Stimulation of the ERK Pathway by GTP-loaded Rap1 Requires the Concomitant Activation of Ras, Protein Kinase C, and Protein Kinase A in Neuronal Cells

Abstract

The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.Since its original establishment by Greene and Tischler (1), the PC12 pheochromocytoma cell line is a widely used model of neuronal differentiation. The first isolated neurotrophin, nerve growth factor (NGF) 1 (2), was shown to induce a neuronal like phenotype characterized by neurite outgrowth (1). The effect of NGF is dependent on a long lasting activation of the Ras-Raf-MEK-ERK pathway (3). Activation of the cAMP pathway was also reported to induce PC12 differentiation (4,5). In line with the demonstrated role of ERK activation in NGF-induced PC12 differentiation, cAMP analogues, and forskolin, a direct activator of adenylate cyclase (AC) was shown to stimulate ERK activity in this cell line (5, 6). Similarly, mobilization of calcium and/or stimulation of the diacylglycerol (DAG) production results in ERK activation and eventually neurite outgrowth (7, 8).The mechanisms responsible for the control of the ERK pathway by receptor tyrosine kinases, cAMP analogues, and calcium/DAG were intensively investigated, and Ras-like small GTP-binding proteins emerged as key elements in this pathway. The products of the H-Ras gene was shown to stimulate the activity of the MEK kinase Raf-1 following activation of receptor tyrosine kinases (9). Cyclic AMP and calcium were also suggested to control the activity of Ras in some cell types (7, 10), resulting in ERK activation. More recently, the Ras superfamily member Rap1 and the protein kinase B-Raf were suggested to link PKA activation by cAMP analogues to MEK1 stimulation in neuronal cells (11). On the other hand, several mechanisms have been proposed for calcium-induced ERK activation, including ac...

Epac Activation Converts cAMP from a Proliferative into a Differentiation Signal in PC12 Cells

“…Furthermore, our findings suggested a role of Rap1 in the sustained phase of cAMP-induced ERK1/2 activation. This is in agreement with recent studies, which have shown that activation of Rap1 is required for the sustained phase of NGF-and PACAP-induced ERK1/2 activation (York et al, 1998;Bouschet et al, 2003). Thus, Rap1 might be essential for the sustained phase of ERK1/2 activation in response to a variety of different stimuli.…”

“…The band migrating above ERK1 is probably identical to ERK1b, a splice variant of ERK1 (Yung et al, 2000(Yung et al, ). al., 2001Enserink et al, 2002;Piiper et al, 2002;Norum et al, 2003;Bouschet et al, 2003).…”

“…To investigate if the EGFR plays a similar role in ERK1/2 activation in response to a physiological stimulus that activates ERK1/2 through stimulation of adenylyl cyclase and subsequent elevation of [cAMP] i , we studied the effects of AG1478 and H-89 on ERK1/2 activation in response to the pituitary neuropeptide hormone PACAP, which stimulates ERK1/2 by PKA-and phospholipase C-dependent pathway (Bouschet et al, 2003). As shown in Figure 6D, inhibition of PKA by H-89 reduced PACAP-induced ERK1/2 phosphorylation.…”

“…AG1478 completely blocked EGF-induced ERK1/2 activation ( Figure 6C), but had no effect on NGF-induced ERK1/2 activation ( Figure 6B), confirming the specificity of this substance for the EGFR tyrosine kinase. These data support the notion that the initial phase of cAMP-induced ERK1/2 activation is strongly EGFR-dependent, whereas the sustained phase is less EGFRdependent.To investigate if the EGFR plays a similar role in ERK1/2 activation in response to a physiological stimulus that activates ERK1/2 through stimulation of adenylyl cyclase and subsequent elevation of [cAMP] i , we studied the effects of AG1478 and H-89 on ERK1/2 activation in response to the pituitary neuropeptide hormone PACAP, which stimulates ERK1/2 by PKA-and phospholipase C-dependent pathway (Bouschet et al, 2003). As shown in Figure 6D, inhibition of PKA by H-89 reduced PACAP-induced ERK1/2 phosphorylation.…”

P2Y 2 receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation

“…These results identify two independent activities of the P2Y 2 receptor for enhancing neurotrophin-mediated differentiation: (i) increase in level of activated neurotrophin receptor and (ii) increased activation of ERK1͞2. In undifferentiated PC12 cells, P2Y 2 receptors increase intracellular calcium levels (56), which may activate PKC, Ras, and Raf, thereby activating ERK1͞2 (57). Activation of Raf by P2Y 2 receptors and TrkA could represent a convergence point for the two signaling pathways.…”