Properties and Regulation of a Transiently Assembled ERK2·Ets-1 Signaling Complex

Abstract

ERK2 is a proline-directed protein kinase that displays a high specificity for a single threonine (Thr-38) on the substrate Ets-1, which lies within the consensus sequence 36phi-chi-Thr-Pro39 (where phi is typically a small hydrophobic residue and chi appears to be unrestricted). Thr-38 lies in a long flexible N-terminal tail (residues 1-52), which also contains a second potential phosphorylation site, Ser-26. How Ets-1 binds ERK2 to promote the phosphorylation of Thr-38 while simultaneously discriminating against the phosphorylation of Ser-26 is unclear. To delineate the details of the molecular recognition of Ets-1 by ERK2, the binding of various mutants and truncations of Ets-1 were analyzed by fluorescence anisotropy. The data that were obtained support the notion that the N-terminal tail contains a previously unrecognized docking site that promotes the phosphorylation of Thr-38. This new docking site helps assemble the complex of Ets-1 and ERK2 and makes a similar contribution to the stabilization of the complex as does the pointed domain of Ets-1. The in vitro activation of ERK2 by MKK1 induces a large conformational transition of the activation segment (DFG-APE), but neither induces self-association of ERK2 nor destabilizes the stability of the ERK2.Ets-1 complex. This latter observation suggests that interactions intrinsic to the active site are not important for complex assembly, a notion further supported by the observation that the substitution of a number of different amino acids for Pro-39 does not destabilize the complex. Mutagenesis of ERK2 within loop 13 suggests that Ets-1 binds the substrate-binding groove. These data suggest that ERK2 uses two weak docking interactions to specifically assemble the complex, perhaps in doing so denying Ser-26 access to the active site. Displacement of residues 1-138 of Ets-1 (EtsDelta138) from ERK2 by the peptide N-QKGKPRDLELPLSPSL-C, derived from Elk-1, suggests that Ets-1 engages the D-recruitment site (beta7-beta8 reverse turn and the alphaD-alphaE helix) of ERK2. Displacement of EtsDelta138 from ERK2 by the peptide N-AKLSFQFPS-C derived from Elk-1 shows that EtsDelta138 communicates with the F-recruitment site of ERK2 also.

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

“…Recently, the D-site peptide QKGRKPRDLELPLSPSL derived from Elk-1, which binds the DRS of ERK2, was shown to displace Ets-1 from both the activated and unactivated forms of ERK2 (23). We hypothesized that rather than displacing Ets-1 through an allosteric mechanism this peptide directly competes with Ets-1 for the DRS of ERK2.…”

“…Despite lacking a canonical D-site, the substrate Ets-1 is displaced from ERK2 by peptides containing a D-site (23). This suggests that Ets-1 may contain a novel or cryptic D-site.…”

“…Although this sequence conforms, in part, to the consensus sequence for a D-site, mutagenesis data suggests that the interaction of this site with ERK2 is primarily mediated by the hydrophobic residue Phe-120, which lies in the Φ B + 4 position, a position not previously noted as being important for the binding of ligands to the DRS of ERK2. Binding studies also indicate that a second site of interaction lies in the highly flexible N-terminus, although this site has not yet been located (23).…”

The PNT domain from Drosophila pointed‐P2 contains a dynamic N‐terminal helix preceded by a disordered phosphoacceptor sequence

“…1(B)]. With the exception of Glu142, Val143, Phe166 and the N-terminal Gly-Ser-His-Met tag, almost complete 1 H N , 15 N, 13 C a , 13 C b , and 13 C 0 resonance assignments were obtained for this construct. These chemical shifts were used to identify the secondary structural elements of PntP2 142-252 with the MICS (Motif Identification by Chemical Shift) algorithm.…”

“…10,11 The docking interfaces on the PNT domain and the kinase have been mapped coarsely through mutagenesis, NMR spectroscopic, chemical footprinting, and competition studies. 10,[12][13][14][15] To both accommodate the proposed docking mechanism and position the phosphoacceptors in the catalytic site of the kinase, a significant conformational change in the ETS1 PNT domain, such as the unfolding of the appended Nterminal helix H0 (residues Lys42-Thr52), appears to be required. 16 Indeed, NMR relaxation and amide hydrogen exchange (HX) measurement have shown that this helix is only marginally stable and structurally flexible.…”