Independent regulation of two cytoplasmic processing stages of the intermediate filament-associated protein filaggrin and role of Ca2+ in the second stage.
One of the final events in cornification of epidermal cells is processing of profilaggrin to the keratin-associated protein filaggrin. Processing involves several proteolytic events and occurs in two discrete proteolytic stages (Resing, K. A., Walsh, K. A., and Dale, B. A. (1984) J. Cell Biol. 99, 1372-1378; Resing, K. A., Walsh, K. A., Haugen-Scofield, J., and Dale, B. A. (1989) J. Biol. Chem. 264, 1837-1846). In a keratinocyte cell line derived from newborn rat epidermis, these two stages are independently regulated. Profilaggrin was expressed when the cells reached confluence; processing to intermediates began 24-36 h later (stage one), with filaggrin appearing at 48 h (stage two). Stage two processing required calcium in the medium with maximum processing occurring at 5-10 mM. Furthermore, stage two processing was inhibited by nifedipine, a calcium channel blocker, suggesting that calcium influx activates this event. Second-stage processing was also inhibited by the protease inhibitor leupeptin, implicating calpain. Confluent cells had higher levels of calpain I than subconfluent cells; in confluent cells, two immunoreactive bands were detected, comigrating with inactive (80 kDa) and activated (78 kDa) calpain I. In cells processing profilaggrin, most of the calpain I was in the 78-kDa form, implying extensive activation, supporting a role for calpain in processing.
Resing
K A
KA
Department of Biochemistry, University of Washington, Seattle 98195.
al-Alawi
N
N
Blomquist
C
C
Fleckman
P
P
Dale
B A
BA
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004817:Epidermis / Q000378:metabolism; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism; D009543:Nifedipine / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D051381:Rats
D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D011480:Protease Inhibitors; D002154:Calpain; D009543:Nifedipine; D002118:Calcium