Ca²⁺/calmodulin-dependent protein kinase II inhibition by heparin in mesangial cells

Abstract

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.