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.
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.
Konstantopoulos
Nicky
N
CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville 3052, Victoria, Australia.
Marcuccio
Seb
S
Kyi
Stella
S
Stoichevska
Violet
V
Castelli
Laura A
LA
Ward
Colin W
CW
Macaulay
S Lance
SL
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000493:pharmacokinetics*; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007701:Kinetin / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics; D000077546:Roscovitine; D014162:Transfection
C516978:CK59 compound; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; D049452:Green Fluorescent Proteins; C090046:olomoucine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D005947:Glucose; D007701:Kinetin