[An experimental study of epidermal keratin phosphorylation --epidermal keratin as a substrate protein of cAMP-dependent protein kinase].
The phosphorylating system by protein kinases in living cells has been emphasized on its biological importance especially in the responses of cells to environmental changes via humoral transmitters. In the epidermal cells, only phosphorylase was clarified to be activated in the phosphorylating system by cyclic AMP-dependent protein kinase (cAMP-PK) through the adenylate cyclase-cAMP system. In the present study, keratin, which is the most abundant and important constituent of the epidermis was focussed whether it could be a substrate protein of phosphorylation by protein kinase. Pig epidermis was separated into basal, lower spinous, upper spinous, and horny cells and keratin was extracted from each layer. Phosphorylation of keratin was determined in cell free assay system by counting the radioactivity of 32P which was incorporated into keratin with the presence of partially purified pig epidermal cAMP-PK, cAMP, and [gamma-32P] ATP. Phosphorylated keratin was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The results were as follows, pig epidermal keratin was phosphorylated by cAMP-PK, and that 60,000 and 51,000 dalton polypeptides constituting basal and spinous cell keratins were phosphorylated but not 65,000 and 56,000 dalton polypeptides which were found only in spinous cell keratin, in addition, there existed a difference of phosphorylation potential among keratins from various strata. Horny cell keratin was most strongly phosphorylated and upper spinous cell, lower spinous cell, basal cell keratins, were phosphorylated in order of intensity. Therefore it was assumed that keratin phosphorylation may play an important role in the dynamics of keratin biosynthesis and its maturation.
D000818:Animals; D001345:Autoradiography; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004817:Epidermis / Q000201:enzymology / Q000378:metabolism*; D066298:In Vitro Techniques; D007633:Keratins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine
D007633:Keratins; D011494:Protein Kinases