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Growth factor-stimulated expression and activation of c-Fos is regulated by the ERK1/2 pathway. However, recent reports have also suggested a prominent role for the closely related ERK5 pathway in regulating the expression, transcriptional activation and nuclear localization of c-Fos. Here we have compared the role of ERK1/2 and ERK5 in regulating c-Fos using a combination of conditional protein kinases, selective biochemical inhibitors and ERK5 null fibroblasts. We demonstrate that activation of the ERK1/2 pathway, but not ERK5, is sufficient for c-Fos phosphorylation and transcriptional activation. Furthermore, growth factor-dependent expression of c-Fos is blocked by low doses of PD184352 that selectively inhibit the ERK1/2 pathway but proceeds normally in ERK5-/- 3T9 cells; in addition, nuclear localization of c-Fos is normal in ERK5-/- cells. ERK5-/- cells are, however, defective for c-Jun expression but this is reversed by re-expression of ERK5. In addition to ERK5, neither the JNK nor p38 pathways can substitute for ERK1/2 in the regulation of c-Fos transcriptional activity. These results demonstrate that c-Fos transcriptional activity is not regulated by the ERK5 pathway; rather, of all the MAPKs and SAPKs, c-Fos activation appears to be predominantly linked to the ERK1/2 pathway.

The duration of ERK1/2 activation influences the nature of the biological response to agonist. Members of the AP-1 transcription factor family are well known targets of the ERK1/2 pathway and are expressed in a temporally coordinated fashion during cell cycle re-entry. In CCl39 fibroblasts, sustained ERK1/2 activation is required for the expression of Fra-1, Fra-2, c-Jun and JunB, whereas expression of c-Fos is still strongly induced even in response to transient ERK activation. However, the significance of this pattern of expression for AP-1 activity has not been addressed. Here we show that growth factor stimulated activation of the C-terminal c-Fos transactivation domain (TAD) serves as a sensor for ERK1/2 signal duration whereas the c-JunTAD is not responsive to growth factors. In addition, sustained ERK1/2 activation determines the duration of increases in AP-1 DNA binding complexes as well as their qualitative make up. Finally, this is reflected in both the duration and quantitative transcriptional output of stably integrated AP-1 reporter constructs, indicating that AP-1 activity is finely tuned to ERK1/2 signal duration. These results provide new insights into the importance of ERK1/2 signal duration in the regulation of AP-1 and provide an explanation for how differences in signal duration can lead to both quantitative and qualitative changes in gene expression.