Nuclear translocation of p90Rsk and phosphorylation of CREB is induced by ionomycin in a Ras-independent manner in PC12 cells.

In the present study we examined the possible role of p90Rsk in pathways leading to neuronal differentiation of PC12 cells induced by nerve growth factor (NGF) and the calcium ionophore ionomycin. PC12-M17 cells, expressing a dominant inhibitory Ras protein, do not undergo neuronal differentiation in response to NGF like wild-type PC12 cells, but exhibit neurite outgrowth when treated with NGF in combination with ionomycin. However, the blockade of Ras in these cells results in failure of activation of mitogen-activated protein kinase (MAPK)/extracellular signal regulation kinase (ERK) (MEK) and ERK activation as well, therefore kinases other than those of the ERK pathway might play a role in the induction of neuronal differentiation in this case. Here we show that p90Rsk translocates to the nucleus in response to ionomycin in both wild-type PC12 and PC12-M17 cells, and this spatial distribution is followed by increased phosphorylation of the cAMP response element binding protein (CREB). Since CREB is believed to be the transcription factor that can integrate Ca2+, growth factor and cAMP-induced signals, we suggest that p90Rsk may be one of the kinases which is able to replace ERKs under certain circumstances, thereby participating in Ras-independent neuronal differentiation induced by NGF plus ionomycin.

Nerve growth factor in combination with second messenger analogues causes neuronal differentiation of PC12 cells expressing a dominant inhibitory Ras protein without inducing activation of extracellular signal-regulated kinases.

In the present work, nerve growth factor (NGF) was used in combination with the calcium ionophore, ionomycin or dibutyryl cyclic AMP (dbcAMP), to study the connection between neuronal differentiation and extracellular signal-regulated kinase (ERK) activation of PC12 rat pheochromocytoma cells expressing a dominant negative, Ha-Ras Asn17 protein. Due to the block of endogenous Ras activity, neurite outgrowth in response to NGF is completely inhibited in these cells. However, this blockade can be bypassed by combined treatment with NGF plus ionomycin or NGF plus dbcAMP. The mitogen-activated protein kinase (MAPK) /ERK kinase inhibitor, PD98059, proved to be insufficient in inhibiting the neurite outgrowth under these conditions. Moreover, although both long-term ERK activation and nuclear translocation of ERKs are believed to be key events in neuronal differentiation, neither detectable ERK phosphorylation, nor nuclear translocation of these enzymes, occurred upon combination treatments in our experimental system. However, the neuritogenesis induced by either the combination of NGF/ionomycin or NGF/dbcAMP was inhibited by the Trk inhibitor, K252a. Ras-independent pathways, originating from the NGF receptor, can thus synergize with second messenger analogues bypassing the ERK cascade but leading to the same biological result--neurite formation.

Intact Ras function is required for sustained activation and nuclear translocation of extracellular signal-regulated kinases in nerve growth factor-stimulated PC12 cells.

PC12 pheochromocytoma cell lines expressing the dominant negative Ha-Ras Asn-17 protein at different levels were used in this study to analyze the relationship between nerve growth factor (NGF)-induced activation of members of the mitogen-activated protein kinase (MAPK) family, and neuritogenesis. In wild-type PC12 cells, NGF rapidly stimulated the extracellular signal-regulated kinases (ERKs). Kinase activation was sustained and was followed by the translocation of ERK 1 and ERK 2 into the nucleus ultimately leading to neurite outgrowth. In cells expressing relatively high levels of the inhibitory Ras protein, NGF stimulation of ERK 1 and ERK 2 as well as nuclear translocation of these protein kinases were greatly inhibited. In contrast, in PC12 subclones expressing low amounts of Ha-Ras Asn-17 the peak of ERK activation was only slightly reduced, but became transient in nature and was not followed by nuclear translocation of ERKs 1 and 2. Since all PC12 subclones expressing detectable levels of the dominant inhibitory Ras protein are resistant to NGF induction of neurite formation, our observations support the notion that sustained activation and translocation of ERKs into the nucleus are essential for NGF-induced neuronal differentiation of PC12 cells.