Characterization of an epidermal phosphatase specific for filaggrin phosphorylated by casein kinase II.
During cellular remodeling that accompanies cornification of epidermal cells, the highly phosphorylated protein, profilaggrin, is dephosphorylated and proteolytically cleaved to filaggrin, the keratin matrix protein. Using rat filaggrin phosphorylated by bovine casein kinase II (CK II) as a substrate, we have partially purified a phosphatase from rat epidermis which dephosphorylates rat profilaggrin in vitro. Anion exchange, hydroxylapatite, and gel filtration chromatography yielded a 100-fold purification of phosphatase from a low-salt extract. Further purification led to loss of activity; therefore, only the partially purified phosphatase was characterized. Two forms of the phosphatase, with molecular weights of approximately 170 and 40 kDa, were resolved during gel filtration. The 170-kDa form could be converted to the 40-kDa form in the presence of dithiothreitol. Both forms had pH optima of 6.6, and were strongly inhibited by NaCl (50% inhibition at 35-40 mM). Neither form hydrolyzed para-nitrophenylphosphate or dephosphorylated casein or the synthetic peptide arg3-glu3-thr-glu3, which were phosphorylated by casein kinase II. The two forms were similarly inhibited by known inorganic phosphatase inhibitors, with 22%-36% inhibition by 0.1 mM Na+/K+ tartrate, 55%-60% inhibition by 0.1 mM NaF, and 75% inhibition by 0.1 mM Na pyrophosphate. Para-chloromercuribenzoate also inhibited the activity, suggesting that reduced thiols may be important in catalysis. One mM calcium chloride altered the activity in a complex manner depending on the pH, suggesting a possible role for calcium in regulating enzyme activity.
Haugen-Scofield
J
J
Department of Periodontics, University of Washington, Seattle 98195.
Resing
K A
KA
Dale
B A
BA
D000135:Acid Phosphatase / Q000032:analysis; D000818:Animals; D047388:Casein Kinases; D004817:Epidermis / Q000201:enzymology*; D000091344:Filaggrin Proteins; D006863:Hydrogen-Ion Concentration; D007381:Intermediate Filament Proteins / Q000302:isolation & purification*; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification*; D011494:Protein Kinases / Q000494:pharmacology*; D011498:Protein Precursors / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D011498:Protein Precursors; D011494:Protein Kinases; D047388:Casein Kinases; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases