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) ...
Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heatstable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC␣ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RI⌬1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.
We describe a novel mechanism for transcriptional regulation, in which docking of p90 ribosomal S6 kinase 2 (Rsk2) to the hormone-binding domain (HBD) of estrogen receptor alpha (ERalpha) induces a conformational change that enhances the transcriptional activation function contained in the HBD. A constitutively active mutant of Rsk2 specifically enhances ERalpha-mediated transcription by phosphorylation of Ser167 in ERalpha and by physically associating with residues 326-394 of the ERalpha HBD. The anti-estrogen 4-hydroxytamoxifen blocks Rsk2-mediated activation of ERalpha, by inducing a conformation of ERalpha in which the Rsk2 docking site is masked. Transcriptional activation and docking are specific for ERalpha and do not occur with the related isoform, ERbeta. ERalpha phosphorylation, docking and transcriptional activation are regulated by the Rsk2 N-terminal kinase domain. The allosteric regulation of a target protein, independent of phosphorylation, may be paradigmatic of a general function for protein kinase docking sites.
p90 ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase, but its biological functions are not well understood. We have now identified the first small-molecule, RSK-specific inhibitor, which we isolated from the tropical plant Forsteronia refracta. We have named this novel inhibitor SL0101. SL0101 shows remarkable specificity for RSK. The major determinant of SL0101-binding specificity is the unique ATP-interacting sequence in the amino-terminal kinase domain of RSK. SL0101 inhibits proliferation of the human breast cancer cell line MCF-7, producing a cell cycle block in G1 phase with an efficacy paralleling its ability to inhibit RSK in intact cells. RNA interference of RSK expression confirmed that RSK regulates MCF-7 proliferation. Interestingly, SL0101 does not alter proliferation of a normal human breast cell line MCF-10A, although SL0101 inhibits RSK in these cells. We show that RSK is overexpressed in ∼50% of human breast cancer tissue samples, suggesting that regulation of RSK has been compromised. Thus, we show that RSK has an unexpected role in proliferation of transformed cells and may be a useful new target for chemotherapeutic agents. SL0101 will provide a powerful new tool to dissect the molecular functions of RSK in cancer cells.
The consensus substrate site for cAMP-dependent protein kinase (PKA) is Arg-Arg-Xaa-Ser(P)-Xaa and the autoinhibitory domain of the PKA type I alpha regulatory subunit (RI subunit) contains a similar sequence, Arg92-Arg-Arg-Arg-Gly-Ala-Ile-Ser-Ala-Glu. The italicized amino acids form a putative pseudosubstrate site (Ser is replaced with Ala), which together with adjacent residues could competitively inhibit substrate phosphorylation by the PKA catalytic subunit (C subunit). The present studies determine the contributions of Arg92-95, Ile98, and Glu101 to inhibitory potency. Amino-terminal truncation of RI subunit through Arg92 (delta1-92) or Arg93 (delta1-93) had no detectable effect on inhibition of C subunit. Truncation through Arg94 (delta1-94), or point mutation of Arg95 within truncated mutants (delta1-93.R95A or delta1-92.R95A), caused a dramatic reduction in inhibitory potency. Truncation through Arg95 (delta1-95) had a greater effect than did replacement or deletion of Arg94 or Arg95 alone. Using full-length RI subunit, the inhibitory potency was reduced by replacing Ile98 with Ala, Gly, or Gln, but not by replacing it with Val. The inhibitory potency of RI subunit was unchanged when Glu101 was replaced with Ala or Gln. It is concluded that Arg94, Arg95 and, to a lesser extent, Ile98 are vital constituents of PKA autoinhibition by type I alpha R subunit.
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...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.