Generation of Constitutively Active p90 Ribosomal S6 Kinasein Vivo

Abstract

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 (⌬␣) 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 ⌬␣ 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 ⌬␣ and Y707A was ϳ4-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 ⌬␣ 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.p90 ribosomal S6 kinase (RSK) 1 is a member of a growing subfamily of mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) that contain two distinct kinase catalytic domains in a single polypeptide chain (see Fig. 1A). The three mammalian isozymes of RSK (RSK1, RSK2, RSK3), encoded by separate genes (1), are phosphorylated and activated in vivo by extracelluar signal-regulated kinase (ERK).The amino-terminal kinase domain (NTD) of RSK, residues 68 -327 of human RSK2 (see Fig. 1A), is most closely related to p70 S6 kinase with regard to primary structure. To date, only the NTD has been shown to phosphorylate exogenous substrates for RSK, including the cAMP response element binding protein (2, 3), c-Fos (4, 5) and the estrogen receptor (6). The list of substrates suggests that RSK plays a role in transcriptional regulation. The carboxyl-terminal kinase domain (CTD) of RSK, residues 422-679 in RSK2 (see Fig. 1A), is related to calmodulin-dependent protein kinases (CaMKs) and autophosphorylates Ser-386 in the linker region between the two kinase domains (7).Activation of RSK in vivo requires interaction between ERK and the ERK-docking site located in the extreme carboxyl terminus of RSK (8,9). RSK activation also requires ERK phosphorylation of Thr-577 in the CTD activation loop and Ser-369 in the linker, as well as autophosphorylation of Ser-386 by the CTD (see Fig. 1A) (7). Attenuation of CTD activity by mutation of Thr-577 or the ATP binding pocket generates an enzyme that cannot be fully activated (7,10,11). Thus...

Ribosomal S6 kinase 2 interacts with and phosphorylates PDZ domain-containing proteins and regulates AMPA receptor transmission

“…RSKs binding to specific PDZ domain proteins allows phosphorylation of these proteins and may regulate RSK targeting to specific subcellular regions. A previous study reported that RSK2 is associated with a NMDA-R-associated multiprotein complex isolated from forebrain extracts (37). Our results suggest a likely molecular explanation for this finding, because RSK2 directly binds Shank, which is then tethered to NMDA-R protein complexes by Shank interactors, such as guanylate kinase-associated protein (30) or Homer.…”

“…However, GFP-RSK2kn might sterically disrupt PDZ domain protein complexes, and this disruption, rather than the inhibition of RSK2 signaling, might reduce mEPSCs. To distinguish between these possibilities, we transfected cortical neurons with a constitutively active RSK2 (GFP-RSK2 Y707A) (37) in which the PDZ ligand remains intact. We confirmed elevated basal kinase activity of GFP-RSK2YA compared with wt GFP-RSK2 in vitro (data not shown).…”

C-terminal Elements Control Location, Activation Threshold, and p38 Docking of Ribosomal S6 Kinase B (RSKB)

“…Studies of RSK1 (27,46), RSK2 (29,31), and RSKB and mitogen-and stress-activated protein kinase type 1 (7,8) demonstrated the important control exerted by elements in the C-terminal sequences of RSKs. For example, a mutation of a C-terminal tyrosine converted RSK2 to a constitutively active enzyme, suggesting that a putative autoinhibitory structure element controlled the basal activity of the enzyme (31).…”

“…nal tail of RSKs, facilitating interaction with upstream MAPKs, in some instances were found to be essential for activation (8,29,30). The profound control exerted by C-terminal tail elements was further demonstrated by the constitutive activity generated by truncation or mutation in the conserved putative autoinhibitory C-terminal helix of RSK2 (31). RSK1-RSK3 interact with ERK independent of activation state and locate both to the cytoplasm and nucleus under resting conditions; upon activation, the complex translocates to the nucleus (32).…”

Exogenously Added Fibroblast Growth Factor 2 (FGF-2) to NIH3T3 CellsInteracts with Nuclear Ribosomal S6 Kinase 2 (RSK2) in a Cell Cycle-dependentManner

“…To ascertain that the FGF-2/RSK2 interaction was direct and specific, we incubated immobilized B-FGF-2 with purified RSK2 (26). As shown in Fig.…”