Detection of nerve growth factor and epidermal growth factor-regulated protein kinases in PC12 cells with synthetic...

Synthetic peptide substrates specific for cAMP-dependent protein kinase, protein kinase C, ribosomal S6 kinase, and Ca2+/calmodulin-dependent protein kinases were used to monitor regulation of these protein kinases in digitonin-permeabilized PC12 cells following treatment with nerve growth factor (NGF) and epidermal growth factor (EGF). cAMP-dependent protein kinase was not activated by NGF and EGF. In addition, neither the Ca2+/calmodulin-dependent nor -independent activity of a protein kinase similar to Ca2+/calmodulin kinase II was affected by growth factor treatment. However, protein kinase C was rapidly and transiently activated and ribosomal S6 kinase activity was persistently elevated. Maximal protein kinase C activity was observed after 2 to 5 min of treatment and, subsequently, returned to control levels within 30 to 40 min. In contrast, S6 kinase activity was maximal within 15 min of NGF and EGF addition and was stably maintained for at least 24 hr. In addition to protein kinase C and S6 kinase, NGF and EGF regulated a protein kinase that was maximally elevated after 15 to 30 min and returned to control levels within 3 to 5 hr. This kinase (approximately 100 kDa) failed to bind to a calmodulin affinity column and eluted from a cation exchange column as a single major species that was distinct from S6 kinase activity, which eluted as multiple peaks. The findings indicate that at least three protein kinases are rapidly activated in PC12 cells following treatment with NGF and EGF. The distinct durations of activation of each kinase implicates significantly different roles for each in growth factor signalling in PC12 cells.