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.
Endocrinology
; 148(1): 374-85, 2007 Jan.
Article
em En
| MEDLINE
| ID: mdl-17008397
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas Quinases Dependentes de Cálcio-Calmodulina
/
Inibidores de Proteínas Quinases
/
Glucose
/
Hipoglicemiantes
/
Insulina
/
Cinetina
Limite:
Animals
Idioma:
En
Ano de publicação:
2007
Tipo de documento:
Article