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John Blenis - One of the best experts on this subject based on the ideXlab platform.
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p90 Ribosomal S6 Kinase rsk phosphorylates myosin phosphatase and thereby controls edge dynamics during cell migration
Journal of Biological Chemistry, 2019Co-Authors: Shiela C Samson, John Blenis, Michelle C. Mendoza, Andrew Elliott, Brian D Mueller, Yung Kim, Keith R Carney, Jared P BergmanAbstract:Cell migration is essential to embryonic development, wound healing, and cancer cell dissemination. Cells move via leading-edge protrusion, substrate adhesion, and retraction of the cell's rear. The molecular mechanisms by which extracellular cues signal to the actomyosin cytoskeleton to control these motility mechanics are poorly understood. The growth factor-responsive and oncogenically activated protein extracellular signal-regulated Kinase (ERK) promotes motility by signaling in actin polymerization-mediated edge protrusion. Using a combination of immunoblotting, co-immunoprecipitation, and myosin-binding experiments and cell migration assays, we show here that ERK also signals to the contractile machinery through its substrate, p90 Ribosomal S6 Kinase (RSK). We probed the signaling and migration dynamics of multiple mammalian cell lines and found that RSK phosphorylates myosin phosphatase-targeting subunit 1 (MYPT1) at Ser-507, which promotes an interaction of Rho Kinase (ROCK) with MYPT1 and inhibits myosin targeting. We find that by inhibiting the myosin phosphatase, ERK and RSK promote myosin II-mediated tension for lamella expansion and optimal edge dynamics for cell migration. These findings suggest that ERK activity can coordinately amplify both protrusive and contractile forces for optimal cell motility.
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p90 Ribosomal S6 Kinase and p70 Ribosomal S6 Kinase Link Phosphorylation of the Eukaryotic Chaperonin Containing TCP-1 to Growth Factor, Insulin, and Nutrient Signaling
The Journal of biological chemistry, 2009Co-Authors: Yuki Abe, Sang-oh Yoon, Kazuishi Kubota, Michelle C. Mendoza, Steven P. Gygi, John BlenisAbstract:Chaperonin containing TCP-1 (CCT) is a large multisubunit complex that mediates protein folding in eukaryotic cells. CCT participates in the folding of newly synthesized polypeptides, including actin, tubulin, and several cell cycle regulators; therefore, CCT plays an important role in cytoskeletal organization and cell division. Here we identify the chaperonin CCT as a novel physiological substrate for p90 Ribosomal S6 Kinase (RSK) and p70 Ribosomal S6 Kinase (S6K). RSK phosphorylates the β subunit of CCT in response to tumor promoters or growth factors that activate the Ras-mitogen-activated protein Kinase (MAPK) pathway. CCTβ Ser-260 was identified as the RSK site by mass spectrometry and confirmed by site-directed mutagenesis. RSK-dependent Ser-260 phosphorylation was sensitive to the MEK inhibitor UO126 and the RSK inhibitor BID-1870. Insulin weakly activates RSK but strongly activates the phosphoinositide 3-Kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and utilizes S6K to regulate CCTβ phosphorylation. Thus, the Ras-MAPK and PI3K-mTOR pathways converge on CCTβ Ser-260 phosphorylation in response to multiple agonists in various mammalian cells. We also show that RNA interference-mediated knockdown of endogenous CCTβ causes impaired cell proliferation that can be rescued with ectopically expressed murine CCTβ wild-type or phosphomimetic mutant S260D, but not the phosphorylation-deficient mutant S260A. Although the molecular mechanism of CCTβ regulation remains unclear, our findings demonstrate a link between oncogene and growth factor signaling and chaperonin CCT-mediated cellular activities.
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quantitative phosphorylation profiling of the erk p90 Ribosomal S6 Kinase signaling cassette and its targets the tuberous sclerosis tumor suppressors
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bryan A. Ballif, John Blenis, Philippe P. Roux, Scott A. Gerber, Jeffrey P. Mackeigan, Steven P. GygiAbstract:Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein Kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated Kinase (ERK)/p90 Ribosomal S6 Kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 Ribosomal S6 Kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological Kinase inhibitors we uncovered a ERK-independent, protein Kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.
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Quantitative phosphorylation profiling of the ERK/p90 Ribosomal S6 Kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bryan A. Ballif, John Blenis, Philippe P. Roux, Scott A. Gerber, Jeffrey P. Mackeigan, Steven P. GygiAbstract:Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein Kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated Kinase (ERK)/p90 Ribosomal S6 Kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 Ribosomal S6 Kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological Kinase inhibitors we uncovered a ERK-independent, protein Kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.
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Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 Ribosomal S6 Kinase
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Philippe P. Roux, Steven P. Gygi, Bryan A. Ballif, Rana Anjum, John BlenisAbstract:Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-Kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 Ribosomal S6 Kinase 1 (S6K1). The mitogen-activated protein Kinase (MAPK)-activated Kinase, p90 Ribosomal S6 Kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.
Zigang Dong - One of the best experts on this subject based on the ideXlab platform.
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tumor necrosis factor receptor associated factor family protein 2 is a key mediator of the epidermal growth factor induced Ribosomal S6 Kinase 2 camp responsive element binding protein fos protein signaling pathway
Journal of Biological Chemistry, 2012Co-Authors: Cong Peng, Feng Zhu, Weihong Wen, Ke Yao, Ann M. Bode, Kangdong Liu, Tatyana A Zykova, Zigang DongAbstract:TRAF2 has an important function in mediating the TNF-R signaling pathway toward activation of NF-κB and JNKs. Here we reveal a novel function of TRAF2 in the epidermal growth factor (EGF) signaling pathway. Knockdown of TRAF2 blocked EGF-induced AP-1 activity and anchorage- independent cell transformation. Notably, we showed that EGF induces Ribosomal S6 Kinase 2 (RSK2) ubiquitination, and knocking down TRAF2 suppresses ubiquitination of RSK2 induced by EGF. We also found that TRAF2 affects RSK2 activity through RSK2 ubiquitination. RSK2 plays a critical role in AP-1 activity mediated through CREB and c-Fos, which regulates anchorage-independent cell transformation. In addition, TRAF2 is overexpressed in colon cancer and required for colon cancer development, suggesting that TRAF2 might be a potential molecular target for cancer prevention and treatment.
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Abstract 4968: Phosphorylation of caspase-8 (Thr263) by Ribosomal S6 Kinase 2 (RSK2) mediates caspase-8 ubiquitination and stability
Molecular and Cellular Biology, 2012Co-Authors: Cong Peng, Yong Yeon Cho, Feng Zhu, Ann M. Bode, Hua Xie, Zigang DongAbstract:The Ribosomal S6 Kinase 2 (RSK2) is a member of the p90 Ribosomal S6 Kinase (p90RSK) family of proteins and plays a critical role in proliferation, cell cycle and transformation. Here, we report that RSK2 phosphorylates caspase-8 and Thr263 was identified as a novel caspase-8 phosphorylation site. Phosphorylation of Thr263 is associated with the ubiquitination and degradation of caspase through the proteasome pathway. Results suggested that RSK2 regulates caspase-8 stability through its phosphorylation of this caspase. These data provide a direct link between RSK2 and caspase-8 and identify a novel molecular mechanism for caspase-8 modulation by RSK2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4968. doi:1538-7445.AM2012-4968
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Phosphorylation of Caspase-8 (Thr-263) by Ribosomal S6 Kinase 2 (RSK2) Mediates Caspase-8 Ubiquitination and Stability
The Journal of biological chemistry, 2010Co-Authors: Cong Peng, Yong Yeon Cho, Feng Zhu, Jishuai Zhang, Weihong Wen, Ke Yao, Ann M. Bode, Zigang DongAbstract:The Ribosomal S6 Kinase 2 (RSK2) is a member of the p90 Ribosomal S6 Kinase (p90RSK) family of proteins and plays a critical role in proliferation, cell cycle, and cell transformation. Here, we report that RSK2 phosphorylates caspase-8, and Thr-263 was identified as a novel caspase-8 phosphorylation site. In addition, we showed that EGF induces caspase-8 ubiquitination and degradation through the proteasome pathway, and phosphorylation of Thr-263 is associated with caspase-8 stability. Finally, RSK2 blocks Fas-induced apoptosis through its phosphorylation of caspase-8. These data provide a direct link between RSK2 and caspase-8 and identify a novel molecular mechanism for caspase-8 modulation by RSK2.
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Ribosomal S6 Kinase 2 Is a Key Regulator in Tumor Promoter–Induced Cell Transformation
Cancer research, 2007Co-Authors: Yong Yeon Cho, Ke Yao, Ann M. Bode, Bong Seok Kang, Hong-gyum Kim, Duo Zheng, Zigang DongAbstract:The Ribosomal S6 Kinase 2 (RSK2), a member of the p90RSK (RSK) family of proteins, is a widely expressed serine/threonine Kinase that is activated by extracellular signal-regulated Kinase 1/2 and phosphoinositide-dependent Kinase 1 in response to many growth factors and peptide hormones. Its activation signaling enhances cell survival. However, the roles of RSK2 in cell transformation have not yet been elucidated. Here, we found that RSK2 is a critical serine/threonine Kinase for the regulation of cell transformation. When cells were stimulated with tumor promoters, such as epidermal growth factor (EGF) or 12- O -tetradecanoylphorbol-13-acetate (TPA), phosphorylation of RSK was increased within 5 min. Cell proliferation was suppressed in RSK2−/− mouse embryonic fibroblasts (MEFs) compared with RSK2+/+ MEFs. Moreover, RSK2−/− MEFs accumulated at the G1 phase of the cell cycle under normal cell culture conditions as well as after stimulation with EGF or TPA. In the anchorage-independent cell transformation assay (soft agar), stable expression of RSK2 in JB6 cells significantly enhanced colony formation in either the presence or absence of tumor promoters. Furthermore, knockdown of RSK2 with small interfering RNA-RSK2 suppressed constitutively active Ras (RasG12V)-induced foci formation in NIH3T3 cells. In addition, kaempferol, an inhibitor of RSK2, suppressed EGF-induced colony formation of JB6 Cl41 cells in soft agar, which was associated with inhibition of histone H3 phosphorylation (Ser10). These results showed that RSK2 is a key regulator for cell transformation induced by tumor promoters such as EGF and TPA. [Cancer Res 2007;67(17):8104–12]
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Ribosomal S6 Kinase 2 is a key regulator in tumor promoter induced cell transformation
Cancer Research, 2007Co-Authors: Yong Yeon Cho, Ke Yao, Ann M. Bode, Bong Seok Kang, Hong-gyum Kim, Duo Zheng, Zigang DongAbstract:The Ribosomal S6 Kinase 2 (RSK2), a member of the p90RSK (RSK) family of proteins, is a widely expressed serine/threonine Kinase that is activated by extracellular signal-regulated Kinase 1/2 and phosphoinositide-dependent Kinase 1 in response to many growth factors and peptide hormones. Its activation signaling enhances cell survival. However, the roles of RSK2 in cell transformation have not yet been elucidated. Here, we found that RSK2 is a critical serine/threonine Kinase for the regulation of cell transformation. When cells were stimulated with tumor promoters, such as epidermal growth factor (EGF) or 12- O -tetradecanoylphorbol-13-acetate (TPA), phosphorylation of RSK was increased within 5 min. Cell proliferation was suppressed in RSK2−/− mouse embryonic fibroblasts (MEFs) compared with RSK2+/+ MEFs. Moreover, RSK2−/− MEFs accumulated at the G1 phase of the cell cycle under normal cell culture conditions as well as after stimulation with EGF or TPA. In the anchorage-independent cell transformation assay (soft agar), stable expression of RSK2 in JB6 cells significantly enhanced colony formation in either the presence or absence of tumor promoters. Furthermore, knockdown of RSK2 with small interfering RNA-RSK2 suppressed constitutively active Ras (RasG12V)-induced foci formation in NIH3T3 cells. In addition, kaempferol, an inhibitor of RSK2, suppressed EGF-induced colony formation of JB6 Cl41 cells in soft agar, which was associated with inhibition of histone H3 phosphorylation (Ser10). These results showed that RSK2 is a key regulator for cell transformation induced by tumor promoters such as EGF and TPA. [Cancer Res 2007;67(17):8104–12]
Steven P. Gygi - One of the best experts on this subject based on the ideXlab platform.
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p90 Ribosomal S6 Kinase and p70 Ribosomal S6 Kinase Link Phosphorylation of the Eukaryotic Chaperonin Containing TCP-1 to Growth Factor, Insulin, and Nutrient Signaling
The Journal of biological chemistry, 2009Co-Authors: Yuki Abe, Sang-oh Yoon, Kazuishi Kubota, Michelle C. Mendoza, Steven P. Gygi, John BlenisAbstract:Chaperonin containing TCP-1 (CCT) is a large multisubunit complex that mediates protein folding in eukaryotic cells. CCT participates in the folding of newly synthesized polypeptides, including actin, tubulin, and several cell cycle regulators; therefore, CCT plays an important role in cytoskeletal organization and cell division. Here we identify the chaperonin CCT as a novel physiological substrate for p90 Ribosomal S6 Kinase (RSK) and p70 Ribosomal S6 Kinase (S6K). RSK phosphorylates the β subunit of CCT in response to tumor promoters or growth factors that activate the Ras-mitogen-activated protein Kinase (MAPK) pathway. CCTβ Ser-260 was identified as the RSK site by mass spectrometry and confirmed by site-directed mutagenesis. RSK-dependent Ser-260 phosphorylation was sensitive to the MEK inhibitor UO126 and the RSK inhibitor BID-1870. Insulin weakly activates RSK but strongly activates the phosphoinositide 3-Kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway and utilizes S6K to regulate CCTβ phosphorylation. Thus, the Ras-MAPK and PI3K-mTOR pathways converge on CCTβ Ser-260 phosphorylation in response to multiple agonists in various mammalian cells. We also show that RNA interference-mediated knockdown of endogenous CCTβ causes impaired cell proliferation that can be rescued with ectopically expressed murine CCTβ wild-type or phosphomimetic mutant S260D, but not the phosphorylation-deficient mutant S260A. Although the molecular mechanism of CCTβ regulation remains unclear, our findings demonstrate a link between oncogene and growth factor signaling and chaperonin CCT-mediated cellular activities.
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quantitative phosphorylation profiling of the erk p90 Ribosomal S6 Kinase signaling cassette and its targets the tuberous sclerosis tumor suppressors
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bryan A. Ballif, John Blenis, Philippe P. Roux, Scott A. Gerber, Jeffrey P. Mackeigan, Steven P. GygiAbstract:Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein Kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated Kinase (ERK)/p90 Ribosomal S6 Kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 Ribosomal S6 Kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological Kinase inhibitors we uncovered a ERK-independent, protein Kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.
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Quantitative phosphorylation profiling of the ERK/p90 Ribosomal S6 Kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bryan A. Ballif, John Blenis, Philippe P. Roux, Scott A. Gerber, Jeffrey P. Mackeigan, Steven P. GygiAbstract:Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein Kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated Kinase (ERK)/p90 Ribosomal S6 Kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 Ribosomal S6 Kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological Kinase inhibitors we uncovered a ERK-independent, protein Kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.
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Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 Ribosomal S6 Kinase
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Philippe P. Roux, Steven P. Gygi, Bryan A. Ballif, Rana Anjum, John BlenisAbstract:Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-Kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 Ribosomal S6 Kinase 1 (S6K1). The mitogen-activated protein Kinase (MAPK)-activated Kinase, p90 Ribosomal S6 Kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.
Bryan A. Ballif - One of the best experts on this subject based on the ideXlab platform.
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quantitative phosphorylation profiling of the erk p90 Ribosomal S6 Kinase signaling cassette and its targets the tuberous sclerosis tumor suppressors
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bryan A. Ballif, John Blenis, Philippe P. Roux, Scott A. Gerber, Jeffrey P. Mackeigan, Steven P. GygiAbstract:Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein Kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated Kinase (ERK)/p90 Ribosomal S6 Kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 Ribosomal S6 Kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological Kinase inhibitors we uncovered a ERK-independent, protein Kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.
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Quantitative phosphorylation profiling of the ERK/p90 Ribosomal S6 Kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bryan A. Ballif, John Blenis, Philippe P. Roux, Scott A. Gerber, Jeffrey P. Mackeigan, Steven P. GygiAbstract:Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein Kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated Kinase (ERK)/p90 Ribosomal S6 Kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 Ribosomal S6 Kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological Kinase inhibitors we uncovered a ERK-independent, protein Kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.
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Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 Ribosomal S6 Kinase
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Philippe P. Roux, Steven P. Gygi, Bryan A. Ballif, Rana Anjum, John BlenisAbstract:Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-Kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 Ribosomal S6 Kinase 1 (S6K1). The mitogen-activated protein Kinase (MAPK)-activated Kinase, p90 Ribosomal S6 Kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.
Philippe P. Roux - One of the best experts on this subject based on the ideXlab platform.
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quantitative phosphorylation profiling of the erk p90 Ribosomal S6 Kinase signaling cassette and its targets the tuberous sclerosis tumor suppressors
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bryan A. Ballif, John Blenis, Philippe P. Roux, Scott A. Gerber, Jeffrey P. Mackeigan, Steven P. GygiAbstract:Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein Kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated Kinase (ERK)/p90 Ribosomal S6 Kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 Ribosomal S6 Kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological Kinase inhibitors we uncovered a ERK-independent, protein Kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.
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Quantitative phosphorylation profiling of the ERK/p90 Ribosomal S6 Kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bryan A. Ballif, John Blenis, Philippe P. Roux, Scott A. Gerber, Jeffrey P. Mackeigan, Steven P. GygiAbstract:Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein Kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated Kinase (ERK)/p90 Ribosomal S6 Kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 Ribosomal S6 Kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological Kinase inhibitors we uncovered a ERK-independent, protein Kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.
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Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 Ribosomal S6 Kinase
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Philippe P. Roux, Steven P. Gygi, Bryan A. Ballif, Rana Anjum, John BlenisAbstract:Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-Kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 Ribosomal S6 Kinase 1 (S6K1). The mitogen-activated protein Kinase (MAPK)-activated Kinase, p90 Ribosomal S6 Kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.
