Wang Q, Somwar R, Bilan P J, Liu Z, Jin J, Woodgett J R, Klip A
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Mol Cell Biol. 1999 Jun;19(6):4008-18. doi: 10.1128/MCB.19.6.4008.
L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBalpha)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Deltap85alpha). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBalpha, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBalpha/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBalpha with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBalpha/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.
用携带胞外myc表位标签的GLUT4 cDNA稳定转染的L6成肌细胞（L6 - GLUT4myc成肌细胞）用于研究蛋白激酶Bα（PKBα）/Akt1在胰岛素诱导的GLUT4向细胞表面转位中的作用。通过对未通透细胞中myc表位进行免疫荧光标记来检测表面的GLUT4myc。胰岛素在20分钟内诱导GLUT4myc显著转位至质膜。这被磷脂酰肌醇（PI）3 -激酶的显性抑制构建体（Deltap85α）的瞬时转染所阻止。通过共转染绿色荧光蛋白来鉴定瞬时转染的细胞。通过在其N端融合病毒Gag蛋白构建的组成型活性PKBα（GagPKB），与相邻未转染细胞相比，增加了GLUT4myc的细胞表面密度。具有K179A（第179位赖氨酸被丙氨酸取代）、T308A和S473A突变的激酶失活、磷酸化缺陷型PKBα/Akt1构建体（AAA - PKB）表现为共转染的野生型血凝素（HA）标签PKB胰岛素依赖性激活的显性负抑制剂。此外，AAA - PKB显著抑制共转染的BAD的胰岛素诱导的磷酸化，表明内源性PKB/Akt受到抑制。在相同条件下，AAA - PKB几乎完全阻断胰岛素依赖性的表面GLUT4myc增加。单独具有丙氨酸取代T308A和S473A（AA - PKB）或K179A（A - PKB）的PKBα作为野生型HA - PKB胰岛素依赖性激活或GLUT4myc转位的抑制剂，其效力低于AAA - PKB。AAA - PKB与四倍过量的HA - PKB DNA共转染可挽救胰岛素刺激的GLUT4myc转位。AAA - PKB不阻止肌动蛋白成束（膜皱襞），尽管这种反应是PI 3 -激酶依赖性的。因此，AAA - PKB不太可能通过抑制PI 3 -激酶信号传导起作用。这些结果概述了PKBα/Akt1在培养的肌肉细胞中胰岛素刺激葡萄糖转运中的重要作用。
