Knez Jozo, Piluso David, Bilan Patricia, Capone John P
Department of Biochemistry and Biomedical Sciences, McMaster University Medical Center, McMaster University, 1200 Main St. W., Hamilton, Ontario, L8N 3Z5, Canada.
Mol Cell Biochem. 2006 Aug;288(1-2):79-90. doi: 10.1007/s11010-006-9122-x. Epub 2006 Apr 22.
Host Cell Factor (HCF-1) is a conserved, essential protein initially identified as a co-regulator for the Herpes Simplex Virus transactivator VP16. HCF-1 is variously involved in regulating transcription, splicing, cell proliferation and cytokinesis; however, its mechanisms of action remain unknown. HCF-1 function is manifested through an increasing assortment of cellular factors that target different regions of the protein. Several HCF-1 partners target the amino-terminal kelch domain of HCF-1 (residues 1-380) via a consensus HCF-binding motif (HBM) comprising the tetrapeptide (D/E)HXY. Searches of sequence databases indicated that this motif is present in E2F1 and E2F4, two members of the E2F family of cell cycle regulators. We show here that E2F4 specifically and directly interacts with HCF-1. Mutational analysis showed E2F4 independently targets the kelch domain and the basic domain (residues 450-902) of HCF-1, both of which are required for normal cell-cycle progression via separate determinants. The HBM-containing domain of E2F4 was necessary for interaction with the kelch domain of HCF-1 but not for interaction with the basic domain. Mutations in the HCF-1 kelch domain known to block cell growth abrogated E2F4 binding to the kelch domain in the absence but not in the presence of the juxtaposed basic region. Functionally, HCF-1 co-activated E2F4/DP-1 in transient transfection assays, while E2F4 blocked HCF-1-dependent rescue of a cell line that harbors a temperature sensitive mutant of HCF-1 that causes growth arrest. Our findings show that HCF-1 and E2F4 interact via multiple determinants and suggest a linkage between E2F4 and HCF-1 cell growth pathways.
宿主细胞因子（HCF-1）是一种保守的必需蛋白，最初被鉴定为单纯疱疹病毒反式激活因子VP16的共调节因子。HCF-1以多种方式参与调节转录、剪接、细胞增殖和胞质分裂；然而，其作用机制仍不清楚。HCF-1的功能通过越来越多靶向该蛋白不同区域的细胞因子得以体现。几个HCF-1结合伴侣通过包含四肽（D/E）HXY的共有HCF结合基序（HBM）靶向HCF-1的氨基末端kelch结构域（第1至380位氨基酸残基）。对序列数据库的搜索表明，该基序存在于细胞周期调节因子E2F家族的两个成员E2F1和E2F4中。我们在此表明，E2F4特异性且直接地与HCF-1相互作用。突变分析表明，E2F4独立靶向HCF-1的kelch结构域和碱性结构域（第450至902位氨基酸残基），这两个结构域通过不同的决定因素对于正常细胞周期进程都是必需的。E2F4含HBM的结构域对于与HCF-1的kelch结构域相互作用是必需的，但对于与碱性结构域的相互作用则不是必需的。已知在HCF-1 kelch结构域中阻断细胞生长的突变在不存在并列碱性区域的情况下消除了E2F4与kelch结构域的结合，但在存在并列碱性区域的情况下则没有消除。在瞬时转染实验中，HCF-1共激活了E2F4/DP-1，而E2F4阻断了HCF-1对一个携带导致生长停滞的HCF-1温度敏感突变体的细胞系的依赖性拯救。我们的研究结果表明，HCF-1和E2F4通过多种决定因素相互作用，并提示E2F4与HCF-1细胞生长途径之间存在联系。
