Stimulation of the Ras pathway results in the activation of ERK and RSK, but the precise mechanisms involved in RSK activation and inactivation remain elusive. In this study, we characterized the ERK binding domain of RSK1 and found that a serine residue located near the docking site (position 749 in avian RSK1) was involved in the regulation of ERK1/2 binding (Fig. to ). We also found that Ser749 was efficiently phosphorylated by the RSK1 N-terminal kinase domain in vitro (Fig. ) and that mutations in the RSK1 kinase domains disrupted ERK dissociation (Fig. ). Analysis of different RSK isoforms revealed that RSK1 and RSK2 activation induced ERK1/2 release but that mitogen stimulation did not affect the interaction of ERK1/2 with RSK3 (Fig. ). We also found that RSK3 remains activated longer than RSK1 and RSK2 (Fig. ), indicating that ERK association promotes RSK activation or antagonizes RSK inactivation.
Our results indicate that the minimal region in RSK1 necessary for ERK1/2 docking consists of residues 739L
, where the boldface amino acids are essential for ERK1/2 interaction. Mutation of residues Lys745 and Lys746 did not affect RSK1 activation but reduced the overall strength of interaction between RSK1 and ERK1/2. It is possible that ERK1/2 docking to the RSK1 K745A and K746A mutants is not altered in vivo but that the interaction is rapidly disrupted under normal cell lysis conditions. Since an arginine or lysine is always present at both positions in all RSK isoforms, it is possible that these residues play roles in the binding of specific ERK isoforms. We have found that RSK1, RSK2, and RSK3 interact with ERK1/2 to similar levels in serum-starved cells but that only RSK1 and RSK2 dissociate from ERK1/2 following mitogen stimulation of HEK293 and NIH 3T3 cells (data not shown). These results are in disagreement with a report by Zhao et al. (34
), who demonstrated that complexes between endogenous ERK1/2 and ectopically expressed RSK1, RSK2, and RSK3 were not affected by activation of ERK1/2 in COS7 cells. Moreover, they found that ERK1/2 associates preferentially with RSK3 and less with RSK2 but could not bind to RSK1 (34
). Our results indicate that each RSK isoform interacts similarly with ERK1/2 in cells that were serum starved prior to stimulation. It is possible, however, that ERK1/2 may appear to preferentially associate with RSK3 in cells maintained in the presence of serum, because RSK3 is the only isoform that does not dissociate from ERK1/2 upon activation. Therefore, we suggest that the state of Ras/ERK pathway activation will determine the ratio of RSK1, RSK2, and RSK3 that interacts with ERK1/2.
RSK activation leads to the phosphorylation of four essential residues (Ser239, Ser381, Ser398, and Thr590) and two additional sites (Thr377 and Ser749) with unknown functions (8
). We have found that Ser749 regulates ERK1/2 association with RSK1 and that phosphorylation of this residue requires an active N-terminal kinase domain, suggesting that Ser749 is an RSK1 autophosphorylation site that releases ERK1/2 in vivo. While ERK1/2 dissociation was inhibited when both RSK1 kinase domains were mutated (K112/464R), ERK dissociation from RSK1 mutants with only an N-terminal (K112R) or C-terminal (K464R) kinase domain inactivation still occurred, suggesting that Ser749 becomes phosphorylated in these mutants. The K464R mutant displays partial N-terminal kinase domain activity, which can account for the ERK1/2 dissociation, but the K112R mutant was found to be completely inactive toward exogenous substrates (Fig. ). This raised the possibility that the RSK K112R mutant retained its ability to phosphorylate Ser749 in cis
, despite its inactivity toward exogenous substrates. To address this, we mutated the APE motif in the activation segment (subdomain VIII) of the N-terminal kinase domain of RSK1 and found that this mutant (RSK1 AAA/D1) completely lost its ability to dissociate from ERK1/2 upon stimulation. These results indicated that, indeed, RSK1 K112R retained the ability to phosphorylate Ser749 in cis
and that the activity of the N-terminal kinase domain of RSK1 is essential to promote the release of ERK1/2 following mitogen stimulation.
ERK is thought to play at least two roles in RSK1 activation. First, activated ERK phosphorylates RSK1 on Thr590, and possibly on Thr377 and Ser381, and second, ERK brings RSK1 into close proximity to membrane-associated kinases that may phosphorylate RSK1 on Ser381 and Ser398 (23
). While ERK interaction is required for both of these functions, the role for a regulated ERK dissociation mechanism remains unknown. On one hand, it has been suggested that ERK substrates need to dissociate from its docking site in order to be phosphorylated (22
), but our results indicate that even RSK1 mutants that interact constitutively with ERK1/2 become activated to the same levels as wt RSK1. On the other hand, we have found that RSK proteins that interact constitutively with ERK1/2 remained activated longer than other RSK isoforms with regulated ERK docking mechanisms, suggesting that ERK association regulates the kinetics of RSK activation. The significance of signal duration in intracellular signaling has recently been underscored by a report showing that cells in the G1
phase of the cell cycle can sense ERK signal duration and respond to it by a specific biological function (18
). In that study, signal duration was sensed through the timely binding of ERK to specific docking sites, called DEF domains, located in the immediate-early c-Fos protein. Therefore, our results would suggest that the duration of activation of the RSK isozymes may similarly regulate specific cellular targets in order to produce precise biological outcomes.
Although ERK may potentiate RSK3 activation through its constitutive interaction throughout the time course, it is also possible that ERK binding to RSK3 reduces its rate of inactivation. ERK and RSK are generally inactivated in a coordinated fashion subsequent to their activation by growth factors, but the phosphatases involved are still unknown. The nuclear phosphatases MKP1 and MKP2 are good candidates for ERK inactivation and possess ERK docking sites (16
). Since these phosphatases bind to the same region on ERK that is recognized by RSK, it is possible that the stable interaction between RSK3 and ERK may repress ERK inactivation by competing for the same docking sites. While this remains a possibility, we cannot exclude the likelihood that ERK binding to RSK suppresses the ability of RSK phosphatases to specifically inactivate RSK.
In conclusion, we have revealed the mechanisms that regulate the interaction between RSK and ERK and found that regulated ERK docking plays important roles in RSK activation kinetics. These results will help in understanding ERK function and the functional differences among the different RSK isozymes, and they suggest that similar mechanisms may also exist in RSK-related kinases, such as the MSKs.