disruption causing additional solubilization of ODC activity (three- to sevenfold above controls). Fractionation and ODC immunoprecipitation from [32P]orthophosphate-labeled NHEK lysates showed that a phosphorylated form of ODC was present in the insoluble fractions. Taken together, these data suggest that two pools of ODC exist in NHEK. The first is the previously described soluble pool, and the second is enriched in phospho-ODC and associated with insoluble cellular material that by immunohistochemistry appears to be organized in conjunction with the keratin cytoskeleton. |
| Databáze: | OpenAIRE |
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