title: 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.
authors.affiliation: Graduate Program in Biochemistry, University of Texas, Austin, Texas 78712-0252, USA.
authors.forename: Kari A
authors.initials: KA
authors.lastname: Callaway
authors.forename: Mark A
authors.initials: MA
authors.lastname: Rainey
authors.forename: Austen F
authors.initials: AF
authors.lastname: Riggs
authors.forename: Olga
authors.initials: O
authors.lastname: Abramczyk
authors.forename: Kevin N
authors.initials: KN
authors.lastname: Dalby
mesh_terms: D000595:Amino Acid Sequence; D005454:Fluorescence Polarization; D006801:Humans; D008027:Light; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D050784:Proto-Oncogene Protein c-ets-1 / Q000737:chemistry / Q000378:metabolism*; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
chemical_list: C494041:ETS1 protein, human; D050784:Proto-Oncogene Protein c-ets-1; D019950:Mitogen-Activated Protein Kinase 1