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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

CSIRO Molecular and Health Technologies (N.K., V.S., L.A.C., C.W.W., S.L.M.), Parkville, Australia 3052; and CSIRO Molecular and Health Technologies (S.M., S.K.), Clayton South, Australia 3169

Address all correspondence and requests for reprints to: Dr. S. Lance Macaulay, CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville 3052, Victoria, Australia. E-mail: lance.macaulay{at}csiro.au.

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 µM. To identify proteins binding to the analog, [³⁵S]-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 - and ß-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 (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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