Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo

Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-ter minal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, an d RSK3) interacted with extracellular signal-regulated kinase (ERK) but not c-Jun-NH2-kinase (JNK) or p38 mitogen-activated protein kinase (MAPK). Wit hin the carboxyl-terminal residues of the RSK isozymes is a region of high conservation corresponding to residues (722)LAQRRVRKLPSTTL(735) in RSK1, Tr uncation of the carboxyl-terminal 9 residues, (VRKLPSTTL735)-V-727, complet ely eliminated the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues (PSTTL735)-P-731 had no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunopre cipitated with hemagglutinin-tagged wild type RSK2 (HA-RSK2) in BHK cell cy tosol, However, ERK did not coimmunoprecipitate with HA-RSK2((1-729)), a mu tant missing the carboxyl-terminal 11 amino acids, similar to the minimal t runcation that eliminated in vitro interaction of ERK with the GST-RSK1 fus ion protein. Kinase activity of HA-RSK2 increased B-fold in response to ins ulin. HA-RSK2((1-729)) had similar basal kinase activity to that of HA-RSK2 but was not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2((1-729)) could be activated to the same extent in vitro by active E RK2, demonstrating that HA-RSK2((1-729)) was properly folded. These data su ggest that the conserved region of the RSK isozymes ((722)LAQRRVRKL(730) Of RSK1) provides for a specific ERK docking site approximately 150 amino aci ds carboxyl-terminal to the nearest identified ERK phosphorylation site (Th r(573)). Complex formation between RSK and ERK is essential for the activat ion of RSK by ERK in vivo, Comparison of the docking site of RSK with the c arboxyl-terminal tails of other MAPK-activated kinases reveals putative doc king sites within each of these MAPK-targeted kinases, The number and place ment of lysine and arginine residues within the conserved region correlate with specificity for activation by ERK and p38 MAPKs in vivo.