Mutational analysis reveals an essential role for the LXXLL motif in the transformation function of the human herpesvirus-8 oncoprotein, kaposin.
Mutational analysis reveals an essential role for the LXXLL motif in the transformation function of the human herpesvirus-8 oncoprotein, kaposin.
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突变分析揭示了 LXXLL 基序在人类疱疹病毒 8 癌蛋白卡波蛋白转化功能中的重要作用。
DOI：
10.1089/dna.2005.24.10
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Srinivasan,Alagarsamy
中科院分区：
文献类型：
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作者：
Tomkowicz,Brian;Singh,SatyaP;Lai,Derhsing;Singh,Anjali;Mahalingham,Sundarasamy;Joseph,Jeymohan;Srivastava,Shiv;Srinivasan,Alagarsamy
人类疱疹病毒-8 （HHV-8）与卡波西肉瘤（KS）有因果关系。基因组的序列分析和随后的研究显示，包括卡波蛋白在内的几个基因在细胞培养中具有转化特性。在本研究中，我们分析了Kaposin A对细胞转化的要求，以了解其对KS发病机制的贡献。Kaposin与其他蛋白的比较分析鉴定出LXXLL基序从残基31-35 （LVCLL）延伸。观察到LXXLL基序存在于介导核受体共激活因子与核受体相互作用的核受体共激活因子中，这促使我们研究该基序与Kaposin功能的相关性。将KaposinA编码序列克隆到具有Flag （FL）表位的真核表达质粒c端（Kap-FL）内框融合的真核表达质粒。为了评估亮氨酸残基在基序中的作用，利用定点诱变，用丙氨酸取代亮氨酸残基（Kap-AXXAA-FL）。Kap- fl和Kap- AXXAA-FL在细胞中的表达水平相似。有趣的是，Kap-AXXAA-FL突变体在两项独立试验中均未表现出转化活性：不依赖锚定生长和焦点形成。免疫荧光（IFA）和FACS分析显示Kap-FL分别定位于细胞核周围和细胞表面。然而，通过IFA检测，Kap-AXXAA-FL呈弥漫性细胞质染色，但在细胞表面仍可通过FACS检测到。具有讽刺意味的是，Kap-FL和Kap-AXXAAFL都能够激活AP-1启动子。这些结果支持LXXLL基序在Kaposin诱导转化的能力中发挥重要作用。
Human herpesvirus-8 (HHV-8) is causally linked to Kaposi's sarcoma (KS). Sequence analysis of the genome and subsequent studies revealed several genes includingkaposin, with transformation properties in cell culture. In this study, we have analyzed the requirement of Kaposin A for cellular transformation in an effort to understand its contribution towards KS pathogenesis. Comparative analysis of Kaposin with other proteins identified the LXXLL motif spanning from residues 31–35 (LVCLL). The observation that the LXXLL motif is present in nuclear receptor coactivators that mediate the interaction of coactivators with nuclear receptors has prompted us to investigate the relevance of this motif for Kaposin's function(s).KaposinA coding sequences were cloned into a eukaryotic expression plasmid with the Flag (FL) epitope fused in-frame at the C-terminus (Kap-FL). To evaluate the role of leucine residues in the motif, site-directed mutagenesis was utilized, whereby alanine was substituted for the leucine residues (Kap-AXXAA-FL). Both Kap-FL and Kap- AXXAA-FL exhibited similar levels of expression in cells. Interestingly, the Kap-AXXAA-FL mutant failed to show transforming activity by two independent assays: anchorage-independent growth, and focus formation. Immunofluorescence (IFA) and FACS analysis indicated that Kap-FL was localized around the nucleus and at the cell surface, respectively. However, Kap-AXXAA-FL exhibited diffuse cytoplasmic staining as measured by IFA yet was still detectable on the cell surface by FACS. Ironically, both Kap-FL and Kap-AXXAAFL were able to activate the AP-1 promoter. These results support an important role for the LXXLL motif in the ability of Kaposin to induce transformation.