Generation of Constitutively Active p90 Ribosomal S6 Kinasein Vivo

p90 ribosomal S6 kinases (RSKs), containing two distinct kinase catalytic domains, 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) autophosphorylates Ser-386. A conserved putative autoinhibitory alpha helix is present in the carboxyl-terminal tail of the RSK isozymes ( 697 HLVKGAMAATYSALNR 712 of RSK2). Here, we demonstrate that truncation (Δα) or mutation (Y707A) of this helix in RSK2 resulted in constitutive activation of the CTD. In vivo , both mutants enhanced basal Ser-386 autophosphorylation by the CTD above that of wild type (WT). The enhanced Ser-386 autophosphorylation was attributed to disinhibition of the CTD because a CTD dead mutation (K451A) eliminated Ser-386 autophosphorylation even in conjunction with Δα and Y707A. Constitutive activity of the CTD appears to enhance NTD activity even in the absence of ERK phosphorylation because basal phosphorylation of S6 peptide by Δα and Y707A was ∼4-fold above that of WT. A RSK phosphorylation motif antibody detected a 140-kDa protein (pp140) that was phosphorylated upon epidermal growth factor or insulin treatment. Ectopic expression of Δα or Y707A resulted in increased basal phosphorylation of pp140 compared with that of WT, presenting the possibility that pp140 is a novel RSK substrate. Thus, it is clear that the CTD regulates NTD activity in vivo as well as in vitro .