ERK2 primarily recognizes substrates through two recruitment sites, which lie outside theactive site cleft of the kinase.  These recruitment sites bind modular-docking sequences called docking sites and are potentially attractive sites for the development of non-ATP competitive inhibitors.  The D-recruitment site (DRS) and the F-recruitment site (FRS) bind D-sites and F-sites, respectively.  To facilitate the design of ERK2 inhibitors it is important to understand how tightly recruitment sites are energetically coupled to other ligand recognition sites within ERK2.   Energetic coupling within ERK2 was investigated using two new modular peptide substrates for ERK2.  Modeling shows that one peptide (Sub-D) recognizes the DRS, while the other peptide (Sub-F) binds the FRS.  A steady-state kinetic analysis reveals little evidence of thermodynamic linkage between peptide substrate and ATP.  Both peptides are phosphorylated through a random-order sequential mechanism with a kcat/Km comparable to Ets-1, a bona fide ERK2 substrate.  Occupancy of the FRS with a peptide containing a modular docking sequence has no effect on the intrinsic ability of ERK2 to phosphorylate Sub-D.  Occupancy of the DRS with a peptide containing a modular docking sequence has a slight effect on the intrinsic ability of ERK2 to phosphorylate Sub-F.  These data suggest that while docking interactions at the DRS and the FRS are energetically uncoupled, the DRS can exhibit weak communication to the active site.  In addition, they suggest that peptides bound to the FRS inhibit the phosphorylation of protein substrates through a steric mechanism.  The modeling and kinetic data suggest that the recruitment of ERK2 to cellular locations via its DRS may facilitate the formation of F-site selective ERK2 signaling complexes, while recruitment via the FRS will likely inhibit ERK2 through a steric mechanism of inhibition.  Such recruitment may serve as an additional level of ERK2 regulation.

Two modular peptide substrates to identify substrate selective MAPK inhibitors

243rd National Meeting (2012)

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