Nerve growth factor treatment or cAMP elevation reduces Ca2+/calmodulin-dependent protein kinase III activity in PC12...

Ca2+/calmodulin-dependent protein kinase III (Ca2+/CaM kinase III) phosphorylates a protein of Mr = 100,000 (the 100-kDa protein), a major substrate for Ca2+/CaM-dependent protein phosphorylation found in many mammalian tissues and cell lines (Nairn, A.C., Baghat, B., and Palfrey, H.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7939-7943). Treatment of PC12 cells with nerve growth factor (NGF) or forskolin resulted in a decrease in the depolarization-dependent phosphorylation of the 100-kDa protein in intact cells and in a decrease in the Ca2+/CaM-dependent phosphorylation of the 100-kDa protein in cytosolic extracts. In experiments using cytosolic extracts, the initial effect of NGF on the phosphorylation of the 100-kDa protein was observed in less than 1 h, was maximal (70% decrease) after 12 h, and began to recover after 24 h. The effect of forskolin was more rapid and the maximal effect was greater (90-95% decrease). Decreased Ca2+/CaM kinase III activity was also found in PC12 cells treated with epidermal growth factor, 2-chloroadenosine plus isobutylmethylxanthine, or dibutyryl cAMP. The effect of forskolin did not reverse unless it was removed. Cycloheximide blocked the recovery of Ca2+/CaM kinase III activity observed following the removal of forskolin but did not affect the ability of forskolin to reduce kinase activity. Short-term treatment with phorbol ester had little effect on Ca2+/CaM kinase III activity; long-term treatment with phorbol ester, which results in the disappearance of enzymatically detectable protein kinase C, had no effect on the ability of NGF or 2-chloroadenosine to reduce Ca2+/CaM kinase III activity. The level of the 100-kDa protein as determined by immunological techniques was not changed by any treatment. These results suggested that the effect of treatment of PC12 cells with NGF or forskolin was to reduce the level of Ca2+/CaM kinase III per se.