The findings presented in this study show how the strength and overall duration of ERK signaling control the global expression of several DEF domain-containing IEG products and how this relates to the expression of second-tier genes and cell cycle progression. First, we show that the putative DEF domains in Fra-1, Fra-2, and c-Myc are indeed functional. Like c-Fos, the DEF domains found in these transcription factors regulate their hyperphosphorylation and are predicted to control their biological function. Second, all DEF domain-containing IEG products examined display the same differential response to the duration of ERK signals, and prolonged ERK signaling is required for the sustained expression of these IEG products through the G1 phase of the cell cycle. Third, a small reduction in ERK signal strength results in significant loss of hyperphosphorylation and stability of these IEG products. Fourth, expression of the AP-1 target gene, cyclin D1, is significantly reduced when ERK signal strength and duration are decreased. Fifth, the IEG expression response to signal duration is tightly correlated with quantitative differences in cell cycle progression. Taken together, these observations argue that the posttranslational regulation of IEG products provides the cell with a mechanism to respond to subtle changes in ERK signal strength and duration, which in turn leads to significant changes in cellular function.
Understanding how sustained ERK and RSK signaling regulates c-Fos has provided a molecular framework to explain how differences in signal duration lead to the generation of a specific biological outcome (26
). During sustained signaling, ERK- and RSK-mediated phosphorylation of the c-Fos COOH terminus primes hyperphosphorylation at Thr325 and Thr331 (26
). Hyperphosphorylation also requires an intact DEF domain, and when mutated, Fos-mediated transformation is inhibited (26
). In the present study, we have specifically shown that Fra-2, Fra-1, and c-Myc also have functional DEF domains that are required for their hyperphosphorylation. Experimental and sequence analyses show that posttranslational control of Fra-2 and Fra-1 is likely very similar to that of c-Fos due to the presence of priming phosphorylation sites and DEF domain-mediated phosphorylation sites in their COOH termini. Interestingly, the transactivation potential of Fra-1 is completely inhibited when Thr231 is mutated to alanine (43
), and our findings indicate that this residue is under the regulation of the DEF domain. In the case of c-Myc, the DEF domain regulates the ERK-dependent phosphorylation of Ser62. Since phosphorylation of Ser62 promotes c-Myc stability, DEF-dependent docking of ERK to c-Myc would lead to c-Myc stabilization. Interestingly, the DEF domain and NH2
-terminal phosphorylation sites Ser62 and Thr58 both flank a region known as Myc box II (MbII). MbII is required for the assembly of a high-molecular-weight complex that contains the transformation-transactivation domain-associated protein (TRRAP) and TIP49/TIP48 (23
). This complex is required for Myc-dependent transformation and may be involved in chromatin modification of Myc target genes (10
). Apart from directly phosphorylating Ser62, ERK docking to c-Myc may also have a role in regulating the recruitment and/or activity of the TRRAP complex.
Several other IEG products have expression patterns that are characteristic of the Fos and Myc family sensors. For example, differences in signal duration can regulate c-Jun expression just like c-Fos. Based on sequence analysis, however, it is predicted that c-Jun does not contain a functional DEF domain (26
). Nevertheless, it is known that the ERK pathway can control the phosphorylation of Ser63 and Ser73 during sustained signaling in PC12 cells (19
). How can c-Jun expression be regulated by sustained ERK signaling in the absence of a DEF domain? One possibility is that transphosphorylation of Ser63 and Ser73 by Fos-associated ERK would result in increased c-Jun stabilization and transcriptional activation. In support of this, JNK-mediated transphosphorylation of c-Jun dimerization partners has been demonstrated (16
). Another possibility is that the stabilization of Fos family proteins indirectly controls the stability of Jun proteins, because Jun/Fos dimers are more stable than Jun/Jun dimers (1
The phosphorylation of Thr102 and Thr104 in JunB is thought to be predominantly regulated by JNK and not p38 or ERK (20
). Interestingly, these amino acids, which are required for JunB-driven interleukin-4 expression in Th2 lymphocytes (20
), are located NH2
terminal to the putative DEF domain in JunB (26
). It is possible that in different cell types and in the presence of an appropriate partner protein, the DEF domain facilitates the ERK-dependent phosphorylation of JunB, and future work is required to investigate this possibility. Finally, ERK-mediated differentiation of PC12 cells requires Egr-1 transcription activity to increase the expression of p35
, an Egr-1 target gene (14
). While the role of the Egr-1 DEF domain is presently unknown in this process, the possibility exists that ERK docking controls the stability and/or transcriptional activity of Egr-1. Interestingly, several putative ERK phosphorylation sites (Ser-Pro) are located NH2
terminal to this DEF domain (26
), and Egr-1 itself is phosphorylated in vivo (6
The observations in this study illuminate a global mechanism that is responsible for dictating cellular responses to ERK signal duration. This mechanism takes into account the effect of both signal kinetics and signal strength. We believe that the presence of DEF domains in several growth factor-induced sensors enables the cell to respond to small increases or decreases in signal strength. This concept is best exemplified by the response of Fra-1 to sustained ERK signals. In Swiss 3T3 fibroblasts, although ERK activity at the G1-S boundary is only 10 to 20% of that measured after 10 min of initial stimulation, it is nevertheless sufficient to promote hyperphosphorylation of Fra-1. This and other evidence strongly suggests that DEF domains locally concentrate the active ERK to Fos, Jun, Myc, and Egr-1 sensors during sustained signaling, and this results in efficient phosphorylation of target phosphoacceptor sites.
The transcriptional induction of fra-1
can be directly regulated by c-Fos (4
) and as a result is compromised in c-fos−/−
osteoclasts and fibroblasts (22
). This suggests that in addition to the role of ERK signaling directly on Fra-1 protein, the effect of signal duration on the c-Fos sensor can also determine the transcriptional induction of fra-1
. Although Fra-1 expression occurs only when ERK signaling is sustained (i.e., cells treated with PDGF but not EGF), c-Fos is still transiently expressed and is hypophosphorylated in EGF-treated cells but is not sufficient to induce Fra-1. This implies that growth factor-induced fra-1
expression requires prior ERK-mediated phosphorylation and/or stabilization of c-Fos. This important observation discriminates between an absolute requirement of c-fos
for the induction of fra-1
expression, as indicated by the behavior of c-fos−/−
cells, and an essential posttranslational role for ERK signaling in this process. Further support for the latter mechanism comes from experiments in which signal strength was reduced. With low doses of PDGF, although the expression of c-Fos was relatively transient, it was nonetheless phosphorylated to a greater extent than that normally associated with EGF treatment, and critically, this preceded Fra-1 expression in Swiss 3T3 cells. We believe that after the initial c-Fos-dependent induction of fra-1
), Fra-1 protein is hyperphosphorylated by ERK, and this primarily contributes to sustained Fra-1 expression in G1
. Thus, the c-Fos-Fra-1 pathway is an example of how two sensors that have overlapping expression profiles provide the cell with temporal information about the amplitude of ERK signaling.
The ability of a single cell to respond to minute changes in the concentration of extracellular stimuli underlies processes that control embryonic development, cell morphology, migration, differentiation, and proliferation. In fibroblasts, a two- to threefold change in PDGF concentration can result in a modest reduction in ERK signal strength. This small change in ERK signaling, however, leads to large differences in the expression kinetics and phosphorylation of IEG-encoded sensors and cell cycle progression. This response to agonist concentration resembles the behavior of Drosophila
embryos to morphogens. In these systems, as little as a two- to fourfold change in morphogen concentration can result in quantitative differences in gene expression and cell fate outcome (13
). Many morphogens, such as Screw and Activin, control rapid activation of “early” target genes independent of ongoing protein synthesis (13
), which resembles the IEG response to extracellular stimuli. Morphogens are also associated with the delayed expression of “late” target genes, and it has been proposed that the combination of intracellular signaling and the presence of early proteins can regulate late gene expression (28
). During embryonic development, an IEG sensor-like mechanism could allow individual cells to interpret their position in a morphogen gradient and therefore would be required to generate a specific cell fate.
In conclusion, the uniform response shown by DEF domain-containing IEG products to differences in ERK signal duration and strength demonstrates that these gene products can collectively act as sensors for ERK signaling. Since IEG products are induced by a diverse range of extracellular stimuli, these proteins may have the general capacity to interpret differences in the kinetics of signaling pathways other than ERK. Therefore, this model in part may help explain how complex intracellular signals can be decoded and ultimately transduced into a phenotypic change in cell behavior.