scholarly journals A Rictor-Myo1c Complex Participates in Dynamic Cortical Actin Events in 3T3-L1 Adipocytes

2008 ◽  
Vol 28 (13) ◽  
pp. 4215-4226 ◽  
Author(s):  
G. Nana Hagan ◽  
Yenshou Lin ◽  
Mark A. Magnuson ◽  
Joseph Avruch ◽  
Michael P. Czech
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Molecular Motor ◽  
Regulatory Protein ◽  
Mammalian Target Of Rapamycin ◽  
Cortical Actin ◽  
Actin Remodeling ◽  
Membrane Ruffling ◽  
Akt Phosphorylation ◽  
Protein Kinase Akt

ABSTRACT Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the Rictor protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the Rictor-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.

scholarly journals Targeting TORC2 in multiple myeloma with a new mTOR kinase inhibitor

Blood ◽  
2010 ◽  
Vol 116 (22) ◽  
pp. 4560-4568 ◽  
Author(s):  
Bao Hoang ◽  
Patrick Frost ◽  
Yijiang Shi ◽  
Eileen Belanger ◽  
Angelica Benavides ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Kinase Inhibitor ◽  
Mammalian Target Of Rapamycin ◽  
Target Of Rapamycin ◽  
Phosphatase And Tensin Homolog ◽  
Akt Phosphorylation ◽  
Tensin Homolog ◽  
Complex Protein ◽  
Preclinical Work

Although preclinical work with rapalogs suggests potential in treatment of multiple myeloma (MM), they have been less successful clinically. These drugs allostearically inhibit the mammalian target of rapamycin kinase primarily curtailing activity of the target of rapamycin complex (TORC)1. To assess if the mammalian target of rapamycin within the TORC2 complex could be a better target in MM, we tested a new agent, pp242, which prevents activation of TORC2 as well as TORC1. Although comparable to rapamycin against phosphorylation of the TORC1 substrates p70S6kinase and 4E-BP-1, pp242 could also inhibit phosphorylation of AKT on serine 473, a TORC2 substrate, while rapamycin was ineffective. pp242 was also more effective than rapamycin in achieving cytoreduction and apoptosis in MM cells. In addition, pp242 was an effective agent against primary MM cells in vitro and growth of 8226 cells in mice. Knockdown of the TORC2 complex protein, rictor, was deleterious to MM cells further supporting TORC2 as the critical target for pp242. TORC2 activation was frequently identified in primary specimens by immunostaining for AKT phosphorylation on serine 473. Potential mechanisms of up-regulated TORC2 activity in MM were stimulation with interleukin-6 or insulin-like growth factor 1, and phosphatase and tensin homolog or RAS alterations. Combining pp242 with bortezomib led to synergistic anti-MM effects. These results support TORC2 as a therapeutic target in MM.

scholarly journals Localization of mTORC2 activity inside cells

2017 ◽  
Vol 216 (2) ◽  
pp. 343-353 ◽  
Author(s):  
Michael Ebner ◽  
Benjamin Sinkovics ◽  
Magdalena Szczygieł ◽  
Daniela Wolfschoon Ribeiro ◽  
Ivan Yudushkin
Keyword(s):  
Plasma Membrane ◽  
Growth Factors ◽  
Intracellular Localization ◽  
Mammalian Target Of Rapamycin ◽  
Pleckstrin Homology Domain ◽  
Growth And Survival ◽  
Membrane Recruitment ◽  
Akt Phosphorylation ◽  
Protein Kinase Akt ◽  
Cell Growth And Proliferation

Activation of protein kinase Akt via its direct phosphorylation by mammalian target of rapamycin (mTOR) complex 2 (mTORC2) couples extracellular growth and survival cues with pathways controlling cell growth and proliferation, yet how growth factors target the activity of mTORC2 toward Akt is unknown. In this study, we examine the localization of the obligate mTORC2 component, mSin1, inside cells and report the development of a reporter to examine intracellular localization and regulation by growth factors of the endogenous mTORC2 activity. Using a combination of imaging and biochemical approaches, we demonstrate that inside cells, mTORC2 activity localizes to the plasma membrane, mitochondria, and a subpopulation of endosomal vesicles. We show that unlike the endosomal pool, the activity and localization of mTORC2 via the Sin1 pleckstrin homology domain at the plasma membrane is PI3K and growth factor independent. Furthermore, we show that membrane recruitment is sufficient for Akt phosphorylation in response to growth factors. Our results indicate the existence of spatially separated mTORC2 populations with distinct sensitivity to PI3K inside cells and suggest that intracellular localization could contribute to regulation of mTORC2 activity toward Akt.

scholarly journals Small molecule H89 renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors

2020 ◽  
Vol 477 (10) ◽  
pp. 1847-1863
Author(s):  
Chase H. Melick ◽  
Jenna L. Jewell
Keyword(s):  
Kinase Activity ◽  
Mtor Inhibitor ◽  
Kinase Inhibitor ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Inhibitors ◽  
Short Term ◽  
Akt Phosphorylation ◽  
Evolutionarily Conserved ◽  
Multiple Cell ◽  
Mtor Complex 1

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr kinase that comprises two complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 phosphorylates S6K1 at Thr 389, whereas mTORC2 phosphorylates AKT at Ser 473 to promote cell growth. As the mTOR name implies it is the target of natural product called rapamycin, a clinically approved drug used to treat human disease. Short-term rapamycin treatment inhibits the kinase activity of mTORC1 but not mTORC2. However, the ATP-competitive catalytic mTOR inhibitor Torin1 was identified to inhibit the kinase activity of both mTORC1 and mTORC2. Here, we report that H89 (N-(2-(4-bromocinnamylamino) ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling.

Phosphorylation of cardiac regulatory proteins by cyclic AMP-dependent protein kinase

1976 ◽  
Vol 231 (5) ◽  
pp. 1330-1336 ◽  
Author(s):  
YS Reddy
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Close Association ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Cardiac myofibrils were purified from canine myocardium, and the regulatory proteins (troponin + tropomyosin) were extracted and shown to contain endogenous cyclic AMP-dependent protein kinase activity. Other cyclic nucleotide stimulated the protein kinase activity but only at higher concentrations. The enzyme was able to catalyze phosphorylation of conventional substrates such as histones and casein as well as a component of the regulatory protein fraction with a molecular weight of 28,000 daltons. Endogenous phosphorylation required the presence of Mg2+ and was inhibited by Ca2+. A protein kinase inhibitor obtained from skeletal muscle inhibited the cyclicAMP-dependent phosphorylation. Escherichia coli alkaline phosphatase dephosphorylated the endogenous substrates. The level of phosphorylation found is severalfold higher than we have previously reported. A protein kinase, with its close association with the regulatory proteins, seems to be well suited to transmitting the message from the cyclic AMP to the regulatory proteins, a phenomenon that may influence the cardiac contractility via the troponin phosphorylation. The inhibitory effect of troponin on actomyosin might be changed by its state of phosphorylation.

Putative Substrates of AKT in Multiple Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5013-5013
Author(s):  
Lee Ping Yew ◽  
Chih-Hao Chen ◽  
Bo-Feng Chu ◽  
Yen-Peng Ho ◽  
Jung-Hsin Hsu
Keyword(s):  
Mass Spectrometry ◽  
Multiple Myeloma ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Maldi Mass Spectrometry ◽  
Myeloma Cell ◽  
Serine Kinase ◽  
Myeloma Cells ◽  
Protein Kinase Akt

Abstract The serine/threonine protein kinase AKT (also known as protein kinase B) is thought to be a key mediator of signal transduction processes. The AKT kinase is activated in myeloma tumor cells. Frequent AKT activation in situ in myeloma cells of patient bone marrow has been demonstrated. IL-6 is a known growth factor of myeloma cells. Recent evidence indicates that AKT plays a critical role in IL-6-dependent expansion of multiple myeloma clones. A method to identify serine kinase substrates has been described. In that method, an antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method has been adopted in identifying putative substrates of AKT in mutiple myeloma cells. AF-10 cells of myeloma cell line were treated with IL-6 to activate AKT, and the putative AKT substrates were isolated by immunoprecipitation with PAS (phosphospecific Ser/Thr AKT substrate) antibody. The isolated proteins were identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In Western blot probed with PAS antibody, intensities of putative AKT substrates increased with IL-6 treatment, but decreased when the cells were preincubated with wortmannin (a specific PI 3-kinase inhibitor) then treated with IL-6. Putative AKT substrates identified include: nonmuscle myosine-9, vimentin, and tubulin beta-2. All these proteins contain appropriate motifs of AKT substrates (R/KXRXXS/T, XXRXXS/T). Conformation studies and studies of the physiological significances of these putative substrates are undergoing. Recently, it has been reported that a novel isocoumarin derivative induces mitotic phase arrest and apoptosis of human mutiple myeloma cells by affecting tubulin assembly.

scholarly journals AKT (protein kinase B) is implicated in meiotic maturation of porcine oocytes

Reproduction ◽  
2009 ◽  
Vol 138 (4) ◽  
pp. 645-654 ◽  
Author(s):  
Jaroslav Kalous ◽  
Michal Kubelka ◽  
Petr Šolc ◽  
Andrej Šušor ◽  
Jan Motlík
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinase B ◽  
Specific Inhibitor ◽  
Germinal Vesicle ◽  
Akt Phosphorylation ◽  
Meiotic Progression ◽  
Significant Acceleration ◽  
Protein Kinase Akt

The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) maturedin vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I- and II-stage (MI- and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 μM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage.

scholarly journals A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

PLoS ONE ◽  
2010 ◽  
Vol 5 (7) ◽  
pp. e11672 ◽  
Author(s):  
Beatrix Steer ◽  
Barbara Adler ◽  
Stipan Jonjic ◽  
James P. Stewart ◽  
Heiko Adler
Keyword(s):  
Protein Kinase ◽  
Regulatory Protein ◽  
Complement Regulatory Protein ◽  
Protein Kinase Akt

Early signalling events of autophagy

2013 ◽  
Vol 55 ◽  
pp. 1-15 ◽  
Author(s):  
Laura E. Gallagher ◽  
Edmond Y.W. Chan
Keyword(s):  
Protein Kinase ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Mammalian Cells ◽  
Mammalian Target Of Rapamycin ◽  
Interacting Protein ◽  
The Core ◽  
Autophagy Gene ◽  
Autophagy Induction ◽  
Cellular Pathways

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1–ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1–ULK1 signalling module.

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