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)