Templeton D J
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106.
Mol Cell Biol. 1992 Feb;12(2):435-43. doi: 10.1128/mcb.12.2.435-443.1992.
The retinoblastoma tumor suppressor gene product (pRb) is a nuclear protein subject to cell cycle-regulated hyperphosphorylation. I constructed a recombinant vaccinia virus vector that expresses both the underphosphorylated and hyperphosphorylated forms of pRb and purified the recombinant protein by using immunoaffinity chromatography directed toward a synthetic carboxy-terminal epitope. To investigate the hypothesis that hyperphosphorylation of pRb is a means of controlling its growth-regulating activity, I tested purified pRb for the ability to be reincorporated into pRb-deficient nuclei in vitro. The underphosphorylated form of pRb efficiently reassociated with nuclei, but the hyperphosphorylated form remained soluble in this assay. Nuclear binding of pRb was enhanced by phosphatase treatment and reduced by phosphorylation of pRb effected by using a preparation of the cell cycle-regulatory kinase p34cdc2. Mutant-encoded proteins with altered E1A-binding domains failed to bind to nuclei. Pretreatment of target nuclei with nucleases and high-salt extraction did not alter the specificity of binding for underphosphorylated pRb. These observations demonstrate that hyperphosphorylation of pRb can regulate its interaction with nuclei, supporting the hypothesis that hyperphosphorylation controls the growth-regulatory activities of pRb. Further, at least one target of pRb binding appears to be an integral component of the nuclear envelope.
视网膜母细胞瘤肿瘤抑制基因产物（pRb）是一种受细胞周期调节的超磷酸化核蛋白。我构建了一种重组痘苗病毒载体，该载体可表达pRb的低磷酸化和超磷酸化形式，并通过针对合成羧基末端表位的免疫亲和层析纯化重组蛋白。为了研究pRb的超磷酸化是控制其生长调节活性的一种方式这一假说，我测试了纯化的pRb在体外重新整合到缺乏pRb的细胞核中的能力。pRb的低磷酸化形式能有效地与细胞核重新结合，但超磷酸化形式在此测定中仍保持可溶状态。用磷酸酶处理可增强pRb与细胞核的结合，而用细胞周期调节激酶p34cdc2制剂对pRb进行磷酸化则会降低这种结合。具有改变的E1A结合域的突变编码蛋白无法与细胞核结合。用核酸酶和高盐提取对靶细胞核进行预处理不会改变低磷酸化pRb结合的特异性。这些观察结果表明，pRb的超磷酸化可调节其与细胞核的相互作用，支持超磷酸化控制pRb生长调节活性的假说。此外，pRb结合的至少一个靶点似乎是核膜的一个组成成分。
