Glutathione S-transferase (GST)-fusion proteins containing the carboxyl-terminal tails of three p90 ribosomal S6 kinase (RSK) isozymes (RSK1, RSK2, and RSK3) interacted with extracellular signal-regulated kinase (ERK) but not c-Jun-NH 2 -kinase (JNK) or p38 mitogenactivated protein kinase (MAPK). Within the carboxylterminal residues of the RSK isozymes is a region of high conservation corresponding to residues 722 LAQRRVR-KLPSTTL 735 in RSK1. Truncation of the carboxyl-terminal 9 residues, 727 VRKLPSTTL 735 , completely eliminated the interaction of the GST-RSK1 fusion protein with purified recombinant ERK2, whereas the truncation of residues 731 PSTTL 735 had no effect on the interaction with purified ERK2. ERK1 and ERK2 co-immunoprecipitated with hemagglutinin-tagged wild type RSK2 (HA-RSK2) in BHK cell cytosol. However, ERK did not coimmunoprecipitate with HA-RSK2(1-729) , a mutant missing the carboxyl-terminal 11 amino acids, similar to the minimal truncation that eliminated in vitro interaction of ERK with the GST-RSK1 fusion protein. Kinase activity of HA-RSK2 increased 6-fold in response to insulin. HA-RSK2(1-729) had a similar basal kinase activity to that of HA-RSK2 but was not affected by insulin treatment. Immunoprecipitated HA-RSK2 and HA-RSK2 Mitogen-activated protein kinases (MAPKs)1 transduce signals from the cell surface to the nucleus, altering the activity and subcellular localization of transcription factors. ERK, JNK, and p38 MAPKs lie in distinct signaling pathways that are activated by distinct stimuli. Whereas the minimal consensus phosphorylation sequence of these proline-directed kinases would suggest promiscuous phosphorylation of many proteins, the kinases play an integral role in the cellular growth machinery; therefore, substrate specificity must be tightly regulated. It is becoming clear that the substrate specificity of MAPKs with regard to transcription factors involves high affinity binding of MAPK to sequences within the substrate that are distinct from the consensus phosphorylation sequence (1, 2). Such an interaction has been described for JNK and the transcription factors c-Jun and activating transcription factor (ATF-2) (3-5). Recently, a sequence within Elk-1 was shown to contain overlapping but distinct interaction sites for ERK and JNK (6). Specific targeting interactions between MAPKs and substrates may not be limited to transcription factors. One possible ERK substrate for which targeting interactions might occur is p90 ribosomal S6 protein kinase (RSK). RSK is phosphorylated and activated by ERK in vitro (7), and inhibition of the ERK pathway with the mitogen-activated protein/ERK kinase-specific inhibitor PD98059 prevents in vivo activation of RSK (8,9). RSK is unusual in that it contains two distinct kinase catalytic domains within a single polypeptide chain (10). In vivo ERK phosphorylation sites within RSK have been identified (11,12) (Fig. 1A). Two of these sites are essential for activation of RSK: 1) Ser 363 in the linker between the two kinase domains, and 2) ...

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1 In cultures of bovine tracheal smooth muscle cells, platelet-derived growth factor-BB (PDGF), bradykinin (BK) and endothelin-1 (ET-1) stimulated the tyrosine phosphorylation and activation of both pp42 and pp44 kDa forms of mitogen-activated protein (MAP) kinase. 2 Both ET-l and PDGF stimulated a sustained activation of MAP kinase whilst the response to BK was transient.