Sackett Dan L, Ozbun Laurent, Zudaire Enrique, Wessner Lisa, Chirgwin John M, Cuttitta Frank, Martínez Alfredo
Laboratory of Integrative and Medical Biophysics, National Cancer Institute, Bethesda, Maryland 20892, USA.
Endocrinology. 2008 Jun;149(6):2888-98. doi: 10.1210/en.2007-1763. Epub 2008 Mar 6.
Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are secretory hormones, but it is not unusual to find them in intracellular compartments. Using yeast-2 hybrid technology, we found interactions between AM and several microtubule-associated proteins (MAPs), and between PAMP and tubulin. Expression of fluorescent-tagged AM and PAMP as well as immunofluorescence for the native peptides showed a complete decoration of the microtubules and colocalization with other MAPs. PAMP, but not AM, bound to tubulin in vitro and destabilized tubulin polymerization. Down-regulation of the gene coding for both AM and PAMP through small interfering RNA technology resulted in morphological changes, microtubule stabilization, increase in posttranslational modifications of tubulin such as acetylation and detyrosination, reduction in cell motility, and partial arrest at the G2 phase of the cell cycle, when compared with cells transfected with the same vector carrying a scrambled sequence. These results show that PAMP is a novel MAP, whereas AM may be exerting more subtle effects in regulating cytoskeleton function.
肾上腺髓质素（AM）和肾上腺髓质素原N端20肽（PAMP）是分泌性激素，但在细胞内区室中发现它们并不罕见。利用酵母双杂交技术，我们发现AM与几种微管相关蛋白（MAPs）之间以及PAMP与微管蛋白之间存在相互作用。荧光标记的AM和PAMP的表达以及天然肽的免疫荧光显示微管完全被标记且与其他MAPs共定位。PAMP而非AM在体外与微管蛋白结合并破坏微管蛋白的聚合。通过小干扰RNA技术下调编码AM和PAMP的基因，与转染携带随机序列的相同载体的细胞相比，导致细胞形态改变、微管稳定、微管蛋白翻译后修饰如乙酰化和去酪氨酸化增加、细胞运动性降低以及细胞周期在G2期部分停滞。这些结果表明PAMP是一种新型的MAP，而AM可能在调节细胞骨架功能方面发挥更微妙的作用。
