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
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.