Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase 2A family.
The aggregation of cellular intermediate filaments is an important step in the terminal differentiation of keratinocytes. It has been shown that epidermal filaggrin can cause intermediate filaments to aggregate in vitro and may also have the same function in vivo. Filaggrin is derived via dephosphorylation and proteolysis from a highly phosphorylated precursor, profilaggrin, which is found in the granular layer of the epidermis. Using casein kinase II phosphorylated filaggrin as substrate, a profilaggrin phosphatase has been partially purified from rat epidermal homogenate by three chromatographic steps (DE52, hydroxylapatite and S200 gel filtration). Profilaggrin phosphatase activity eluted from the last column has a Km of 0.12 mM and a Vmax of 8 nmol/mg/min with respect to phosphofilaggrin. Results obtained by initial rate analysis showed that the enzymatic activity is not affected by phospho-tyrosyl phosphatase inhibitors and the active fractions preferentially dephosphorylate the alpha subunit of phosphorylase kinase which has been phosphorylated by cAMP-dependent kinase. These results suggest that epidermal profilaggrin phosphatase is not a phospho-tyrosyl phosphatase or a type 1 phospho-seryl/phospho-threonyl phosphatase. Dephosphorylation is not affected by EDTA, calcium or magnesium, but is very sensitive to okadaic acid inhibition (IC50 = 80 pM), suggesting that the enzymatic activity is related to that of the protein phosphatase 2A (PP2A).(ABSTRACT TRUNCATED AT 250 WORDS)
Kam
E
E
Department of Oral Biology, University of Washington, Seattle 98195.
Resing
K A
KA
Lim
S K
SK
Dale
B A
BA
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004817:Epidermis / Q000201:enzymology*; D004988:Ethers, Cyclic / Q000494:pharmacology; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D005810:Multigene Family; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D054648:Protein Phosphatase 2; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D014638:Vanadates / Q000494:pharmacology
D004988:Ethers, Cyclic; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D010750:Phosphoproteins; D011498:Protein Precursors; D019319:Okadaic Acid; D014638:Vanadates; D012965:Sodium Chloride; D004492:Edetic Acid; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D008274:Magnesium; D002118:Calcium; D007460:Iodoacetamide