Ca(2+)/calmodulin-dependent protein kinase II inhibition by heparin in mesangial cells.
Heparin exerts an antiproliferative effect in smooth muscle cells, and the Ca(2+)/calmodulin-dependent protein kinase (CaMK) signaling pathway is heparin sensitive. Here, we report that transfection with a truncated 326-amino acid fragment of CaMK-IIalpha increases basal activity of CaMK-II in mesangial cells. Ionomycin increased CaMK-II activity in both transfected and untransfected cells, with a concomitant increase in activated Ca(2+)/calmodulin. Heparin (1 microg/ml), but not chondroitin or dermatan sulfate, significantly attenuated both serum- or ionomycin-induced CaMK-II activity, and attendant c-fos mRNA expression, but did not affect upstream Ca(2+)/calmodulin. Autophosphorylation of Thr286 generates an autonomously active CaMK-II. Both serum and ionomycin increased phosphorylation at this site and increased CaMK-II activity in antiphosphothreonine immunoprecipitates. Heparin (1 microg/ml) did not inhibit phosphorylation of Thr286 (although much higher concentrations did). Replacement of Thr286 with Asp produces a constitutively active mutant that was insensitive to ionomycin but was inhibited by heparin maximally at 1 microg/ml. These results suggest that heparin at physiological concentrations acts at or downstream of CaMK-II to suppress its activity independent of an effect on autophosphorylation.
Xiao
Weiqun
W
Department of Laboratory Medicine and Pathobiology, Medical Sciences Bldg. Rm. 6302, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8.
Liu
Ying
Y
Templeton
Douglas M
DM
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D002227:Carbazoles / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000494:pharmacology; D003871:Dermatan Sulfate / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D006493:Heparin / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D014162:Transfection
D002227:Carbazoles; D007211:Indoles; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; C063769:KT 5926; D003871:Dermatan Sulfate; D015759:Ionomycin; D006493:Heparin; D002809:Chondroitin Sulfates; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases