Generation of constitutively active p90 ribosomal S6 kinase in vivo - Implications for the mitogen-activated protein kinase-activated protein kinase family

p90 ribosomal S6 kinases (RSKs), containing two distinct kinase catalytic d omains, are phosphorylated and activated by extracellular signal-regulated kinase (ERK), The amino-terminal kinase domain (NTD) of RSK phosphorylates exogenous substrates, whereas the carboxyl-terminal kinase domain (CTD) aut ophosphorylates Ser-386. A conserved putative autoinhibitory alpha helix is present in the carboxyl-terminal. tail of the RSK isozymes ((697)HLVKGAMAA TYSALNR(712) Of RSK2) Here, we demonstrate that truncation (Delta alpha) or mutation (Y707A) of this helix in RSK2 resulted in constitutive activation of the CTD. In vivo, both mutants enhanced basal Ser-386 autophosphorylati on by the CTD above that of wild type (WT), The enhanced Ser-386 autophosph orylation was attributed to disinhibition of the CTD because a CTD dead mut ation (K451A) eliminated Ser-386 autophosphorylation even in conjunction wi th Delta alpha and Y707A. Constitutive activity of the CTD appears to enhan ce NTD activity even in the absence of ERK phosphorylation because basal ph osphorylation of S6 peptide by Delta alpha and Y707A was similar to 4-fold above that of WT. A RSK phosphorylation motif antibody detected a 140-kDa p rotein (pp140) that was phosphorylated upon epidermal growth factor or insu lin treatment. Ectopic expression of Delta alpha or Y707A resulted in incre ased basal phosphorylation of pp140 compared with that of WT, presenting th e possibility that pp140 is a novel RSK substrate. Thus, it is clear that t he CTD regulates NTD activity in vivo as well as in vitro.