The Insulin-sensitive Glucose Transporter, GLUT4, Interacts Physically with Daxx
The Insulin-sensitive Glucose Transporter, GLUT4, Interacts Physically with Daxx
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胰岛素敏感葡萄糖转运蛋白 GLUT4 与 Daxx 发生物理相互作用
DOI：
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发表时间：
2002
影响因子：
4.8
通讯作者：
I. Sandoval
中科院分区：
文献类型：
--
作者：
Vassiliki S. Lalioti;Silvia Vergarajauregui;D. Pulido;I. Sandoval
在这项研究中，我们使用酵母双杂交系统来鉴定与胰岛素敏感的葡萄糖转运蛋白GLUT4(C-GLUT4)的羧基-细胞质结构域(残基464-509)相互作用的蛋白质。以C-GLUT4为诱饵，我们分离了与Fas和II型转化生长因子-β(T-βRII)受体(1，2)相关的接头蛋白Daxx的羧基结构域(C-Daxx)。通过体外翻译的C-GLUT4与体外翻译的全长Daxx和C-Daxx的相互作用验证了C-GLUT4和C-Daxx之间的双杂交相互作用。C-Daxx不与GLUT1的C-细胞质结构域相互作用，GLUT1是一种与GLUT4同源的普遍存在的葡萄糖转运蛋白。丙氨酸和丝氨酸对二亮氨酸对(Leu489-Leu490)的替代对将GLUT4从跨高尔基网络靶向到核周细胞内储存区以及通过内吞作用使其表面内化至关重要，可抑制C-GLUT4与Daxx的相互作用2倍。从稳定转染GLUT4的3T3-L1成纤维细胞和表达这两种蛋白生理水平的3T3-L1脂肪细胞的裂解产物中用GLUT4免疫共沉淀物拉低Daxx。同样，GLUT4可以用抗Daxx免疫沉淀物恢复。使用已建立的细胞分离程序，我们提出了在细胞核和低密度微粒子中存在两个不同的细胞内Daxx池的证据。共聚焦免疫荧光显微镜研究将Daxx定位于早幼粒细胞白血病的核体和点状的细胞质结构，通常排列成串状并在质膜下。Daxx和GLUT4通过它们与抗SUMO1抗体的反应以及这种抗体拖累Daxx和GLUT4的能力来证明是SUMOL。
In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxyl-cytoplasmic domain (residues 464–509) of the insulin-sensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-β (TβRII) receptors (1, 2). The two-hybrid interaction between C-GLUT4 and C-Daxx is validated by the ability ofin vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu489-Leu490) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with anti-Daxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.
影响因子：
3.3
作者：
MELUH, PB;KOSHLAND, D
通讯作者：
KOSHLAND, D
影响因子：
56.9
作者：
Chang, HY;Nishitoh, H;Baltimore, D
通讯作者：
Baltimore, D