Chen Xiaoli, Al-Hasani Hadi, Olausson Torbjorn, Wenthzel Ann-Marie, Smith Ulf, Cushman Samuel W
EDMNS/DB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 8 Center Dr MSC 0842, Bethesda, MD 20892-0842, USA.
J Cell Sci. 2003 Sep 1;116(Pt 17):3511-8. doi: 10.1242/jcs.00675. Epub 2003 Jul 22.
In this study, fusion of the kinase domain of Akt2 to the cytosolic C terminus of exofacially-HA-tagged GLUT4 is used to investigate the activity, phosphorylation state and subcellular localization of Akt2 specifically targeted to the GLUT4-trafficking pathway in rat adipose cells. Fusion of wild-type (wt) Akt2, but not a kinase-dead (KD) mutant results in constitutive targeting of the HA-GLUT4 fusion protein to the cell surface to a level similar to that of HA-GLUT4 itself in the insulin-stimulated state. Insulin does not further enhance the cell-surface level of HA-GLUT4-Akt2-wt, but does stimulate the translocation of HA-GLUT4-Akt2-KD. Cell-surface HA-GLUT4-Akt2-wt is found to be phosphorylated on Ser474 in both the absence and presence of insulin, and mutation of Ser474 to Ala reduces the increased basal cell-surface localization of the fusion protein. While Ser474 phosphorylation of HA-GLUT4-Akt2-KD is detected only in the insulin-stimulated state, trapping this fusion protein on the cell surface by coexpression of a dominant negative mutant dynamin does not induce Ser474 phosphorylation. Phosphorylation on Thr309 is not detectable in either HA-GLUT4-Akt2-wt or HA-GLUT4-Akt2-KD, in either the basal or insulin-stimulated state, and mutation of Thr309 to Ala does not influence the insulin-independent increases in cell-surface localization and Ser474 phosphorylation. Expression of HA-GLUT4-Akt2-wt stimulates the translocation of cotransfected myc-GLUT4 to a level similar to that in the insulin-stimulated state; this increase is moderately reduced by mutation of Ser474 to Ala and absent with the kinase-dead mutant. These results demonstrate that targeting Akt2 to the GLUT4-trafficking pathway induces Akt2 activation and GLUT4 translocation. Ser474 phosphorylation is an autocatalytic reaction requiring an active kinase, and kinase activity is associated with a plasma membrane localization. Fusion of Akt2 to the C terminus of GLUT4 appears to substitute for Thr309 phosphorylation in activating the autocatalytic process.
在本研究中，将Akt2的激酶结构域与胞外HA标签化的GLUT4的胞质C末端融合，用于研究特异性靶向大鼠脂肪细胞中GLUT4转运途径的Akt2的活性、磷酸化状态和亚细胞定位。野生型（wt）Akt2的融合，而非激酶失活（KD）突变体的融合，导致HA-GLUT4融合蛋白组成性靶向细胞表面，其水平与胰岛素刺激状态下HA-GLUT4自身的水平相似。胰岛素不会进一步提高HA-GLUT4-Akt2-wt的细胞表面水平，但会刺激HA-GLUT4-Akt2-KD的转位。发现在有无胰岛素的情况下，细胞表面的HA-GLUT4-Akt2-wt在Ser474位点均被磷酸化，并且将Ser474突变为Ala会降低融合蛋白基础细胞表面定位的增加。虽然仅在胰岛素刺激状态下检测到HA-GLUT4-Akt2-KD的Ser474磷酸化，但通过共表达显性负性突变体发动蛋白将该融合蛋白捕获在细胞表面并不会诱导Ser474磷酸化。在基础或胰岛素刺激状态下，HA-GLUT4-Akt2-wt或HA-GLUT4-Akt2-KD中均未检测到Thr309的磷酸化，并且将Thr309突变为Ala不会影响细胞表面定位和Ser474磷酸化的胰岛素非依赖性增加。HA-GLUT4-Akt2-wt的表达将共转染的myc-GLUT4的转位刺激到与胰岛素刺激状态相似的水平；这种增加在Ser474突变为Ala时适度降低，而在激酶失活突变体中则不存在。这些结果表明，将Akt2靶向GLUT4转运途径可诱导Akt2激活和GLUT4转位。Ser474磷酸化是一种需要活性激酶的自催化反应，并且激酶活性与质膜定位相关。将Akt2与GLUT4的C末端融合似乎在激活自催化过程中替代了Thr309磷酸化。
