A purine analog kinase inhibitor, calcium/calmodulin-dependent protein kinase II inhibitor 59, reveals a role for calcium/calmodulin-dependent protein kinase II in insulin-stimulated glucose transport
A purine analog kinase inhibitor, calcium/calmodulin-dependent protein kinase II inhibitor 59, reveals a role for calcium/calmodulin-dependent protein kinase II in insulin-stimulated glucose transport
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DOI：
10.1210/en.2006-0446
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发表时间：
2007-01-01
期刊：
影响因子：
4.8
通讯作者：
Macaulay, S. Lance
中科院分区：
文献类型：
--
作者：
Konstantopoulos, Nicky;Marcuccio, Seb;Macaulay, S. Lance
Olomoucine是一种细胞周期蛋白依赖性激酶抑制剂。我们发现奥洛莫辛阻断胰岛素刺激葡萄糖转运的能力。这样做不会影响已知胰岛素信号蛋白的活性。为了鉴定对奥洛莫星敏感的激酶，我们制备了可以固定在亲和树脂上以分离结合蛋白的类似物。所产生的类似物之一抑制胰岛素刺激的葡萄糖摄取，与奥洛莫辛相比具有增加的灵敏度。抑制胰岛素刺激的葡萄糖摄取的IC 50发生在低至0.1 μ M的类似物浓度。为了鉴定与类似物结合的蛋白质，将从3 T3-L1脂肪细胞制备的[S-35]标记的细胞裂解物与化学交联至树脂支持物的类似物一起孵育，并通过SDS-PAGE分析结合蛋白。主要结合物质为50-60 kDa的双联体，通过N端肽分析鉴定为钙/钙调蛋白依赖性蛋白激酶II（CaMKII），并通过基质辅助激光解吸电离质谱法确认为δ和β样亚型。为了研究CaMKII参与胰岛素刺激的葡萄糖摄取，用编码绿色荧光蛋白（GFP）-血凝素标签（HA）标记的CaMKII野生型或ATP结合突变体K42 M的逆转录病毒感染3 T3-L1脂肪细胞。GFP-HA-CaMKII K42 M细胞的激酶活性低于表达野生型GFP-HAcaMKII的细胞。胰岛素刺激的葡萄糖转运显着减少（类似于80%）GFP-HA-CaMKII K42 M细胞，与未转染的细胞，细胞表达GFP-HA-CaMKII或GFP-HA。与单独的GFP-HA相比，GFP-HA-CaMKII K42 M细胞中胰岛素刺激的GLUT 4易位没有伴随减少。然而，与GFP-HA或GFP-HA-CaMKII K42 M细胞相比，GFP-HA-CaMKII细胞中胰岛素刺激的GLUT 4易位显著更高。我们的研究结果暗示CaMKII参与葡萄糖转运的许可作用。
Olomoucine is known as a cyclin-dependent kinase inhibitor. We found that olomoucine blocked insulin's ability to stimulate glucose transport. It did so without affecting the activity of known insulin signaling proteins. To identify the olomoucine-sensitive kinase(s), we prepared analogs that could be immobilized to an affinity resin to isolate binding proteins. One of the generated analogs inhibited insulin-stimulated glucose uptake with increased sensitivity compared with olomoucine. The IC50 for inhibition of insulin-stimulated glucose uptake occurred at analog concentrations as low as 0.1 mu M. To identify proteins binding to the analog, [S-35]-labeled cell lysates prepared from 3T3-L1 adipocytes were incubated with analog chemically cross-linked to a resin support and binding proteins analyzed by SDS-PAGE. The major binding species was a doublet at 50-60 kDa, which was identified as calcium/calmodulin-dependent protein kinase II (CaMKII) by N-terminal peptide analysis and confirmed by matrix-assisted laser desorption ionization-mass spectrometry as the delta- and beta-like isoforms. To investigate CaMKII involvement in insulin-stimulated glucose uptake, 3T3-L1 adipocytes were infected with retrovirus encoding green fluorescent protein (GFP)-hemagluttinin tag (HA)-tagged CaMKII wild-type or the ATP binding mutant, K42M. GFP-HA-CaMKII K42M cells had less kinase activity than cells expressing wild-type GFP-HACaMKII. Insulin-stimulated glucose transport was significantly decreased (similar to 80%) in GFP-HA-CaMKII K42M cells, compared with nontransfected cells, and cells expressing either GFP-HA-CaMKII or GFP-HA. There was not a concomitant decrease in insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII K42M cells when compared with GFP-HA alone. However, insulin-stimulated GLUT4 translocation in GFP-HA-CaMKII cells was significantly higher, compared with either GFP-HA or GFP-HA-CaMKII K42M cells. Our results implicate the involvement of CaMKII in glucose transport in a permissive role.