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A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1

2000; Springer Nature; Volume: 19; Issue: 12 Linguagem: Inglês

10.1093/emboj/19.12.2924

1460-2075

Morten Frödin, Claus J. Jensen, Karine Mérienne, Steen Gammeltoft,

NF-κB Signaling Pathways

Article15 June 2000free access A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1 Morten Frödin Corresponding Author Morten Frödin Department of Clinical Biochemistry, Glostrup Hospital, DK-2600 Glostrup, Denmark Search for more papers by this author Claus J. Jensen Claus J. Jensen Department of Clinical Biochemistry, Glostrup Hospital, DK-2600 Glostrup, Denmark Search for more papers by this author Karine Merienne Karine Merienne IGBMC Parc d'Innovation, 1 rue Laurent Fries, 67404 Illkirch, Cu Strasbourg, France Search for more papers by this author Steen Gammeltoft Steen Gammeltoft Department of Clinical Biochemistry, Glostrup Hospital, DK-2600 Glostrup, Denmark Search for more papers by this author Morten Frödin Corresponding Author Morten Frödin Department of Clinical Biochemistry, Glostrup Hospital, DK-2600 Glostrup, Denmark Search for more papers by this author Claus J. Jensen Claus J. Jensen Department of Clinical Biochemistry, Glostrup Hospital, DK-2600 Glostrup, Denmark Search for more papers by this author Karine Merienne Karine Merienne IGBMC Parc d'Innovation, 1 rue Laurent Fries, 67404 Illkirch, Cu Strasbourg, France Search for more papers by this author Steen Gammeltoft Steen Gammeltoft Department of Clinical Biochemistry, Glostrup Hospital, DK-2600 Glostrup, Denmark Search for more papers by this author Author Information Morten Frödin 1, Claus J. Jensen1, Karine Merienne2 and Steen Gammeltoft1 1Department of Clinical Biochemistry, Glostrup Hospital, DK-2600 Glostrup, Denmark 2IGBMC Parc d'Innovation, 1 rue Laurent Fries, 67404 Illkirch, Cu Strasbourg, France ‡M.Frödin and C.J.Jensen contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2924-2934https://doi.org/10.1093/emboj/19.12.2924 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The 90 kDa ribosomal S6 kinase-2 (RSK2) is a growth factor-stimulated protein kinase with two kinase domains. The C-terminal kinase of RSK2 is activated by ERK-type MAP kinases, leading to autophosphorylation of RSK2 at Ser386 in a hydrophobic motif. The N-terminal kinase is activated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) through phosphorylation of Ser227, and phosphorylates the substrates of RSK. Here, we identify Ser386 in the hydrophobic motif of RSK2 as a phosphorylation-dependent docking site and activator of PDK1. Treatment of cells with growth factor induced recruitment of PDK1 to the Ser386-phosphorylated hydrophobic motif and phosphorylation of RSK2 at Ser227. A RSK2-S386K mutant showed no interaction with PDK1 or phosphorylation at Ser227. Interaction with Ser386-phosphorylated RSK2 induced autophosphorylation of PDK1. Addition of a synthetic phosphoSer386 peptide (RSK2373–396) increased PDK1 activity 6-fold in vitro. Finally, mutants of RSK2 and MSK1, a RSK-related kinase, with increased affinity for PDK1, were constitutively active in vivo and phosphorylated histone H3. Our results suggest a novel regulatory mechanism based on phosphoserine-mediated recruitment of PDK1 to RSK2, leading to coordinated phosphorylation and activation of PDK1 and RSK2. Introduction The 90 kDa ribosomal S6 kinases (RSK1–4) are a family of broadly expressed serine/threonine kinases that are activated by extracellular signal-regulated protein kinase (ERK1 and ERK2) in response to many growth factors, peptide hormones and neurotransmitters (reviewed in Frödin and Gammeltoft, 1999; Nebreda and Gavin, 1999). RSK is an important effector of ERK in the regulation of cell division and survival. In Xenopus laevis oocytes, RSK regulates G2–M phase progression in meiosis I through phosphorylation of the p34cdc2-inhibitory kinase Myt1 (Palmer et al., 1998). During meiosis II, RSK induces metaphase arrest to prevent cell division in the unfertilized egg (Bhatt and Ferrell, 1999; Gross et al., 1999). In cerebellar granule neurons, RSK phosphorylates and inactivates the pro-apoptotic protein BAD (Bonni et al., 1999). RSK has been proposed to phosphorylate and activate transcription factors like estrogen receptor-α (Joel et al., 1998) and CREB (Xing et al., 1996), and to bind to and modulate the function of CBP and p300, which are transcriptional co-activators (Nakajima et al., 1996). Moreover, RSK2 phosphorylates histone H3 at Ser10, which may cause chromatin remodeling and facilitate gene transcription (Sassone-Corsi et al., 1999). Inactivating mutations in the RSK2 gene are responsible for human Coffin–Lowry syndrome, which is characterized by severe mental retardation and progressive skeletal deformations (Trivier et al., 1996). Mutations in RSK4 may also lead to mental retardation (Yntema et al., 1999). Recently, two mitogen- and stress-activated protein kinases (MSK) were discovered that are homologous to RSK (Deak et al., 1998; Pierrat et al., 1998). MSK is activated by ERK as well as by p38 MAP kinase in response to growth factors and various cellular stress stimuli. MSK appears to be a major growth factor-activated CREB kinase (Deak et al., 1998; Pierrat et al., 1998) and has been associated with phosphorylation of histone H3 and HMG-14 (Thomson et al., 1999), suggesting a role of MSK in transcriptional regulation. RSK is composed of two kinase domains connected by an ∼100-amino-acid linker region (Figure 1). The N-terminal kinase (NTK) phosphorylates the substrates of RSK and its activity is regulated by the C-terminal kinase (CTK) and the linker. Activation of RSK requires phosphorylation at four sites (Dalby et al., 1998). As a probable sequence of events, ERK phosphorylates Ser369 in the linker (using mouse RSK2 numbering) and Thr577 in the activation loop of the CTK, leading to its activation (Fisher and Blenis, 1996; Dalby et al., 1998). The CTK then phosphorylates Ser386 in the linker (Vik and Ryder, 1997). Finally, the NTK requires phosphorylation at Ser227 in the activation loop, which is catalyzed by 3-phosphoinositide-dependent protein kinase-1 (PDK1) (Jensen et al., 1999; Richards et al., 1999; Williams et al., 2000). While phosphorylation at Ser227 directly activates the NTK (Jensen et al., 1999), it is not known how phosphorylation at Ser386 modulates the activity of the NTK. Figure 1.Structure and regulatory phosphorylation sites of RSK2. RSK is composed of two kinase domains connected by a regulatory linker region. The C-terminal tail contains a docking site for ERK (Gavin and Nebreda, 1999; Smith et al., 1999). The locations of five phosphorylation sites and the kinases that phosphorylate these sites are shown. Phosphorylation at Ser227, Ser369, Ser386 and Thr577 regulates kinase activity, whereas the role of Thr365 phosphorylation is unclear (mouse RSK2 numbering). Amino acid sequences show the PDK1 consensus phosphorylation motif and the hydrophobic motif with conserved residues in bold. The alignment illustrates that both motifs are present in many growth factor-activated kinases, including RSK, MSK, p70 S6K, PKBα, PKCδ, SGK and PRK2. Serines/threonines that require phosphorylation for kinase activation are shown in red. Sequences are human, except RSK1 (rat), RSK2 and PKCδ (both mouse). Download figure Download PowerPoint PDK1 plays a central role in the activation of several growth factor-induced protein kinases (reviewed in Belham et al., 1999), which include protein kinase B (PKB) (Alessi et al., 1997a; Stephens et al., 1998), p70 S6 kinase (Alessi et al., 1997b; Pullen et al., 1998), several PKC isotypes (Dutil et al., 1998; Le Good et al., 1998) and serum and glucocorticoid-induced kinase (SGK) (Kobayashi and Cohen, 1999; Park et al., 1999). These kinases are all phosphorylated by PDK1 at a serine or threonine in the activation loop analogous to Ser227 in RSK2 (Figure 1). In addition, activation of these kinases requires phosphorylation at a serine or threonine residue located C-terminally to the kinase domain analogous to Ser386 in RSK. This phosphorylation site is situated in a characteristic hydrophobic motif (Figure 1), first recognized in p70 S6 kinase (Pearson et al., 1995). The mechanisms that determine the specificity of PDK1 action towards its multiple kinase substrates are only partly characterized. Activation of PKB by PDK1 requires co-localization at the plasma membrane, mediated by 3-phosphoinositide binding of the pleckstrin homology (PH) domain of the two kinases (Andjelkovic et al., 1997; Anderson et al., 1998; Currie et al., 1999). In addition, 3-phosphoinositides convert PKB into a good substrate for PDK1, possibly through a conformational change (Alessi et al., 1997a; Stokoe et al., 1997). Phosphorylation of p70 S6 kinase and SGK by PDK1 depends on phosphorylation events occurring elsewhere in these kinases, including the hydrophobic motif (Alessi et al., 1997b; Dennis et al., 1998; Pullen et al., 1998; Kobayashi and Cohen, 1999). The catalytic activity of PDK1 is not known to be regulated. Thus, the apparently constitutive activity of PDK1 was not increased by growth factor treatment of cells or by interaction of 3-phosphoinositides with the PH domain of PDK1 (Alessi et al., 1997a,b; Pullen et al., 1998). In this study, we have investigated the interaction and regulation of RSK2 and PDK1. We report a mechanism for activation of PDK1 and specificity of PDK1 action that is based on phosphoserine recognition. Thus, growth factor-induced phosphorylation at Ser386 in the hydrophobic motif of RSK2 creates a binding site that recruits PDK1 and stimulates its catalytic activity towards Ser227 in RSK2. Results Ser386 in the hydrophobic motif of RSK2 is required for phosphorylation of Ser227 and activation of the NTK domain To study the role of the linker in regulation of RSK2, we generated a series of mutants composed of the NTK domain with increasing portions of the linker region. The mutants were expressed in COS7 cells and their kinase activity was determined and compared with full-length RSK2. The deletion mutants RSK21–360, RSK21–373 and RSK21–380 were nearly inactive in cells (Figure 2). In contrast, RSK21–389 showed 2.5-fold higher kinase activity than full-length RSK2 from unstimulated cells, thus behaving as a constitutively active RSK2 mutant. Residues 381–389 include the hydrophobic motif and Ser386, which is phosphorylated by the CTK. Finally, RSK21–417 and RSK21–422 possessed roughly the same activity as RSK21–389 when corrected for their lower expression (Figure 2). RSK21–360, RSK21–373 and RSK21–380 were not inactive due to misfolding, since they could be activated to the same level as RSK21–389 by co-expression with PDK1 (data not shown). In conclusion, the hydrophobic motif is capable of activating the isolated NTK domain of RSK2 in vivo. Figure 2.Kinase activity of RSK2 deletion mutants in vivo. COS7 cells were transfected with plasmids expressing HA epitope-tagged full-length RSK2 or deletion mutants. After 48 h and a final 3 h serum starvation period, cells were lysed and RSK was precipitated with Ab to the HA epitope tag and subjected to kinase assay (lower panel). Data are expressed as a percentage of full-length RSK2 from unstimulated cells and are the mean ± SD of three independent experiments performed in duplicate. The activities of RSK21–389 and unstimulated RSK2 were different (p <0.001) when compared by non-paired t-test. After the kinase assay, RSK was subjected to SDS–PAGE and immunoblotting with Ab to the HA epitope tag (upper panel). Download figure Download PowerPoint We investigated the phosphorylation of RSK21–389 using phospho-specific antibodies (Abs) to the four phosphorylation sites present in this mutant: Ser227, Thr365, Ser369 and Ser386. Surprisingly, RSK21–389 was heavily phosphorylated at Ser386, even though the mutant contains no CTK domain (Figure 3B, lanes 3 and 4). Apparently, phosphorylation of Ser386 is required for the activity of RSK21–389, since the mutant RSK21–389S386A showed greatly reduced kinase activity (Figure 3A, lanes 5 and 6). Exposure of the cells to epidermal growth factor (EGF) did not increase phosphorylation at Ser386 or kinase activity of RSK21–389, in contrast to full-length RSK2 (Figure 3A and B). Interestingly, the PDK1 site, Ser227, was also highly phosphorylated in RSK21–389, but only marginally phosphorylated in RSK21–389S386A and not phosphorylated in RSK21–380 (Figure 3C). Phosphorylation at Ser227 was strongly induced by EGF in full-length RSK2, but not in the mutants (Figure 3C). Finally, none of the RSK2 mutants showed any phosphorylation at the two ERK phosphorylation sites in the linker (Thr365 and Ser369) under basal or EGF-stimulated conditions. In contrast, full-length RSK2 showed EGF-induced phosphorylation at these sites (Figure 3D and E). All constructs were present at roughly similar levels in the precipitates, as shown by immunoblotting for their hemagglutinin (HA) epitope tag (Figure 3F). These data suggest that RSK21–389 is active in cells because it is constitutively phosphorylated at Ser386 and Ser227. Moreover, the two phosphorylation sites exhibit a hierarchical relationship since phosphorylation at Ser386 is required for the phosphorylation of Ser227. Figure 3.Role of the hydrophobic motif in regulation of phosphorylation sites in the N-terminal kinase of RSK2. COS7 cells were transfected with plasmids expressing HA epitope-tagged wild-type RSK2 or mutant RSK2. After 48 h and a final 3 h serum starvation period, cells were exposed, or not, to 20 nM EGF for 20 min and lysed. Thereafter, RSK was precipitated from the cell lysates with Ab to the HA epitope. (A) The kinase activity of RSK was determined and expressed as a percentage of basal RSK21–389 activity. Data are the mean ± SD of four independent experiments performed in duplicate. The data of the following bars were different when compared by non-paired t-test: 3 versus 5 (p <0.001); 3 versus 7 (p 90% of PDK1 in the in vitro phosphorylation reaction (Figure 9B). Figure 9.Autophosphorylation of PDK1 induced by an RSK2 peptide containing phospho-Ser386 in vitro. Immunopurified myc-PDK1 was pre-incubated for 20 min alone (no addition) or with 10 μM S6 peptide or RSK2 peptide, residues 373–396, which was unphosphorylated (Ser386 peptide) or phosphorylated at Ser386 (pSer386 peptide). (A) PDK1 was allowed to autophosphorylate for 20 min in the presence of [γ-32P]ATP, whereafter the reactions were subjected to SDS–PAGE and autoradiography. (B) PDK1 was allowed to autophosphorylate for 35 min in the presence or absence of ATP, whereafter the reactions were subjected to SDS–PAGE and immunoblotting with Ab to the myc tag in PDK1. The experiments were performed twice with similar results. Download figure Download PowerPoint We next investigated whether the increase in autophosphorylation of PDK1 was associated with catalytic activation of PDK1. Immunopurified PDK1 was subjected to in vitro kinase assay using bacterially expressed, kinase-deficient RSK21–373 as a substrate. Incubation with pSer386 peptide increased the kinase activity of PDK1 6-fold as compared with incubation alone, with S6 peptide or with Ser386 peptide (Figure 10A and B). Phosphorylation of NTK by PDK1 occurred exclusively at Ser227, since the mutant RSK21–360S227E was not phosphorylated by PDK1, in contrast to RSK21–360 (Figure 10C). Activation of PDK1 by pSer386 peptide was also observed when using S6 peptide as a substrate (data not shown), although S6 peptide is a rather poor substrate for PDK1, since it does not contain a PDK1 consensus phosphorylation sequence. Figure 10.Activation of PDK1 by a RSK2 peptide containing phosphoSer386 in vitro. Immunopurified myc-PDK1 was pre-incubated for 20 min alone (no addition) or with 10 μM S6 peptide, Ser386 peptide or pSer386 peptide. (A and C) PDK1 was then incubated for 10 min with [γ-32P]ATP and 2 μg of RSK21–373KD or 0.5 μg of RSK21–360 as a substrate, as indicated in each panel, whereafter the reactions were subjected to SDS–PAGE and autoradiography. (B) PDK1 activity in (A) was determined by quantitation of radioactivity incorporated into RSK21–373KD using a PhosphorImager. Data are expressed as a percentage of PDK1 activity in the presence of pSer386 peptide and are the mean ± SD of three independent experiments. The data of the following bars were not different when compared by non-paired t-test: 1 versus 2 or 3 (p >0.2). The data from bars 1 and 4 were different when compared by non-paired t-test (p <0.001). Download figure Download PowerPoint These data suggest that PDK1 is activated upon interaction with phosphoSer386 in the hydrophobic motif of RSK2, leading to intramolecular autophosphorylation of PDK1 and phosphorylation of RSK2 at Ser227. This represents a novel regulatory mechanism for PDK1, in which PDK1 is activated through interaction with its substrate RSK2 and thus may be indirectly activated by the ERK signaling pathway. Constitutively active mutants of RSK2 and MSK1 phosphorylate histone H3 in vivo We used the new insight into the activation mechanism of RSK2 to generate constitutively active kinases of RSK2 and MSK1. RSK21–389 was not phosphorylated at the two ERK phosphorylation sites in the linker (see Figure 3D and E), raising the possibility that the introduction of negatively charged amino acids at these sites would further increase its activity. Indeed, RSK21–389T365E and RSK21–389S369E showed 60–80% higher kinase activity than RSK21–389 (Figure 11A). The increased activity of RSK21–389T365E provides the first evidence for a role of Thr365 phosphorylation in activation of RSK. Mutation of both ERK sites generated a mutant, RSK21–389T365E/S369E, with the same activity as EGF-stimulated wild-type RSK2. Finally, we attempted to activate RSK2 by enhancing the docking of PDK1 to the linker. Recently, the hydrophobic motif of protein kinase C-related kinase-2 (PRK2) was isolated as a PDK1-interacting fragment in a yeast two-hybrid screen (Balendran et al., 1999a). The hydrophobic motif of PRK2 contains the three characteristic aromatic residues, but in addition it contains two Asp residues,