Microtubule-associated Protein/Microtubule Affinity-regulating Kinase 4 (MARK4) Is a Negative Regulator of the Mammalian Target of Rapamycin Complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a central cell growth regulator. It resides in two protein complexes, which in mammals are referred to as mTORC1 and mTORC2. mTORC1, which is directly inhibited by rapamycin, promotes cell growth by stimulating protein synthesis and inhibiting autophagy. A wide range of extra and intracellular signals, including growth factors, nutrients, energy levels, and various stress conditions, regulates mTORC1. Dysregulation of mTORC1 contributes to many human diseases, including cancer, cardiovascular disease, autoimmunity, and metabolic disorder. In this study, we identified MARK4, an AMP-activated kinase-related kinase, as a negative regulator of mTORC1. In Drosophila S2 cells and mammalian cells, knockdown of MARK family member increased mTORC1 activity, whereas overexpression of MARK4 in mammalian cells significantly inhibited mTORC1 activity. Interestingly, MARK4 selectively inhibits mTORC1 activation by Rag GTPases, which are involved in amino acid signaling, but does not inhibit the effect of Rheb, which directly binds to and activates mTORC1. In addition, we found that MARK4 phosphorylates Raptor, a key component of mTORC1, and this phosphorylation may interfere with Raptor-Rag interaction. Our data demonstrate MARK4 as a new negative regulator of mTORC1.     

Phosphatidic acid activates mammalian target of rapamycin complex 1 (mTORC1) kinase by displacing FK506 binding protein 38 (FKBP38) and exerting an allosteric effect

Phosphatidic acid (PA) is a critical mediator of mitogenic activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, a master regulator of mammalian cell growth and proliferation. The mechanism by which PA activates mTORC1 signaling has remained unknown. Here, we report that PA selectively stimulates mTORC1 but not mTORC2 kinase activity in cells and in vitro. Furthermore, we show that PA competes with the mTORC1 inhibitor, FK506 binding protein 38 (FKBP38), for mTOR binding at a site encompassing the rapamycin-FKBP12 binding domain. This leads to PA antagonizing FKBP38 inhibition of mTORC1 kinase activity in vitro and rescuing mTORC1 signaling from FKBP38 in cells. Phospholipase D 1, a PA-generating enzyme that is an established upstream regulator of mTORC1, is found to negatively affect mTOR-FKBP38 interaction, confirming the role of endogenous PA in this regulation. Interestingly, removal of FKBP38 alone is insufficient to activate mTORC1 kinase and signaling, which require PA even when the FKBP38 level is drastically reduced by RNAi. In conclusion, we propose a dual mechanism for PA activation of mTORC1: PA displaces FKBP38 from mTOR and allosterically stimulates the catalytic activity of mTORC1.     

The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.     

An Atg13 protein-mediated self-association of the Atg1 protein kinase is important for the induction of autophagy

Autophagy pathways in eukaryotic cells mediate the turnover of a diverse set of cytoplasmic components, including damaged organelles and abnormal protein aggregates. Autophagy-mediated degradation is highly regulated, and defects in these pathways have been linked to a number of human disorders. The Atg1 protein kinase appears to be a key site of this control and is targeted by multiple signaling pathways to ensure the appropriate autophagic response to changing environmental conditions. Despite the importance of this kinase, relatively little is known about the molecular details of Atg1 activation. In this study we show that Atg13, an evolutionarily conserved regulator of Atg1, promotes the formation of a specific Atg1 self-interaction in the budding yeast, Saccharomyces cerevisiae. The appearance of this Atg1-Atg1 complex is correlated with the induction of autophagy, and conditions that disrupt this complex result in diminished levels of both autophagy and Atg1 kinase activity. Moreover, the addition of a heterologous dimerization domain to Atg1 resulted in elevated kinase activity both in vivo and in vitro. The formation of this complex appears to be an important prerequisite for the subsequent autophosphorylation of Thr-226 in the Atg1 activation loop. Previous work indicates that this modification is necessary and perhaps sufficient for Atg1 kinase activity. Interestingly, this Atg1 self-association does not require Atg17, suggesting that this second conserved regulator might activate Atg1 in a manner mechanistically distinct from that of Atg13. In all, this work suggests a model whereby this self-association stimulates the autophosphorylation of Atg1 within its activation loop.     

Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.     

Alterations at dispersed sites cause phosphorylation and activation of SNF1 protein kinase during growth on high glucose

The SNF1/AMP-activated protein kinases are central energy regulators in eukaryotes. SNF1 of Saccharomyces cerevisiae is inhibited during growth on high levels of glucose and is activated in response to glucose depletion and other stresses. Activation entails phosphorylation of Thr(210) on the activation loop of the catalytic subunit Snf1 by Snf1-activating kinases. We have used mutational analysis to identify Snf1 residues that are important for regulation. Alteration of Tyr(106) in the αC helix or Leu(198) adjacent to the Asp-Phe-Gly motif on the activation loop relieved glucose inhibition of phosphorylation, resulting in phosphorylation of Thr(210) during growth on high levels of glucose. Substitution of Arg for Gly(53), at the N terminus of the kinase domain, increased activation on both high and low glucose. Alteration of the ubiquitin-associated domain revealed a modest autoinhibitory effect. Previous studies identified alterations of the Gal83 (β) and Snf4 (γ) subunits that relieve glucose inhibition, and we have here identified a distinct set of Gal83 residues that are required. Together, these results indicate that alterations at dispersed sites within each subunit of SNF1 cause phosphorylation of the kinase during growth on high levels of glucose. These findings suggest that the conformation of the SNF1 complex is crucial to maintenance of the inactive state during growth on high glucose and that the default state for SNF1 is one in which Thr(210) is phosphorylated and the kinase is active.     

Distinct docking mechanisms mediate interactions between the Msg5 phosphatase and mating or cell integrity mitogen-activated protein kinases (MAPKs) in Saccharomyces cerevisiae

MAPK phosphatases (MKPs) are negative regulators of signaling pathways with distinct MAPK substrate specificities. For example, the yeast dual specificity phosphatase Msg5 dephosphorylates the Fus3 and Slt2 MAPKs operating in the mating and cell wall integrity pathways, respectively. Like other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains. These include D-motifs, which contain basic residues that interact with acidic residues in the common docking (CD) domain of MAPKs. Here we show that Msg5 interacts not only with Fus3, Kss1, and Slt2 but also with the pseudokinase Slt2 paralog Mlp1. Using yeast two-hybrid and in vitro interaction assays, we have identified distinct regions within the N-terminal domain of Msg5 that differentially bind either the MAPKs Fus3 and Kss1 or Slt2 and Mlp1. Whereas a canonical D-site within Msg5 mediates interaction with the CD domains of Fus3 and Kss1, a novel motif ((102)IYT(104)) within Msg5 is involved in binding to Slt2 and Mlp1. Furthermore, mutation of this site prevents the phosphorylation of Msg5 by Slt2. This motif is conserved in Sdp1, another MKP that dephosphorylates Slt2, as well as in Msg5 orthologs from other yeast species. A region spanning amino acids 274-373 within Slt2 and Mlp1 mediates binding to this Msg5 motif in a CD domain-independent manner. In contrast, Slt2 uses its CD domain to bind to its upstream activator Mkk1. This binding flexibility may allow MAPK pathways to exploit additional regulatory controls in order to provide fine modulation of both pathway activity and specificity.     

Calmodulin kinase II constitutively binds, phosphorylates, and inhibits brush border Na+/H+ exchanger 3 (NHE3) by a NHERF2 protein-dependent process

The epithelial brush border (BB) Na(+)/H(+) exchanger 3 (NHE3) accounts for most renal and intestinal Na(+) absorption. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibits NHE3 activity under basal conditions in intact intestine, acting in the BB, but the mechanism is unclear. We now demonstrate that in both PS120 fibroblasts and polarized Caco-2BBe cells expressing NHE3, CaMKII inhibits basal NHE3 activity, because the CaMKII-specific inhibitors KN-93 and KN-62 stimulate NHE3 activity. This inhibition requires NHERF2. CaMKIIγ associates with NHE3 between aa 586 and 605 in the NHE3 C terminus in a Ca(2+)-dependent manner, with less association when Ca(2+) is increased. CaMKII inhibits NHE3 by an effect on its turnover number, not changing surface expression. Back phosphorylation demonstrated that NHE3 is phosphorylated by CaMKII under basal conditions. This overall phosphorylation of NHE3 is not affected by the presence of NHERF2. Amino acids downstream of NHE3 aa 690 are required for CaMKII to inhibit basal NHE3 activity, and mutations of the three putative CaMKII phosphorylation sites downstream of aa 690 each prevented KN-93 stimulation of NHE3 activity. These studies demonstrate that CaMKIIγ is a novel NHE3-binding protein, and this association is reduced by elevated Ca(2+). CaMKII inhibits basal NHE3 activity associated with phosphorylation of NHE3 by effects requiring aa downstream of NHE3 aa 690 and of the CaMKII-binding site on NHE3. CaMKII binding to and phosphorylation of the NHE3 C terminus are parts of the physiologic regulation of NHE3 that occurs in fibroblasts as well as in the BB of an intestinal Na(+)-absorptive cell.     

Intestinal cell kinase (ICK) promotes activation of mTOR complex 1 (mTORC1) through phosphorylation of Raptor Thr-908

Intestinal cell kinase (ICK), named after its cloning origin, the intestine, is actually a ubiquitously expressed and highly conserved serine/threonine protein kinase. Recently we reported that ICK supports cell proliferation and G(1) cell cycle progression. ICK deficiency significantly disrupted the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling events. However, the biological substrates that mediate the downstream signaling effects of ICK in proliferation and the molecular mechanisms by which ICK interacts with mTORC1 are not well defined. Our prior studies also provided biochemical evidence that ICK interacts with the mTOR/Raptor complex in cells and phosphorylates Raptor in vitro. In this report, we investigated whether and how ICK targets Raptor to regulate the activity of mTORC1. Using the ICK substrate consensus sequence [R-P-X-S/T-P/A/T/S], we identified a putative phosphorylation site, RPGT908T, for ICK in human Raptor. By mass spectrometry and a phospho-specific antibody, we showed that Raptor Thr-908 is a novel in vivo phosphorylation site. ICK is able to phosphorylate Raptor Thr-908 both in vitro and in vivo and when Raptor exists in protein complexes with or without mTOR. Although expression of the Raptor T908A mutant did not affect the mTORC1 integrity, it markedly impaired the mTORC1 activation by insulin or by overexpression of the small GTP-binding protein RheB under nutrient starvation. Our findings demonstrate an important role for ICK in modulating the activity of mTORC1 through phosphorylation of Raptor Thr-908 and thus implicate a potential signaling mechanism by which ICK regulates cell proliferation and division.     

Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants

Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs.     

Regulation of protein kinase C-related protein kinase 2 (PRK2) by an intermolecular PRK2-PRK2 interaction mediated by Its N-terminal domain

Protein kinase C-related protein kinases (PRKs) are effectors of the Rho family of small GTPases and play a role in the development of diseases such as prostate cancer and hepatitis C. Here we examined the mechanism underlying the regulation of PRK2 by its N-terminal region. We show that the N-terminal region of PRK2 prevents the interaction with its upstream kinase, the 3-phosphoinositide-dependent kinase 1 (PDK1), which phosphorylates the activation loop of PRK2. We confirm that the N-terminal region directly inhibits the kinase activity of PRK2. However, in contrast to previous models, our data indicate that this inhibition is mediated in trans through an intermolecular PRK2-PRK2 interaction. Our results also suggest that amino acids 487-501, located in the linker region between the N-terminal domains and the catalytic domain, contribute to the PRK2-PRK2 dimer formation. This dimerization is further supported by other N-terminal domains. Additionally, we provide evidence that the region C-terminal to the catalytic domain intramolecularly activates PRK2. Finally, we discovered that the catalytic domain mediates a cross-talk between the inhibitory N-terminal region and the activating C-terminal region. The results presented here describe a novel mechanism of regulation among AGC kinases and offer new insights into potential approaches to pharmacologically regulate PRK2.     

Gcn1 and actin binding to Yih1: implications for activation of the eIF2 kinase GCN2

Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.     

Fission yeast receptor of activated C kinase (RACK1) ortholog Cpc2 regulates mitotic commitment through Wee1 kinase

In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G(1)/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G(2)/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G(1)/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.     

Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 μM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.     

Multiple site acetylation of Rictor stimulates mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt protein

The serine/threonine protein kinase Akt is a critical regulator of cell growth and survival in response to growth factors. A key step in Akt activation is phosphorylation at Ser-473 by the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). Although Rictor is required for the stability and activity of mTORC2, little is known about functional regions or post-translational modifications within Rictor that are responsible for regulating mTORC2. Here, we demonstrate that Rictor contains two distinct central regions critical for mTORC2 function. One we refer to as the stability region because it is critical for interaction with Sin1.1 and LST8, and a second adjacent region is required for multisite acetylation. p300-mediated acetylation of Rictor increases mTORC2 activity toward Akt, whereas site-directed mutants within the acetylation region of Rictor exhibit reduced insulin-like growth factor 1 (IGF-1)-stimulated mTORC2 kinase activity. Inhibition of deacetylases, including the NAD+-dependent sirtuins, promotes Rictor acetylation and IGF-1-mediated Akt phosphorylation. These results suggest that multiple-site acetylation of Rictor signals for increased activation of mTORC2, providing a critical link between nutrient-sensitive deacetylases and mTORC2 signaling to Akt.     

Raf kinase inhibitor protein (RKIP) dimer formation controls its target switch from Raf1 to G protein-coupled receptor kinase (GRK) 2

Proteins controlling cellular networks have evolved distinct mechanisms to ensure specificity in protein-protein interactions. Raf kinase inhibitor protein (RKIP) is a multifaceted kinase modulator, but it is not well understood how this small protein (21 kDa) can coordinate its diverse signaling functions. Raf1 and G protein-coupled receptor kinase (GRK) 2 are direct interaction partners of RKIP and thus provide the possibility to untangle the mechanism of its target specificity. Here, we identify RKIP dimer formation as an important mechanistic feature in the target switch from Raf1 to GRK2. Co-immunoprecipitation and cross-linking experiments revealed RKIP dimerization upon phosphorylation of RKIP at serine 153 utilizing purified proteins as well as in cells overexpressing RKIP. A functional phosphomimetic RKIP mutant had a high propensity for dimerization and reproduced the switch from Raf1 to GRK2. RKIP dimerization and GRK2 binding, but not Raf1 interaction, were prevented by a peptide comprising amino acids 127-146 of RKIP, which suggests that this region is critical for dimer formation. Furthermore, a dimeric RKIP mutant displayed a higher affinity to GRK2, but a lower affinity to Raf1. Functional analyses of phosphomimetic as well as dimeric RKIP demonstrated that enhanced dimerization of RKIP translates into decreased Raf1 and increased GRK2 inhibition. The detection of RKIP dimers in a complex with GRK2 in murine hearts implies their physiological relevance. These findings represent a novel mechanistic feature how RKIP can discriminate between its different interaction partners and thus advances our understanding how specific inhibition of kinases can be achieved.