mTORC1‐ and mTORC2‐interacting proteins keep their multifunctional partners focused

Bracho-Valdés, Ismael; Moreno-Álvarez, Paola; Valencia-Martínez, Israel; Robles-Molina, Evelyn; Chávez-Vargas, Lydia; Vázquez‐Prado, José

doi:10.1002/iub.558

Cited by 69 publications

(81 citation statements)

References 204 publications

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“…Interestingly, chronic static and chronic dynamic cultures exhibited equal Ser2448 phosphorylation of mTOR, which is present in both mTORC1 and mTORC2 [83]. The elevated mTORC1 activity (S6K phospho-Thr389) in dynamic culture and elevated mTORC2 activity (Akt phospho-Ser473) [84] in static culture generally suggest that dynamic culture facilitates growth signaling, as opposed to survival signaling in static culture [85, 86]. The most important changes in mTORC1-related signaling by dynamic culture are summarized in Fig.…”

Section: Discussionmentioning

confidence: 99%

Dynamic culture yields engineered myocardium with near-adult functional output

2016

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Engineered cardiac tissues hold promise for cell therapy and drug development, but exhibit inadequate function and maturity. In this study, we sought to significantly improve the function and maturation of rat and human engineered cardiac tissues. We developed dynamic, free-floating culture conditions for engineering “cardiobundles”, 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes or human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in fibrin-based hydrogel. Compared to static culture, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (~2.1-fold), contractile force (~3.5-fold), and conduction velocity of action potentials (~1.4-fold). The average contractile force per cross-sectional area (59.7 mN/mm2) and conduction velocity (52.5 cm/sec) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions, which was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation and was blocked by rapamycin. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy. Optimized dynamic culture conditions also augmented function of human cardiobundles made reproducibly from cardiomyocytes derived from multiple hPSC lines, resulting in significantly increased contraction force (~2.5-fold) and conduction velocity (~1.4-fold). The average specific force of 23.2 mN/mm2 and conduction velocity of 25.8 cm/sec approached the functional metrics of adult human myocardium. In conclusion, we have developed a versatile methodology for engineering cardiac tissues with a near-adult functional output without the need for exogenous electrical or mechanical stimulation, and have identified mTOR signaling as an important mechanism for advancing tissue maturation and function in vitro.

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Section: Discussionmentioning

confidence: 99%

Dynamic culture yields engineered myocardium with near-adult functional output

2016

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“…One of the initial steps leading to the activation of Akt involves PI3K, which via phosphatidylinositol 3,4,5-trisphosphate (PIP3) recruits PDK1 and Akt to the cell membrane [9]. PIP3 has been shown to directly stimulate the kinase activity of mTORC2 [37].…”

Section: Discussionmentioning

confidence: 99%

“…mTORC1 is uniquely defined by the proteins raptor and PRAS40, while mTORC2 is identified by the proteins rictor, protor1/2 and mSin1 [8]. mTORC1 responds to growth factors and nutrients and phosphorylates the 4E-BPs and S6Ks [9]. The link between receptor activation and mTORC2 is poorly defined and has even been referred to as “the black box [10],” but it is clear that mTORC2 is responsible for the direct phosphorylation of Akt at S473, SGK1 and PKC-α [10,11].…”

Section: Introductionmentioning

confidence: 99%

mTORC2 mediates CXCL12-induced angiogenesis

Ziegler

Hatch

et al. 2016

Angiogenesis

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The chemokine CXCL12, through its receptor CXCR4, positively regulates angiogenesis by promoting endothelial cell (EC) migration and tube formation. However, the relevant downstream signaling pathways in EC have not been defined. Similarly, the upstream activators of mTORC2 signaling in EC are also poorly defined. Here we demonstrate for the first time that CXCL12 regulation of angiogenesis requires mTORC2 but not mTORC1. We find that CXCR4 signaling activates mTORC2 as indicated by phosphorylation of serine 473 on Akt, and does so through a G-protein- and PI3K-dependent pathway. Significantly, independent disruption of the mTOR complexes by drugs or multiple independent siRNAs reveals that mTORC2, but not mTORC1, is required for microvascular sprouting in a 3D in vitro angiogenesis model. Importantly, in a mouse model both tumor angiogenesis and tumor volume are significantly reduced only when mTORC2 is inhibited. Finally, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), which is a key regulator of glycolytic flux, is required for microvascular sprouting in vitro, and its expression is reduced in vivo when mTORC2 is targeted. Taken together, these findings identify mTORC2 as a critical signaling nexus downstream of CXCL12/CXCR4 that represents a potential link between mTORC2, metabolic regulation and angiogenesis.

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“…This RacGEF is activated by G␤␥ and phosphatidylinositol-3,4,5-trisphosphate (28 -32), by growth factor receptors, and regulators of neuronal morphology (33)(34)(35)(36) as well as by direct interaction with mTORC2, a fundamental multimeric kinase with affinity for P-Rex1 DEP domains (20,37) and by protein phosphatase 1␣, which dephosphorylates P-Rex1 at Ser-1165 (38). In endothelial cells, in which this guanine nucleotide exchange factor (GEF) is among the most highly expressed RhoGEFs (39), it influences changes in cell morphology in response to PDGF (28,38) and participates in the chemotactic response to sphingosine 1-phosphate and SDF-1 (23,27).…”

Section: Morphology Of Migrating Cells Is Regulated By Rho Gtpases Anmentioning

confidence: 99%

Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

Chávez-Vargas¹,

Adame-García

Cervantes‐Villagrana³

et al. 2016

Journal of Biological Chemistry

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Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation.PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RI␣, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cellderived factor 1). The P-Rex1 DEP 1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP 1 -PDZ 2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA.Rho guanine exchange factors (RhoGEFs) 4 are mechanistically linked to fundamental cellular processes, such as migration, adhesion, and morphogenesis. Based on their ability to integrate signaling inputs that result in the activation of Rho GTPases, RhoGEFs indirectly contribute to establish nucleation sites for actin polymerization, thus exerting a tight control on cytoskeleton dynamics (1, 2). RhoGEFs can also play a role as scaffolds of different signaling cascades, thus representing regulatory nodes for spatial and temporal control of signaling (3). These fundamental processes are fine-tuned by phosphorylation of RhoGEFs. However, the complex interplay between RhoGEFs and kinases is not completely understood; relevant examples include RhoGEFs known to be activated or inhibited by phosphorylation, some of them by PKA (4, 5).The ability of PKA to modulate the activity of RhoGEFs might be further facilitated by direct interactions that would also influence the subcellular localization of PKA and its accessibility to specific substrates. The paradigmatic example of this situation is AKAP-Lbc, which is a RhoGEF activated by G 12 -coupled receptors that promotes polymerization of actin filaments downstream of Rho and also interacts with multiple kinases regulating mitogenic inputs (6, 7). AKAP-Lbc ...

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mTORC1‐ and mTORC2‐interacting proteins keep their multifunctional partners focused

Cited by 69 publications

References 204 publications

Dynamic culture yields engineered myocardium with near-adult functional output

Dynamic culture yields engineered myocardium with near-adult functional output

mTORC2 mediates CXCL12-induced angiogenesis

Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

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