Konstantopoulos Nicky, Marcuccio Seb, Kyi Stella, Stoichevska Violet, Castelli Laura A, Ward Colin W, Macaulay S Lance
CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville 3052, Victoria, Australia.
Endocrinology. 2007 Jan;148(1):374-85. doi: 10.1210/en.2006-0446. Epub 2006 Sep 28.
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 IC(50) for inhibition of insulin-stimulated glucose uptake occurred at analog concentrations as low as 0.1 microM. To identify proteins binding to the analog, [(35)S]-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-HA-CaMKII. Insulin-stimulated glucose transport was significantly decreased (approximately 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.
olomoucine是一种细胞周期蛋白依赖性激酶抑制剂。我们发现olomoucine能阻断胰岛素刺激葡萄糖转运的能力。它在不影响已知胰岛素信号蛋白活性的情况下做到了这一点。为了鉴定对olomoucine敏感的激酶，我们制备了可以固定到亲和树脂上以分离结合蛋白的类似物。所产生的一种类似物与olomoucine相比，以更高的敏感性抑制胰岛素刺激的葡萄糖摄取。抑制胰岛素刺激的葡萄糖摄取的IC(50)在低至0.1微摩尔的类似物浓度下出现。为了鉴定与该类似物结合的蛋白质，将从3T3-L1脂肪细胞制备的[(35)S]标记的细胞裂解物与化学交联到树脂支持物上的类似物一起孵育，并通过SDS-PAGE分析结合蛋白。主要的结合条带是一条50 - 60 kDa的双峰带，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在胰岛素刺激的葡萄糖摄取中的作用，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K42M的逆转录病毒感染3T3-L1脂肪细胞。GFP-HA-CaMKII K42M细胞的激酶活性低于表达野生型GFP-HA-CaMKII的细胞。与未转染细胞以及表达GFP-HA-CaMKII或GFP-HA的细胞相比，GFP-HA-CaMKII K42M细胞中胰岛素刺激的葡萄糖转运显著降低（约80%）。与单独的GFP-HA相比，GFP-HA-CaMKII K42M细胞中胰岛素刺激的GLUT4转位没有相应降低。然而，与GFP-HA或GFP-HA-CaMKII K42M细胞相比，GFP-HA-CaMKII细胞中胰岛素刺激的GLUT4转位显著更高。我们的结果表明CaMKII在葡萄糖转运中起允许作用。
