In this study, we show that NGF activation of B-Raf and ERK signaling in both DRG cultures and PC12 cells is blocked by inhibition of PI3-K. Multiple methods of inhibiting PI3-K, using both pharmacological and molecular agents, all had the same effect. Furthermore, this requirement of PI3-K was specific for NGF; the ability of cAMP to activate ERKs in both DRGs and PC12 cells did not require PI3-K.
We have previously demonstrated that both Ras and Rap1 pathways contribute to the activation of ERKs by NGF. Indeed, the co-ordinated signaling via these two pathways accounts for the ability of NGF to induce both sustained activation of ERKs and neuronal differentiation (106
). Interestingly, in this study, we demonstrate that the requirement for PI3-K in ERK activation by NGF may reflect distinct actions on signaling via Ras and Rap1. Thus, PI3-K inhibition blocked activation of Rap1 but not Ras. However, PI3-K did block the ability of activated Ras to couple to B-Raf and ERKs. Both actions of PI3-K prevent NGF from utilizing either Ras- or Rap1-dependent signals to activate ERKs.
Our data demonstrating the requirement for PI3-K in the activation of Rap1 may reflect a role in the endocytosis of activated TrkA receptors. We found that inhibition of PI3-K activity blocked TrkA internalization after NGF stimulation. Other blockers of endocytosis had the same effect as PI3-K inhibitors, preventing activation of Rap1 but not Ras. Taken together, we propose a model in which TrkA activation can stimulate Ras at the plasma membrane but requires PI3-K-dependent internalization to activate Rap1 (Fig. ).
FIG. 10 Model of NGF signaling to ERKs. In PC12 cells, NGF activates Ras and Rap1 to mediate the rapid and sustained activation of ERKs, respectively. Both TrkA internalization and Rap1 activation require PI3-K. Clathrin-mediated endocytosis is also required (more ...)
A role for endocytosis in NGF signaling is consistent with the emerging view that internalization of active TrkA receptors into signaling vesicles is required for the downstream actions of neurotrophins (109
). For example, the ability of both NGF and TrkA to be transported in a retrograde fashion from the nerve terminal to the cell body has been demonstrated by many groups (5
). Recent studies demonstrate that this retrograde transport process delivers active TrkA to the cell body which may be required for the stimulation of gene expression by NGF (5
). These conclusions are supported by studies in which signaling-competent vesicles containing active NGF-TrkA complexes were isolated from PC12 cells following clathrin-mediated endocytosis of TrkA (28
). Furthermore, these data are consistent with those of studies examining other growth factor receptors, as well as G-protein-coupled receptors, where clathrin-mediated endocytosis has been implicated in ERK activation (10
). Therefore, receptor trafficking may serve an important signaling function in addition to simply mediating receptor down-regulation via lysosomal degradation. It is possible that the sorting determinants and mechanisms for targeting proteins to signaling vesicles versus lysosomes may be differentially regulated for different ligand-receptor complexes (46
). Whether such sorting events impart specificity in the ability of growth factors to activate Rap1-dependent pathways remains to be determined.
A proposed role for endocytosis is to bring activated receptors to the location of downstream signaling molecules (9
). Accordingly, the localization of Ras and Rap1 to distinct membrane compartments may account for the differential roles of PI3-K and endocytosis in Ras and Rap1 activation. Our data showing that Rap1 resides with vesicular membranes are consistent with the localization of Rap1 to endosomal compartments. In contrast, Ras is at the plasma membrane itself and can be activated by TrkA without additional endocytic events. Membrane targeting of Ras-like molecules is determined by postranslational modifications of their C termini. Differences in the C-terminal motifs found in Rap1 and Ras may account for their different membrane distribution (11
). Importantly, these differences in membrane distribution of Ras family members influence downstream signaling actions (69
). Our data suggest that their location may also determine how these small G proteins become activated.
PI3-K-dependent TrkA internalization may control differential activation of Ras versus Rap1 signaling to dictate the specificity of NGF action. Our lab has previously shown that in PC12 cells Rap1 is activated by NGF (106
), but not EGF (98
). Furthermore, we have shown that Rap1-dependent signals contribute to growth factor specificity by mediating both the sustained phase of ERK activation by NGF and aspects of neuronal differentiation (106
). Differences in the kinetics of receptor internalization have also been proposed to impart specificity to NGF versus EGF signaling in PC12 cells (32
). In this study, Huang et al. have shown that TrkA remains associated with caveola-like membranes following NGF treatment, whereas EGF treatment results in the rapid depletion of the EGF receptor from this membrane population. Consistent with these results, we have observed a more rapid internalization of the EGF receptor compared to TrkA following ligand stimulation (data not shown).
The slower internalization of TrkA following NGF treatment may allow for the assembly of signaling complexes which subsequently lead to Rap1 activation within the mature signaling endosome. It has been shown that the phosphorylation of the adapter molecule FRS2 recruits additional adapter molecules that contribute to the sustained ERK activation seen following treatment with NGF (43
). One of these adapter molecules which binds phosphorylated FRS2 is Crk (55
). Crk has been shown to contribute to Rap1 activation via its association with C3G, the Rap1-specific exchanger (33
). Accelerating TrkA internalization by incubating PC12 cells with an NGF-antibody complex results in a transient activation of ERKs similar to that seen with EGF treatment (75
). Interestingly, acceleration of TrkA internalization resulted in a TrkA signaling complex that lacked critical phosphorylations, including phosphorylation of FRS2, that are required for sustained activation of ERKs. Here, we propose that the contributions of Rap1 signaling and endocytosis to sustained ERK activation are intimately connected; Rap1 participates in the late phase of ERK activation by NGF because Rap1 activation by NGF requires endocytic events.
One finding of this study was that the ability of Ras to couple to its downstream effector, B-Raf, was also blocked by inhibitors of PI3-K. This action explains the contribution of PI3-K to the early (Ras-dependent) activation of ERKs. This inhibition of Ras-dependent signaling downstream of Ras activation may also reflect a requirement of PI3-K for endocytic events. B-Raf is localized to vesicles within DRG neurons and PC12 cells, and this localization did not appear to change following NGF treatment (data not shown). Interestingly, Grimes and coworkers (29
) have shown that NGF treatment leads to the redistribution of TrkA immunostaining to a pattern similar to that seen for B-Raf. Therefore, it is possible that endocytosis and subsequent vesicular fusion are required to bring Ras in contact with B-Raf, consistent with the ability of MDC to disrupt Ras–B-Raf complexes. This model is consistent with recent evidence suggesting that Ras is internalized into endocytic vesicles following growth factor stimulation (72
). The requirement of PI3-K in Ras-dependent activation of B-Raf may also depend on a second action of PI3-K. For example, recent studies suggest that phosphorylation of Raf-1 by kinases downstream of PI3-K may stimulate Raf kinase activity (74
). In contrast, the direct phosphorylation of Raf-1 by the PI3-K target, Akt, has been shown to be inhibitory in some cells (79
). However, this ability of Akt to inhibit Raf-1 appears to depend on the cell type and cellular context (73
). A similar study examining B-Raf has also been reported (30
), although no studies have been performed with neuronal cells. In any case, the data presented here demonstrate that PI3-K has indirect actions that enhance signaling through B-Raf.
The major significance of this study is that we show PI3-K is required for NGF activation of ERKs through its specific action in regulating TrkA internalization. Both PI3-K and ERKs are well-studied targets downstream of NGF. A prevailing view is that they represent the end points of distinct linear pathways and trigger distinct physiological actions. However, recent reports suggest that these pathways may be interconnected (34
). Here, we provide a biochemical basis for a model that these two pathways share overlapping functions within neurons and suggest that the regulation of endocytosis by PI3-K may provide an important mechanism to modulate ERK signaling cascades.