Phosphorylated human keratinocyte ornithine decarboxylase is preferentially associated with insoluble cellular proteins

Citation
Mm. Pomidor et al., Phosphorylated human keratinocyte ornithine decarboxylase is preferentially associated with insoluble cellular proteins, MOL BIOL CE, 10(12), 1999, pp. 4299-4310
Citations number
48
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cell & Developmental Biology
Journal title
MOLECULAR BIOLOGY OF THE CELL
ISSN journal
1059-1524 → ACNP
Volume
10
Issue
12
Year of publication
1999
Pages
4299 - 4310
Database
ISI
SICI code
1059-1524(199912)10:12<4299:PHKODI>2.0.ZU;2-G
Abstract
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 l ocalization that becomes more diffuse under conditions that alter actin arc hitecture. We have thus asked whether ODC colocalizes with a component of t he NHEK cytoskeleton. Confocal immunofluorescence showed that ODC distribut ion in NHEK was primarily perinuclear; upon disruption of the-actin cytoske leton with cytochalasin D, ODC distribution was diffuse. The ODC distributi on 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 wit h actin and minimal overlap with tubulin filament systems were observed. Su bcellular fractionation by sequential homogenizations and centrifugations o f NHEK lysates or detergent and salt extractions of NHEK in situ revealed t hat 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 i mmunoprecipitation from [P-32]orthophosphate-labeled NHEK lysates showed th at a phosphorylated form of ODC was present in the insoluble fractions. Tak en together, these data suggest that two pools of ODC exist in NHEK. The fi rst is the previously described soluble pool, and the second is enriched in phospho-ODC and associated with insoluble cellular material that by immuno histochemistry appears to be organized in conjunction with the keratin cyto skeleton.