Stirdivant S M, Huber H E, Patrick D R, Defeo-Jones D, McAvoy E M, Garsky V M, Oliff A, Heimbrook D C
Department of Cancer Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Mol Cell Biol. 1992 May;12(5):1905-14. doi: 10.1128/mcb.12.5.1905-1914.1992.
The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.
人乳头瘤病毒E7基因可转化鼠类成纤维细胞，并在转化原代细胞培养物中与其他病毒癌基因协同作用。与E7蛋白相关的一种生化特性是与视网膜母细胞瘤肿瘤抑制基因产物（pRB）结合。与pRB相关的生化特性包括与病毒转化蛋白（E1A、大T抗原和E7）结合、与细胞蛋白（E2F和Myc）结合以及与DNA结合。E7刺激细胞生长的机制尚不确定。然而，E7与pRB的结合会抑制细胞蛋白与pRB的结合，并且似乎会阻断pRB的生长抑制活性。我们发现E7还会抑制pRB与DNA的结合。在网织红细胞翻译反应或细菌中产生的60 kDa版本的pRB（pRB60）能定量结合到DNA纤维素上。与pRB60以1:1或10:1摩尔比使用的重组E7蛋白分别阻断了50%或大于95%的pRB60 DNA结合活性。一种与pRB60结合活性降低的突变E7蛋白（E7-Ala-24）在阻断pRB60与DNA结合方面也表现出相应降低。一种能阻断E7蛋白与pRB60结合的E7（20-29）肽在有E7存在的情况下恢复了pRB60的DNA结合活性。肽E7（2-32）不阻断pRB60与DNA的结合，而肽E7（20-57）和一个包含1至60位残基的E7片段部分阻断了DNA结合。包含3至75位残基的E7物种在阻断pRB60与DNA结合方面完全有效。这些研究表明E7蛋白特异性地阻断pRB60与DNA的结合，并表明负责此特性的E7区域位于32至75位残基之间。这些观察结果的功能意义尚不清楚。然而，我们发现pRB60中的一个点突变损害了DNA结合活性，同时也阻断了pRB60抑制细胞生长的能力。这种相关性表明视网膜母细胞瘤蛋白的DNA结合活性有助于其生物学特性。
