We have previously reported the identification of an NGF-inducible, Ras-dependent protein kinase that phosphorylates CREB at Ser-133 (24
), a phosphorylation event that is necessary for CREB-dependent transcriptional activation. Purification, sequencing, and biochemical characterization of this CREB kinase revealed that it is identical to a member of the pp90RSK family, RSK2 (76
). Identification of the NGF-inducible CREB kinase as RSK2 raised the question of the role of the other two members of the RSK family in mediating NGF-induced CREB Ser-133 phosphorylation. The three members of the RSK family are more than 80% identical in overall amino acid sequence and are even more highly conserved within their two catalytic kinase domains (1
). They also have similar substrate specificity and are known to translocate to the nucleus upon activation (6
), where they would have access to the potential substrate CREB. In this study, we show that all three members of the RSK family are activated in response to NGF and other growth factors and that they phosphorylate CREB Ser-133 both in vitro and in vivo. Our results indicate that RSK1, RSK2, and RSK3 function as a family of growth factor-inducible CREB Ser-133 kinases.
Several lines of evidence suggest that the RSK family members are not totally redundant in their in vivo function. First, the RSK family members display different patterns of tissue-specific gene expression (1
). The expression of RSK2 appears to be widespread, whereas the expression of RSK1 and RSK3 is restricted to specific tissues (52
). Second, mutations within the RSK2 gene have recently been found to be associated with Coffin-Lowry syndrome (CLS), an X-linked disorder characterized by mental retardation, facial and digital dysmorphisms, and skeletal deformations (69
). Since RSK1 and RSK3 are expressed at normal levels in CLS patients, the presence of RSK1 and RSK3 does not appear to fully compensate for the loss of RSK2 function in CLS patients. We show here that a physiological role of the RSK family members is the phosphorylation of CREB Ser-133 in response to growth factor stimulation. It is possible that the contribution of individual RSK family members to growth factor-induced CREB Ser-133 phosphorylation in vivo varies depending on the tissue type or developmental stage.
Several factors could have influenced our previous identification of RSK2, instead of RSK1 or RSK3, as an NGF-inducible CREB Ser-133 kinase in PC12 cells. First, available evidence suggests that RSK2 is expressed in PC12 cells at higher levels than RSK1 (data not shown). Consistent with the idea that individual members of the RSK family are expressed at different levels in a given cell type, we failed to detect RSK1 protein or kinase activity in NIH 3T3 cells, either before or after growth factor stimulation (data not shown). Second, we cannot exclude the possibility that our methods for preparation and fractionation of cell extracts favored the extraction and preservation of activities of certain RSK family members over others. It is worth noting that most of the HA-RSK3 expressed in COS cells could be extracted only under harsh denaturing conditions (reference 78
and data not shown). In addition, RSK3 immunoprecipitated from PC12 cells exhibited a higher basal activity (in the absence of NGF) than did RSK1 and RSK2, which accounts, at least partially, for the lower fold activation of RSK3 observed with NGF stimulation (see Fig. ).
Although the RSK family of Ser/Thr kinases is activated by NGF and other growth factors, and although RSK1, RSK2, and RSK3 phosphorylate CREB at Ser-133, inhibition of the ERK/RSK pathway did not completely block NGF induction of CREB Ser-133 phosphorylation. This finding suggested that there are other pathways in addition to the ERK/RSK pathway that mediate NGF induction of CREB Ser-133 phosphorylation. We have excluded the possible involvement of protein kinase A, CaMK, or pp70S6K in NGF-induced phosphorylation of CREB Ser-133 (24
; see above). However, our results indicate that another member of the MAPK superfamily, the p38 MAPK, is activated along with ERK MAPKs in NGF-treated PC12 cells. The p38 MAPK is known to be strongly activated by the proinflammatory cytokines tumor necrosis factor alpha and interleukin-1 and by environmental stresses such as UV irradiation, heat, and osmotic stress (32
). However, recent evidence suggests that certain growth factors activate p38 MAPK as well. For example, FGF activates the p38 MAPK pathway in SK-N-MC cells (66
), and p38 MAPK is also activated by hematopoietic growth factors including colony-stimulating factor type 1, granulocyte-macrophage colony-stimulating factor, and interleukin-3 in hematopoietic cells (20
). In addition to NGF, we found that EGF activates p38 MAPK in 3T3 cells. These results suggest the possibility that activation of the p38 MAPK signaling pathway is a general mechanism by which growth factors elicit cellular responses and that the p38 MAPK might play a general role in mediating growth factor-induced CREB Ser-133 phosphorylation as well as CREB-dependent transcription (41a
). We have found, in preliminary studies, that inhibition of p38 MAPK with SB 203580 partially blocks NGF-induced CREB-dependent IEG transcription.
It is likely that p38 MAPK activation leads to CREB Ser-133 phosphorylation through the activation of MAPKAP kinase 2 or the closely related kinase MAPKAP kinase 3. It has been previously shown that MAPKAP kinase 2 can phosphorylate CREB Ser-133 in vitro (66
). Taken together with our findings that NGF treatment of PC12 cells stimulates the ability of MAPKAP kinase 2 to phosphorylate CREBtide and that NGF activation of MAPKAP kinase 2 is blocked by the p38 MAPK inhibitor SB 203580, these results suggest that NGF activates a pathway in which the p38 MAPK phosphorylates and activates MAPKAP kinase 2, which then directly phosphorylates CREB Ser-133. Another recently identified kinase termed MNK1 (MAP kinase-interacting kinase 1) is activated by both ERKs and p38 MAPK (21
); however, the potential ability of this kinase to phosphorylate CREB Ser-133 has not yet been determined. It is possible that in addition to MAPKAP kinase 2, MNK1 or other unknown kinases activated by p38 MAPK also contribute to CREB phosphorylation in response to NGF.
The signaling pathway(s) which leads to p38 MAPK activation in response to growth factor stimulation is still not completely understood. An important mediator of NGF signaling is the guanine nucleotide-binding protein Ras (67
). NGF induction of CREB Ser-133 phosphorylation is completely blocked by a dominant interfering form of Ras (24
), and CREB Ser-133 phosphorylation is potentiated by the expression of a constitutively active form of Ras (19a
). These results imply that the signaling pathways that lead to CREB Ser-133 phosphorylation in NGF-stimulated PC12 cells function downstream of Ras activity. It has been well established that activation of the ERK-RSK pathway is dependent on Ras activity. We have found in preliminary experiments that NGF induction of p38 MAPK in PC12 cells may also be dependent on the activity of Ras, since expression of a dominant interfering form of Ras blocked NGF-induced p38 MAPK activation (data not shown). The suggestion that the two signaling pathways that mediate growth factor-induced CREB Ser-133 phosphorylation, the ERK/RSK pathway and the p38/MAPKAP kinase 2 pathway, may both be Ras dependent is consistent with the requirement of Ras activity for NGF-induced CREB Ser-133 phosphorylation.
The mechanism by which Ras might trigger p38 MAPK activation in response to growth factor stimulation is not clear. One possibility is that Ras regulates p38 MAPK by activating members of the Rho family of guanine nucleotide binding proteins (72
). Rho proteins, including Rho, Rac1, and Cdc42, regulate the activation of p38 MAPK (12
). The active, GTP-bound form of Rho family members binds and activates the Pak (p21-activated kinase) family of Ser/Thr kinases (46
). The activation of Pak by Rho family members may involve membrane localization of Pak, a process that has been suggested to be mediated by the SH2/SH3 domain-containing adapter protein Nck (45
). Paks have been proposed to be upstream activators of the MAPK kinase kinases (MKKKs) or MEK kinases (MEKKs) (72
), which in turn phosphorylate and activate the MAPK kinases. Several MAPK kinases, including MKK3, MKK4, and MKK6, directly phosphorylate and activate p38 MAPK (16
). Although MKK4 also activates JNK, MKK3 and MKK6 appear to activate p38 MAPK exclusively.
Our finding that both the ERK pathway and the p38 pathway contribute to NGF-induced CREB Ser-133 phosphorylation raises the question of why there are two separate MAPK pathways that lead to CREB phosphorylation. One possibility is that both the ERK and p38 pathways are required to produce the maximal amount of CREB Ser-133 phosphorylation in response to NGF. Consistent with this idea, preincubation of PC12 cells with either PD 098059 to inhibit the ERK pathway or SB 203580 to inhibit the p38 MAPK pathway reduced the level of CREB Ser-133 phosphorylation in NGF-stimulated PC12 cells. Another possibility is that differences in the kinetics of ERK and p38 MAP kinase activation in NGF-treated PC12 cells allow for temporal changes in the extent of CREB phosphorylation. Support for this possibility comes from the finding that NGF activates the ERK/RSK pathway with prolonged kinetics while activation of the p38 MAPK pathway is transient. Thus, at early time points after NGF treatment, both ERK and p38 MAPK pathways contribute to CREB Ser-133 phosphorylation, but at later time points, the ERK/RSK pathway is likely to be solely responsible for CREB phosphorylation. Consistent with this idea, blocking the ERK/RSK pathway with PD 098059 changes the kinetics of NGF-induced CREB Ser-133 phosphorylation from prolonged to transient (data not shown). Therefore, at early times after growth factor stimulation, activation of both the ERK and p38 MAPK pathways may be necessary for maximal CREB Ser-133 phosphorylation and the full induction of IEGs that have CREs within their promoters. However, at later times after NGF addition, the ERK pathway alone may be sufficient to maintain CREB Ser-133 phosphorylation at a somewhat lower level that may be sufficient for the transcription of delayed response genes that contain CREB binding sites.
To conclude, CREB Ser-133 phosphorylation is a critical event for transcriptional activation that is induced by a variety of growth factors and neurotrophins. Despite the critical importance for CREB Ser-133 phosphorylation in growth factor signaling, the mechanisms by which CREB phosphorylation is induced by growth factors were not clear. Previous reports suggested the involvement of the ERK/RSK2, p38/MAPKAP kinase 2, and pp70S6K
pathways in mediating CREB Ser-133 phosphorylation in NGF-treated PC12 cells, bFGF-treated SK-N-MC cells, and serum-treated NIH 3T3 cells, respectively (14
). In this study, we have shown that both the ERK/RSK and the p38/MAPKAP kinase 2 pathways contribute to CREB Ser-133 phosphorylation in NGF-treated PC12 cells and that inhibition of both of these pathways is necessary to completely abolish NGF-induced CREB Ser-133 phosphorylation in these cells. In addition to their role in NGF-treated PC12 cells, the ERK and p38 MAPK pathways may function to mediate CREB Ser-133 phosphorylation in neurons and in nonneuronal cell lines. Our preliminary results show that p38 MAPK is activated in NGF-treated superior cervical ganglion neurons. In addition, p38 MAPK is activated by growth factors such as EGF and FGF in PC12 cells and NIH 3T3 cells. Thus, the ERK/RSK and p38/MAPKAP kinase 2 pathways are likely to play general roles in mediating CREB Ser-133 phosphorylation and IEG induction in response to a variety of neurotrophins and growth factors in a wide range of cell types.