Identification of hematoxylin‐stainable protein in epidermal keratohyalin granules as phosphorylated cystatin α by protein kinase C

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Abstract
The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin α. However, only its pi value (4.7) differed from that of cystatin α (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [γ-32P]ATP was incorporated into recombinant cystatin α by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin α and was inhibited significantly by H-7. a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin α. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin α and that the phosphorylation is catalyzed specifically by PKC.