Interaction of phospholipids, retinoids and PMA with calcium, phospholipid-dependent protein kinase-catalyzed reaction in skin.

Previous investigations suggested that a mechanism independent of cAMP may be associated with the action of some retinoids. An alternative pathway involving calcium, phospholipid-dependent protein kinase (C-kinase), was therefore studied. In order to demonstrate this, C-kinase was partially purified from skin of hairless, Balb/c normal and Balb/c nude mouse. Interaction and effects of various response modifiers such as phospholipids, retinoids and phorbol ester tumor promoters showed both major and minor differences among these enzymes. In general, retinal, retinoic acid, 13-cis-retinoic acid and etretinate stimulated skin enzyme activity in the absence of the natural stimulants, phosphatidyl serine and diacylglycerol (DAG). However in their presence the C-kinases were inhibited by retinoids. Our data further indicated that the active retinoids may compete with DAG for binding sites on the enzyme. However, the high concentrations of retinoids needed to elicit these effects suggested a pharmacological role for retinoid action as a result of hydrophobic interaction with lipid domains on the enzyme. These investigations also revealed some of the complexity associated with retinoid effects on C-kinase. Tumor promoter, phorbol-12-myristate 13-acetate (PMA) interacted with its receptor (C-kinase) from hairless and normal mouse skin and stimulated enzyme activity. However, PMA-dependent stimulation of nude mouse C-kinase was about half of that noted with the other two C-kinases. Furthermore, unlike its effect on hairless and Balb/c normal C-kinases, PMA was unable to potentiate the retinoid-stimulated activity of nude mouse skin enzyme. This behavior suggested that nude mouse C-kinase may be a variant form of the normal enzyme. The presence of this variant C-kinase may, therefore, be responsible for the lack of phorbol ester-induced tumor promotion observed earlier in nude mouse skin by other investigators. Endogenous substrate phosphorylation catalyzed by C-kinase from hairless and Balb/c normal mice resulted in 32P incorporation into four target polypeptides of molecular weights 75-78, 47-50, 25-29 and 14-18 kilodaltons. However, with the nude mouse enzyme, only the 75- to 78-kilodalton protein served as the target supporting the suggestion that this may be a variant C-kinase. Neither retinoic acid (10(-3) M) nor PMA (10(-6) M) seemed to affect the phosphorylation of any of the four polypeptides.(ABSTRACT TRUNCATED AT 400 WORDS)