Kumar R, Holian O, Fiedler V C
Department of Dermatology, University of Illinois at Chicago, College of Medicine.
Dermatologica. 1987;175 Suppl 1:56-66.
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)
先前的研究表明，一种独立于环磷酸腺苷（cAMP）的机制可能与某些类视黄醇的作用有关。因此，对一条涉及钙、磷脂依赖性蛋白激酶（C激酶）的替代途径进行了研究。为了证实这一点，从无毛、Balb/c正常和Balb/c裸鼠的皮肤中部分纯化了C激酶。各种反应调节剂如磷脂、类视黄醇和佛波酯肿瘤促进剂的相互作用及作用在这些酶之间显示出主要和次要的差异。一般来说，视黄醛、视黄酸、13-顺式视黄酸和依曲替酯在没有天然刺激物磷脂酰丝氨酸和二酰基甘油（DAG）的情况下刺激皮肤酶活性。然而，在它们存在的情况下，类视黄醇会抑制C激酶。我们的数据进一步表明，活性类视黄醇可能与DAG竞争酶上的结合位点。然而，引发这些效应所需的高浓度类视黄醇表明，类视黄醇作用具有药理学作用，这是由于其与酶上的脂质结构域发生疏水相互作用的结果。这些研究还揭示了类视黄醇对C激酶作用的一些复杂性。肿瘤促进剂佛波醇-12-肉豆蔻酸酯13-乙酸酯（PMA）与来自无毛和正常小鼠皮肤的受体（C激酶）相互作用并刺激酶活性。然而，PMA对裸鼠C激酶的刺激作用约为其他两种C激酶的一半。此外，与它对无毛和Balb/c正常C激酶的作用不同，PMA无法增强裸鼠皮肤酶的类视黄醇刺激活性。这种行为表明裸鼠C激酶可能是正常酶的一种变体形式。因此，这种变体C激酶的存在可能是其他研究者先前在裸鼠皮肤中观察到缺乏佛波酯诱导的肿瘤促进作用的原因。来自无毛和Balb/c正常小鼠的C激酶催化的内源性底物磷酸化导致32P掺入分子量为75 - 78、47 - 50、25 - 29和14 - 18千道尔顿的四种靶多肽中。然而，对于裸鼠酶，只有75至78千道尔顿的蛋白质作为靶标，支持了这可能是一种变体C激酶的观点。视黄酸（10⁻³ M）和PMA（10⁻⁶ M）似乎都不影响这四种多肽中任何一种的磷酸化。（摘要截短至400字）
