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

Frödin, Morten; Jensen, Claus Munk; Mérienne, Karine; Gammeltoft, Steen

doi:10.1093/emboj/19.12.2924

Cited by 282 publications

(285 citation statements)

References 48 publications

(73 reference statements)

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“…MSK1 was originally identified [14] through a search for novel AGC kinases that might be regulated by PDK1 (phosphoinositide-dependent kinase 1), a 'master' kinase required for the activation of a subset of AGC kinases including RSK. PDK1 acts by phosphorylating a specific residue in the T-loop of its substrates, and the phosphorylation of this site is essential for kinase activation [18][19][20]. Work using PDK1 −/− embryonic stem cells confirmed that PDK1 was required for the activation of RSK1, RSK2 and RSK3 [20].…”

Section: Introductionmentioning

confidence: 99%

Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS

McCoy

Macdonald

Morrice

et al. 2007

Biochemical Journal

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MSK1 (mitogen-and stress-activated kinase 1) is a dual kinase domain protein that acts downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPK (mitogenactivated protein kinase) signalling pathways in cells. MSK1, and its related isoform MSK2, phosphorylate the transcription factors CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor 1), and the chromatin proteins histone H3 and HMGN1 (high-mobility-group nucleosomalbinding protein 1) in response to either mitogenic stimulation or cellular stress. MSK1 activity is tightly regulated in cells, and activation requires the phosphorylation of MSK1 by either ERK1/2 or p38α. This results in activation of the C-terminal kinase domain, which then phosphorylates further sites in MSK1, leading to the activation of the N-terminal kinase domain and phosphorylation of substrates. Here, we use precursor ion scanning MS to identify five previously unknown sites in MSK1: Thr 630 , Ser 647 , Ser 657 , Ser 695 and Thr 700 . One of these sites, Thr 700 , was found to be a third site in MSK1 phosphorylated by the upstream kinases ERK1/2 and p38α. Mutation of Thr 700 resulted in an increased basal activity of MSK1, but this could be further increased by stimulation with PMA or UV-C radiation. Surprisingly, however, mutation of Thr 700 resulted in a dramatic loss of Thr 581 phosphorylation, a site essential for activity. Mutation of Thr 700 and Thr 581 to an alanine residue resulted in an inactive kinase, while mutation of both sites to an aspartic acid residue resulted in a kinase with a significant basal activity that could not be further stimulated. Together these results are consistent with a mechanism by which Thr 700 phosphorylation relieves the inhibition of MSK1 by a C-terminal autoinhibitory helix and helps induce a conformational shift that protects Thr 581 from dephosphorylation.

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Section: Introductionmentioning

confidence: 99%

Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS

McCoy

Macdonald

Morrice

et al. 2007

Biochemical Journal

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“…Akt and RSK1 share some common substrates and both can be regulated by PDK1 (Alessi et al, 1997). While RSK and Akt are both substrates of PDK1, recruitment of PDK1 by RSK2 was shown to cause phosphorylation and activation of PDK1 that in turn would activate both Akt and RSK2 again illustrating a close relationship between RSK and Akt activation (Frodin et al, 2000).…”

Section: Discussionmentioning

confidence: 89%

“…The proteins, that mediate KGF-induced Akt activation, however, are not known. It was recently shown that RSK2, a member of the RSK family of proteins recruits PDK1 and promotes coordinated phosphorylation and activation of PDK1 and RSK2 (Frodin et al, 2000). PDK1 is a kinase that is critical for the activation of Akt and related enzymes, including RSK, that regulate many physiological processes such as cell survival, proliferation, and gene expression.…”

Section: Rsk1 Is Required For Akt Activation By the Kgfrmentioning

confidence: 99%

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Ribosomal S6 Kinase as a Mediator of Keratinocyte Growth Factor-induced Activation of Akt in Epithelial Cells

Pan

Devaux

Ray

2004

MBoC

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The keratinocyte growth factor receptor (KGFR) is a member of the fibroblast growth factor receptor (FGFR) superfamily. The proximal signaling molecules of FGFRs are much less characterized compared with other growth factor receptors. Using the yeast two-hybrid assay, we have identified ribosomal S6 kinase (RSK) to be a protein that associates with the cytoplasmic domain of the KGFR. The RSK family of kinases controls multiple cellular processes, and our studies for the first time show association between the KGFR and RSK. Using a lung-specific inducible transgenic system we have recently demonstrated protective effects of KGF on the lung epithelium and have demonstrated KGF-induced activation of the prosurvival Akt pathway both in vivo and in vitro. Here we show that a kinase inactive RSK mutant blocks KGF-induced Akt activation and KGF-mediated inhibition of caspase 3 activation in epithelial cells subjected to oxidative stress. It was recently shown that RSK2 recruits PDK1, the kinase responsible for both Akt and RSK activation. When viewed collectively, it appears that the association between the KGFR and RSK plays an important role in KGF-induced Akt activation and consequently in the protective effects of KGF on epithelial cells. INTRODUCTIONThe keratinocyte growth factor receptor (KGFR) is a member of the FGFR (fibroblast growth factor receptor) family. The KGFR is expressed only in epithelial cells and it plays critical roles in the proliferation, migration, and morphogenesis of epithelial cells (Ulich et al., 1994;Wilson et al., 1994;Post et al., 1996;Buckley et al., 1997). The KGFR also plays important roles in skin wound healing and lung epithelial cell survival during injury (Werner et al., 1994;Panos et al., 1995;Yi et al., 1996;Barazzone et al., 1999;Das and Olsen, 2001;Ray et al., 2003). The KGFR is activated by FGF-1, FGF-3, KGF/FGF-7, and FGF-10, whereas FGFR2 is mainly activated by FGF-2/bFGF (Bottaro et al., 1990;Miki et al., 1991Miki et al., , 1992Orr-Urtreger et al., 1993). In contrast to information on signaling by other growth factor receptors, the proximal signaling molecules of FGFRs are much less characterized.To characterize the KGFR-induced signaling pathways, we screened for proteins interacting with the KGFR cytoplasmic domain using the yeast two-hybrid assay. RSK1 is one of the proteins we identified and this interaction was confirmed in mammalian epithelial cells. The RSK (or p90RSK) family includes three members, RSK1-3, which show 75-80% similarity at the amino acid level (Frodin and Gammeltoft, 1999). RSK family members contain two heterologous kinase domains (Fisher and Blenis, 1996). The Nterminal kinase (NTK) domain belongs to the AGC group of kinases, which includes PKA, PKB/Akt, PKC, and PKG, and is the kinase domain that phosphorylates the substrates of RSKs. The C-terminal kinase (CTK) domain and the linker are involved in the activation of the RSK NTK domain (Frodin and Gammeltoft, 1999). At the carboxyl-terminus, there is an ERK-binding site conserved in all three RSK ...

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“…However, Src, Fyn, and Abl tyrosine kinases have been shown to phosphorylate PDK1 in vitro and overexpression of v-Src in mammalian cells results in tyrosine phosphorylation and activation of PDK1 [15][16][17]. In addition, mammalian PDK1 is affected by interactions with PDK1-associating proteins, including PKC-related kinase 1 (PRK1), and Hsp90 [18][19][20][21], suggesting that its activity is controlled by interactions with other proteins as well. Furthermore, human PDK1 has been shown to interact with 14-3-3 h and g and a 14-3-3 recognition site has been identified at Ser-241 [22].…”

Section: Introductionmentioning

confidence: 99%

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

et al. 2006

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The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp-167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro target of AtPDK1. Our data clearly show that Asp-167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident.

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

Cited by 282 publications

References 48 publications

Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS

Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS

Ribosomal S6 Kinase as a Mediator of Keratinocyte Growth Factor-induced Activation of Akt in Epithelial Cells

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

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