IKK Interacts With Rictor And Regulates MTORC2

Background:The mammalian target of rapamycin (mTOR) is a conserved serine/threonine protein kinase that plays a central role in controlling cell growth, size and metaboli-sm. It elicits its pleiotropic functions in the context of two functionally distinct sign-aling complexes termed as mTOR complex1(mTORC1) and complex2(mTORC2). mTORC1, which contains mTOR, mLST8/GβL, Raptor, and PRAS40, is sensitive to immunosuppressive drug rapamycin.mTORC2shares mTOR and mLST8/GβL with mTORC1, but processes three unique components, rictor, mSinl, and PRR5/Protor. Despite presence of mTOR, mTORC2is not inhibited by acute treatment of rapam-ycin.mTORC1activity is regulated by an array of diverse intracellular and extracell-ular cues, including growth factors, nutrient conditions, energy levels and stresses. Upon receiving and integrating upstream signals, mTORCl in turn controls a wide range of growth-related cellular processes, including translation, transcription, autop-hagy and hypoxic adaptation.mTORC2is also activated by growth factors and thus phosphorylates its down substrate AKT at Ser473, while the mechanisms are not well characterized. Besides its functions of controlling AKT phosphorylation, mTORC2has been demonstrated to play key roles in various biological processes, including cell survival, metabolism, proliferation and cytoskeleton organization. Studies have suggested that mTORC2controlsthe actin cytoskeleton by promoting protein kinase C(PKCa) phosphorylation, phosphorylation of paxillin and its relocalization to focal adhesions, and the GTP loa-ding of RhoA and Racl,while the molecular mechanism by which mT0RC2regula-tes these processes has not been determined.IκB kinase (IKK) complex is a key component in the tumor necrosis factor (TNFα)/NFκB signaling pathway that is involved in systemic inflammation. It consists of three tightly associated subunits, IKKa, IKKβ and IKKy, of which IKKa and IKKβ serve as the catalytic subunits and IKKy is the regulatory subunit IKK is activated by proinflammatory cytokines, such as TNFa and lipopolysaccharide (LPS).Resent studies have shown that IKK could activate and regulate mTORC1signaling. LEE et al found that in response to TNFa treatment, IKKa interacts with TSC1and phosphorylates it at Ser487and Ser511. The phosphorylation represses the function of the TSC1/TSC2complex, resulting in mTORCl activation. In addition, the IKK directed phosphorylation has been found to correlate with TNFa induced VEGF production in multiple tumor types, indicating that TNFa promotes VEGF expression, angiogenesis through IKK dependent mTORCl activation. Moreover, studies from Baldwin’s group have revealed an association between IKKa and mTORCl (raptor) in PTEN deficient cancer cells. The association is regulated by Akt and is required for Akt stimulated mTORCl activation. Overexpression of IKKa has been found to enhance mTORCl kinase activity in vitro, suggesting that IKKa is a positive regulator of mTORCl. While there are no report about the relationship of IKK and mTORC2.Here, we report for the first time that IKK physically interacts with rictor (the unique component of mTORC2) and regulate mTORC2activity. IKK specific inhibi-tor Bay11-7082and siRNA suppresses mTORC2kinase activity in vitro and in vivo. Inhibited IKKaand IKKβ have a strong association with rictor, which results in a weaker interacts of rictor with mTOR. Moreover, suppressed IKK inhibits protein kinase C(PKCa) phosphorylation and affects cell actin cytoskeleton in NIH3T3cell line.Objectives:While there are no report about the relationship of IKK and mTORC2. In this study, we report for the first time that IKK physically interacts with rictor (the unique component of mTORC2) and regulate mTORC2activity in a variety of cell lines. Our study will provide rationales for targeting of IKK and mTORC2related disease pathogenesisMethods:A variety of cell lines were used in in vitro studies. To inhibit or activated IKK activity by IKK inhibitor Bay11-7082, IKK siRNA, kinase dead IKK plasmids or kinase IKK plasmids, the interaction of IKKα/β with rictor increases, while IKK could regulate mTORC2activity including phosphorylating AKT at serine473site and influencing actin cytoskeleton. And the mTOR-rictor association attenuates.Results:Whereas much is known about how mTORCl can be regulated by upstream signals, such as amino acids and growth factors, we are only beginning to understand how mTORC2activity can be triggered by these signals. In this assay, we demonstr-ate for the first time that IKK could interact with rictor (the unique component of mTORC2) and regulate mTORC2activity. In our coimmunoprecipitation experim-ents, not only endogenous but also exogenous overexpressed IKK a and IKK β phy-sically interact with endogenous or exogenous overexpressed rictor. In the next in vitro binding assays, rictor is divided into five segments at random, and we determine that IKKa and IKKp interact with rictor4(amino acids999-1397).To inhibit IKK activity by IKK inhibitor Bayl1-7082, IKK siRNA or kinase dead IKK plasmids, the interaction of IKKα/β with rictor increases, while the mTOR-rictor association atten-uates. We next find that IKK could regulate mTORC2activity including phosphory-lating AKT at serine473site and influencing actin cytoskeleton. Not only IKK speci-fic inhibitor but also IKK siRNA suppresses mTORC2activity of phosphorylating AKT(S473) obviously in diverse cell lines. Furthermore, IKK specific inhibitor Bay11-7082significantly inhibits mTORC2in vitro kinase activity in HEK293cell. We also find that IKK is required for insulin stimulated mTORC2activation in NIH3T3and MCF-7cell line. In NIH3T3cell line,P-PKCa(S657) is inhibited obviously by Bayl1-7082, and the cell actin cytoskeleton is affected when IKK activity is inhib-ited by IKK inhibitor, IKKa and IKKβ siRNA or KD IKKa and KD IKKβ We alsor found In HEK293cell line, P-rictor(T1135) was inhibited by Bayl1-7082in a time and dose dependent manner. In contrast, P-AKT(S473) was inhibited in a time and dose dependent manner, either, instead of stimulation by IKK specific inhibitor Bay11-7082. It suggests that P-AKT(S473) suppression by IKK specific inhibitor is not mediated by P-rictor(T1135).We conclude that IKK could regulate mTORC2func-tions including phosphorylating AKT(S473) and actin cytoskeleton. through inter-acting with rictor and affecting the stability of mTORC2.Suppressed IKK interacts intensely with rictor, which results in a loose interaction of rictor with mTOR, and thus inhibits mTORC2activity. Phosphorylation of rictor at Thr1135is not involved in IKK regulated mTORC2. 1IKK physically interacts with mTORC2IκBkinase(IKK) has been reported to regulate mTORCl activity by interacting with TSC1/2complex or raptor(unique component of mTORC1). while it is not known if IKK could associate with or regulate mTORC2.In our coimmunoprecipi-tation experiments, we find for the first time that IKKa and IKKP interact with rictor (component of mTORC2)in HEK293and MCF-7cell line.To further determine what exact domains or regions of rictor have an association with IKKa and IKKβ, five segments (amino acids1-398; amino acids335-733; amino acids667-1067; amino acids999-1397; amino acids1323-1708) of rictor named rictor1-5were amplified using PRK5-myc-Rictor as a template by the PCR method and cloned into the Escherichia coli GST-tagged expression vector p-GEX-6p1-GST (Amersham).In vitro binding assays, exogenous IKKa and IKKp interact with myc-rictor4(amino acids999-1397).These data show that IKKa and IKKp physically interact with rictor in the regions of amino acids999-1397.2IKK inhibitor suppresses mTORC2activityAs we find that IKK could interact with rictor, we next examined if IKK could affect the mTORC2activity. We find that specific IKK inhibitor Bay11-7082supp-ress the levels of P-AKT(S473) in a dose and time dependent manner in vivo in sev-eral cell lines such as NIH3T3,HEK293and HEK293T. Bay11-7082also inhibits mTORC2kinase activity in in vitro kinase assay. The results show that mT0RC2activity of phosphorylating P-AKT(S473) is suppressed by IKK inhibitor.3Inhibition of mTORC2by IKK siRNAIn order to further test the direct regulation of mT0RC2activity by IKK, IKKa and IKKβ specific siRNA were used. The results show that P-AKT(S473) is down-regulated by IKKa and IKKβ specific siRNA, which suppresses IKKa and IKKβ protein levels. 4IKK regulates PKC phosphorylation and actin cytoskeletonStudies have suggested that mTORC2controls the actin cytoskeleton by prom-oting protein kinase Ca(PKCa) phosphorylation.In this study, we examined if IKK could affect P-PKCa(S657)and the actin cytoskeleton under a confocal microscope. We found that P-PKCa(S657) were inhibited markedly by IKK specific inhibitor Bay11-7082and the cell actin altered obviously in NIH3T3cell line. In control cells, actin localizes to the cell cortex as well as diffusely throughout the cell cytoplasm. In contrast, in the cells with suppressed IKK activity by Bayll-7082,IKK specific siRNA, IKKa kinase-dead(IKK-a KD) and IKKp kinase-dead (IKK-βKD) vectors, thick actin fibers localizes to much of the cytoplasm and cortical actin is less promi-nent. These data show that IKK could regulate mTORC2in controlling actin cytos-keleton and PKC phosphorylation.5IKK is required for insulin-stimulated mTORC2mTORC2has been reported to be activated by growth factors,such as insulin. In order to determine whether insulin-stimulated mTORC2is IKK dependent, IKK spec-ific inhibitor Bay11-7082was used in NIH3T3and MCF-7cell line. We found that activated P-AKT(S473) by insulin was suppressed by Bay11-7082throughly.6IKK regulates association of rictor with mTORRictor is one of the unique components of mTORC2, and the association of rictor with mTOR is critical for mTORC2kinase activity. In order to explore how IKK regulates mTORC2activity,we first examined if IKK could affect the associ-ation of rictor with mTOR. The results show that the interactions of rictor with mTOR are attenuated, when IKK activity is inhibited by Bayl1-7082or IKKa/p KD vectors, while suppressed IKK has a stronger association with rictor. The association of mTOR with raptor doesn’t change, but suppressed IKK has a weaker interaction with raptor. There has been suggested that mTORC2activity could be negatively regulated via rictor phosphorylation at Thr1135site. We then detected if IKK influences P-rictor(T1135).In HEK293cell line, P-rictor(T1135) was inhibited by Bay11-7082in a time and dose dependent manner.In contrast, P-AKT(S473) was inhibited in a time and dose dependent manner, either, instead of stimulation by IKK specific inhibitor Bay11-7082. It suggests that P-AKT(S473) suppression by IKK specific inhibitor is not mediated by P-rictor(T1135).mTOR can be directly regulated via phosphorylation. Ser2481is an autophosp-horylation site. More recently, phosphorylation of this site was demonstrated to be a marker for intact mTORC2. Phosphorylation at Ser2448is mediated by p70riboso-mal S6kinase (S6K) and occurs predominantly to mTOR in mTORC1.In this study, both P-mTOR(S2481) and P-mTOR(S2448) were suppressed by Bay11-7082. P-mTOR(S2481) suppression indicates that mTORC2assembly is inhibited by IKK specific inhibitor Bay11-7082. P-mTOR(S2448) suppression by IKK inhibitor is consistent with P-S6K suppression by IKK inhibitor in other studies.Conclusion:We conclude that IKK could regulate mTORC2functions including phosphor-ylating AKT(S473) and actin cytoskeleton. through interacting with rictor and affe-cting the stability of mTORC2.Suppressed IKK interacts intensely with rictor, which results in a loose interaction of rictor with mTOR, and thus inhibits mTORC2activity. Phosphorylation of rictor at Thr1135is not involved in IKK regulated mTORC2.