de Graan P N, Gispen W H, van de Veerdonk F C
Mol Cell Endocrinol. 1985 Sep;42(2):119-25. doi: 10.1016/0303-7207(85)90099-1.
To investigate a possible role of protein phosphorylation in the mechanism of action of alpha-MSH, excised tail-fins of Xenopus tadpoles were incubated with or without alpha-MSH. After homogenization, in vitro endogenous protein phosphorylation was assayed using [gamma-32P]ATP. alpha-MSH treatment of intact tail-fins, producing full pigment dispersion, resulted in a 5-fold increase in 32P-incorporation into a 53 kDa protein band. This increase in 53 kDa phosphorylation was completely reversible. The increase was not found in homogenates from the melanophore-free part of the alpha-MSH-treated tail-fins. Phosphorylation of the 53 kDa protein could be detected in homogenates of alpha-MSH-treated primary cultured melanophores. Incubation of tail-fins with ACTH1-24, an alpha-MSH-like peptide producing full pigment dispersion, also induced an increase in 53 kDa phosphorylation. A structurally related peptide (ACTH15-24) and an unrelated peptide (LH-RH), neither of which induced pigment dispersion, were ineffective in stimulating 53 kDa phosphorylation. Injection of white adapted tadpoles with 1 micrograms of alpha-MSH or adaptation of tadpoles to a black background also resulted in a significant increase in 53 kDa phosphorylation. alpha-MSH added to the homogenates did not affect 53 kDa phosphorylation, indicating that alpha-MSH acts through a receptor-mediated mechanism. The increase in 53 kDa phosphorylation measured in vitro (post hoc), most likely reflects an alpha-MSH-induced decrease in 53 kDa phosphorylation in vivo. Our results strongly suggest that a decrease in 53 kDa phosphorylation is involved in the mechanism of action of alpha-MSH on melanophores.
为了研究蛋白质磷酸化在α-促黑素（α-MSH）作用机制中可能发挥的作用，将非洲爪蟾蝌蚪切除的尾鳍在有或无α-MSH的情况下进行孵育。匀浆后，使用[γ-32P]ATP检测体外内源性蛋白质磷酸化情况。对完整尾鳍进行α-MSH处理，使其产生完全的色素扩散，结果显示，向一条53 kDa蛋白带中掺入的32P增加了5倍。53 kDa磷酸化的这种增加是完全可逆的。在经α-MSH处理的尾鳍不含黑素细胞的部分匀浆中未发现这种增加。在经α-MSH处理的原代培养黑素细胞的匀浆中可检测到53 kDa蛋白的磷酸化。用促肾上腺皮质激素1-24（一种能产生完全色素扩散的α-MSH样肽）孵育尾鳍，也可诱导53 kDa磷酸化增加。一种结构相关肽（促肾上腺皮质激素15-24）和一种不相关肽（促性腺激素释放激素），二者均未诱导色素扩散，在刺激53 kDa磷酸化方面无效。给白色适应状态的蝌蚪注射1微克α-MSH或使蝌蚪适应黑色背景，也会导致53 kDa磷酸化显著增加。向匀浆中添加α-MSH并不影响53 kDa磷酸化，这表明α-MSH通过受体介导的机制发挥作用。体外测量的53 kDa磷酸化增加（事后分析），很可能反映了α-MSH在体内诱导的53 kDa磷酸化减少。我们的结果强烈表明，53 kDa磷酸化减少参与了α-MSH对黑素细胞的作用机制。
