Pleckstrin Homology Domain Of Akt Research Articles

A non-canonical role of endothelin converting enzyme 1 (ECE1)in promoting lung cancer development via directly targetingprotein kinase B (AKT).

Lung cancer is the second most common malignancy in the world, and lung adenocarcinoma (LUAD) in particular is the leading cause of cancer death worldwide. Endothelin converting enzyme 1 (ECE1) is a membrane-bound metalloprotease involved in endothelin-1 (ET-1) processing and regulates vasoconstriction. However, very few studies have reported the involvement of ECE1 in regulating tumor cell proliferation, and the mechanism remains poorly understood. Therefore, we aimed to determine the role of ECE1 in lung cancer development. The Cancer Genome Atlas database and Kaplan-Meier plotter were used to assess the association between ECE1 and lung cancer. The expression of ECE1 was detected using immunohistochemistry staining and western blotting. A variety of in vitro assays were performed to evaluate the effects of ECE1 on the colony formation, proliferation, migration and invasion using ECE1 knockdown lung cancer cells. The gene expression profiles regulated by ECE1 were investigated by RNA sequencing. An immunoprecipitation assay and immunofluorescence assay were used to evaluate the mechanism underlying the regulatory effect of ECE1 on protein kinase B (AKT). The effect of ECE1 on tumor development was assessed by xenografted lung cancer cells in either C57BL/6 mice or nude mice. ECE1 was upregulated in LUAD and correlated with the poor prognosis of patients with LUAD. Functional studies showed that knockdown of ECE1 retarded the progression of tumors formed by lung cancer cells at least partly by inhibiting tumor cell proliferation. Moreover, ECE1 accelerated tumor cell proliferation through promoting AKT activation dispensable of its canonical target ET-1. Mechanically, ECE1 interacted with the pleckstrin homology (PH) domain of AKT and facilitated its translocation to the plasma membrane for activation. Furthermore, the inhibition of AKT activity counteracted the lung cancer cell growth inhibition observed both in vitro and in xenografts caused by ECE1 suppression. The present study reveals a non-canonical function of ECE1 in regulating AKT activation and cell proliferation, which provides the basis for the development of a novel strategy for the intervention of cancer including LUAD by abrogating ECE1-AKT signaling.

Glucuronic acid metabolites of phenolic acids target AKT-PH domain to improve glucose metabolism

ObjectivePhenolic acids widely exist in the human diet and exert beneficial effects such as improving glucose metabolism. It is not clear whether phenolic acids or their metabolites play a major role in vivo. In this study, caffeic acid (CA) and ferulic acid (FA), the two most ingested phenolic acids, and their glucuronic acid metabolites, caffeic-4′-O-glucuronide (CA4G) and ferulic-4′-O-glucuronide (FA4G), were investigated. MethodsThree insulin resistance models in vitro were established by using TNF-α, insulin and palmitic acid (PA) in HepG2 cells, respectively. We compared the effects of FA, FA4G, CA and CA4G on glucose metabolism in these models by measuring the glucose consumption levels. The potential targets and related pathways were predicted by network pharmacology. Fluorescence quenching measurement was used to analyze the binding between the compounds and the predicted target. To investigate the binding mode, molecular docking was performed. Then, we performed membrane recruitment assays of the AKT pleckstrin homology (PH) domain with the help of the PH-GFP plasmid. AKT enzymatic activity was determined to compare the effects between the metabolites with their parent compounds. Finally, the downstream signaling pathway of AKT was investigated by Western blot analysis. ResultsThe results showed that CA4G and FA4G were more potent than their parent compounds in increasing glucose consumption. AKT was predicted to be the key target of CA4G and FA4G by network pharmacology analysis. The fluorescence quenching test confirmed the more potent binding to AKT of the two metabolites compared to their parent compounds. The molecular docking results indicated that the carbonyl group in the glucuronic acid structure of CA4G and FA4G might bind to the PH domain of AKT at the key Arg-25 site. CA4G and FA4G inhibited the translocation of the AKT PH domain to the membrane, while increasing the activity of AKT. Western blot analysis demonstrated that the metabolites could increase the phosphorylation of AKT and downstream glycogen synthase kinase 3β in the AKT signaling pathway to increase glucose consumption. ConclusionIn conclusion, our results suggested that the metabolites of phenolic acids, which contain glucuronic acid, are the key active substances and that they activate AKT by targeting the PH domain, thus improving glucose metabolism.

White spot syndrome virus directly activates mTORC1 signaling to facilitate its replication via polymeric immunoglobulin receptor-mediated infection in shrimp.

Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus, which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture.

Dietary Flavone Baicalein Combinate with Genipin Attenuates Inflammation Stimulated by Lipopolysaccharide in RAW264.7 Cells or Pseudomonas aeruginosa in Mice via Regulating the Expression and Phosphorylation of AKT.

Mounting evidence has shown that single-targeted therapy might be inadequate to achieve satisfactory effects. Thus, drug combinations are gaining attention as they can regulate multiple targets to obtain more beneficial effects. Heat shock protein 90 (HSP90) is a molecular chaperone that assists the protein assembly and folding of client proteins and maintains their stability. Interfering with the interaction between HSP90 and its client proteins by inhibiting the latter’s activity may offer a new approach toward combination therapy. The HSP90 client protein AKT plays an important role in the inflammatory response syndrome caused by infections. In this study, the dietary flavone baicalein was identified as a novel inhibitor of HSP90 that targeted the N-terminal ATP binding pocket of HSP90 and hindered the chaperone cycle, resulting in AKT degradation. Combining baicalein with genipin, which was extracted from Gardenia jasminoides, could inhibit the pleckstrin homology domain of AKT, significantly increasing the anti-inflammatory effects both in vitro and in vivo. This synergistic effect was attributed to the reduction in AKT expression and phosphorylation. Thus, elucidating the mechanism underlying this effect will provide a new avenue for the clinical application and development of synergistic anti-inflammatory drugs.

The Pleckstrin Homology Domain of PLCδ1 Exhibits Complex Dissociation Properties at the Inner Leaflet of Plasma Membrane Sheets.

Using total internal reflection fluorescence microscopy, we followed the dissociation of GFP-tagged pleckstrin homology (PH) domains of AKT and PLCδ1 from the plasma membranes of rapidly unroofed cells. We found that the AKT-PH-GFP and PLCδ1-PH-GFP dissociation kinetics can be distinguished by their effective koff values of 0.39 ± 0.05 and 0.56 ± 0.16 s-1, respectively. Furthermore, we identified substantial rebinding events in measurements of PLCδ1-PH-GFP dissociation kinetics. By applying inositol triphosphate (IP3) to samples during the unroofing process, we measured a much larger koff of 1.54 ± 0.42 s-1 for PLCδ1-PH-GFP, indicating that rebinding events are significantly suppressed through competitive action by IP3 for the same PH domain binding site as phosphatidylinositol 4,5-bisphosphate (PIP2). We discuss the complex character of our PLCδ1-PH-GFP fluorescence decays in the context of membrane receptor and ligand theory to address the question of how free PIP2 levels modulate the interaction between membrane-associated proteins and the plasma membrane.

The GATOR2–mTORC2 axis mediates Sestrin2-induced AKT Ser/Thr kinase activation

Sestrins represent a family of stress-inducible proteins that prevent the progression of many age- and obesity-associated disorders. Endogenous Sestrins maintain insulin-dependent AKT Ser/Thr kinase (AKT) activation during high-fat diet-induced obesity, and overexpressed Sestrins activate AKT in various cell types, including liver and skeletal muscle cells. Although Sestrin-mediated AKT activation improves metabolic parameters, the mechanistic details underlying such improvement remain elusive. Here, we investigated how Sestrin2, the Sestrin homolog highly expressed in liver, induces strong AKT activation. We found that two known targets of Sestrin2, mTOR complex (mTORC) 1 and AMP-activated protein kinase, are not required for Sestrin2-induced AKT activation. Rather, phosphoinositol 3-kinase and mTORC2, kinases upstream of AKT, were essential for Sestrin2-induced AKT activation. Among these kinases, mTORC2 catalytic activity was strongly up-regulated upon Sestrin2 overexpression in an in vitro kinase assay, indicating that mTORC2 may represent the major link between Sestrin2 and AKT. As reported previously, Sestrin2 interacted with mTORC2; however, we found here that this interaction occurs indirectly through GATOR2, a pentameric protein complex that directly interacts with Sestrin2. Deleting or silencing WDR24 (WD repeat domain 24), the GATOR2 component essential for the Sestrin2-GATOR2 interaction, or WDR59, the GATOR2 component essential for the GATOR2-mTORC2 interaction, completely ablated Sestrin2-induced AKT activation. We also noted that Sestrin2 also directly binds to the pleckstrin homology domain of AKT and induces AKT translocation to the plasma membrane. These results uncover a signaling mechanism whereby Sestrin2 activates AKT through GATOR2 and mTORC2.

원형질막에서 약한 막결합부위와 PI(4,5)P₂결합에 의한 Membrane Blebbing 형성

Plasma membrane blebs are dynamic cellular cytoskeleton remodeling events involved in apoptosis and cell movement. Phosphoinositide (PI) is one of the phospholipid components within inner leaflet of the plasma membrane, but the contribution of PI on membrane blebbing remains elusive. Here, we found that if the phosphatidylinositol-4-phosphate (PI4P)-specific binding pleckstrin homology (PH) domain of various proteins including OSBP, OSH1, or OSH2 was combined with the N-terminal hydrophobic domain of Aplysia phosphodiesterase 4 short-form (S(N30)), membrane blebbing occurred on the plasma membrane. However, unexpectedly, this membrane blebbing was mediated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) binding rather than PI4P binding. To further elucidate this, we combined S(N30) with selective PI(4,5)P₂ binding PLCδ1 PH domain (S(N30)-GFP-PH(PLCδ1)), selective phosphatidylinositol 3,4,5-bisphosphate (PI(3,4,5)P₃) binding AKT1 PH domain (S(N30)-GFP-PH(AKT1)), or selective PS binding Lact-C2 domain (S(N30)-GFP-Lact-C2). Interestingly, overexpression of S(N30)-GFP-PH(PLCδ1) but not S(N30)-GFP-PH(AKT1) or S(N30)-GFP-Lact-C2 could strongly induce membrane blebbing. Our results clearly indicate that impairment of PI(4,5)P₂ binding in the plasma membrane is associated with cellular process of the plasma membrane blebbing.

Mcl-1 Interacts with Akt to Promote Lung Cancer Progression.

Mcl-1 is a unique antiapoptotic Bcl2 family protein that functions as a gatekeeper in manipulating apoptosis and survival in cancer cells. Akt is an oncogenic kinase that regulates multiple cellular functions and its activity is significantly elevated in human cancers. Here we discovered a cross-talk between Mcl-1 and Akt in promoting lung cancer cell growth. Depletion of endogenous Mcl-1 from human lung cancer cells using CRISPR/Cas9 or Mcl-1 shRNA significantly decreased Akt activity, leading to suppression of lung cancer cell growth in vitro and in xenografts. Mechanistically, Mcl-1 directly interacted via its PEST domain with Akt at the pleckstrin homology (PH) domain. It is known that the interactions between the PH domain and kinase domain (KD) are important for maintaining Akt in an inactive state. The binding of Mcl-1/PH domain disrupted intramolecular PH/KD interactions to activate Akt. Intriguingly, Mcl-1 expression correlated with Akt activity in tumor tissues from patients with non-small cell lung cancer. Using the Mcl-1-binding PH domain of Akt as a docking site, we identified a novel small molecule, PH-687, that directly targets the PH domain and disrupts Mcl-1/Akt binding, leading to suppression of Akt activity and growth inhibition of lung cancer in vitro and in vivo. By targeting the Mcl-1/Akt interaction, this mechanism-driven agent provides a highly attractive strategy for the treatment of lung cancer. SIGNIFICANCE: These findings indicate that targeting Mcl-1/Akt interaction by employing small molecules such as PH-687 represents a potentially new and effective strategy for cancer treatment.

A natural AKT inhibitor swertiamarin targets AKT-PH domain, inhibits downstream signaling, and alleviates inflammation.

Swertiamarin (SW), a representative component in Flos LoniceraeJaponicae, has been reported to exert significant activity in preventing infections. In this research, we aim to clarify the details of SW and its target to explore SW's underlying anti-inflammatory mechanisms. An azide labeled SW probe was synthesized for protein target fishing, and the results demonstrated that AKT could be captured specifically. Immunofluorescence colocalization with AKT was implemented by a click reaction of the SW probe and alkynyl CY5. The result showed that AKT was one of the targets of SW. Then, a competitive combination experiment using a set of AKT inhibitors and a membrane translocation experiment confirmed that SW might target the pleckstrin homology (PH) domain of AKT. This specific binding directly deactivated the phosphorylation of AKT on both Ser473 and Thr308, which induced the dephosphorylation of IKK and NF-κB. Finally, proinflammatory cytokines (TNF-α, IL-6, and IL-8) were suppressed both in cells and in acute lung injury animal model by targeting AKT-PH domain. This study demonstrated that SW functions as a natural AKT inhibitor and presents significant anti-inflammatory activity by directly regulating the AKT-PH domain and inhibiting downstream inflammatory molecules.

The Clinical and Functional Relevance of the Transforming AKT E17K Mutation in T-ALL

INTRODUCTION. The importance of IL-7R signaling has been thoroughly studied in the context of both normal T-cell development and leukemogenesis. Upon IL-7 induced heterodimerization of the IL-7Rα and the γc chain, downstream signaling routes regulate survival and proliferation of differentiating T-cells. JAK-STAT, MAPK-ERK and PI3K-AKT pathways are aberrantly activated in T-cell acute lymphoblastic leukemia (T-ALL) patients by recurrent gain-of-function mutations of the IL-7R signaling pathway. IL-7R signaling mutations are a result of mutations in IL7Ra, JAK1/3, PTPN11, NF1, STAT5B, N/KRAS, PI3K or AKT that are found in nearly 35% of pediatric T-ALL patients and are associated with steroid resistance and inferior event-free survival. Here we focus on downstream AKT activation in relation to cellular transformation and steroid resistance. We previously demonstrated that AKTE17K transforms IL3-dependent BaF3 cells in contrast to wild type AKT, while wild type AKT drives steroid resistance in SUP-T1 T-ALL cells in contrast to mutant AKTE17K. AIM. Our aim was to identify functional differences between AKT and AKTE17K molecules in respect to cellular transformation and steroid resistance. We assessed activation level, cellular localization and downstream signaling events that may explain these functional differences. RESULTS. For this study, we used doxycycline-inducible SUP-T1 cell lines that overexpress wild type AKT or mutant AKTE17K molecules. Using immunohistochemistry, we revealed abundant cytoplasmic localization of wild type AKT but predominant plasma membrane localization of AKTE17K and extends previous findings by others. Furthermore, we found evidence for phosphorylation of wild type AKT on serine-473 (S473) and threonine-308 (T308) residues in both cytoplasmic and nuclear fractions, indicating that part of induced AKT becomes activated. In contrast, expression of AKTE17K was accompanied with abundant phosphorylation of T308 while having relatively low levels of S473 phosphorylation. The T308/S473 phosphorylation ratio was 5 times higher for AKTE17K compared to wild type AKT. These results are in line with current views on AKT activation that S473 phosphorylation facilitates the opening of the AKT pleckstrin homology domain and membrane docking, after which AKT is phosphorylated (activated) on the T308 residue by PDK1. The constitutive plasma membrane localization and open conformation of AKTE17K as a result of its E17K pleckstrin homology domain mutation leaves it independent of S473 phosphorylation and results in enhanced T308 phosphorylation by PDK1. Consequently, AKTE17K mutant cells may exhibit reduced cellular sensitivity to allosteric AKT inhibitors but not to type I ATP-competitive inhibitors. Indeed, SUP-T1 cells expressing wild type AKT or mutant AKTE17K are equally sensitivity to the ATP-competitive AZD5363 inhibitor, but AKTE17K cells display reduced sensitivity to the MK2206 allosteric inhibitor compared to AKT wild type cells. In an attempt to explain enhanced steroid resistance by expression of wild type AKT but not mutant AKTE17K, we found no differences in AKT-mediated S134 phosphorylation of the NR3C1 steroid receptor or impaired nuclear import of NR3C1 following steroid treatment (Piovan et al., Cancer Cell 2013). This indicates that AKT induces steroid resistance by other mechanisms in our SUP-T1 cell system. CONCLUSIONS. In contrast to wild type AKT overexpression, we demonstrated that AKTE17K has (i) transforming activity, (ii) does not yield cellular resistance to steroid treatment, (iii) is predominantly localized at the plasma membrane, (iv) has high T308 over S473 phosphorylation and (v) is less sensitive to the MK2206 allosteric AKT-inhibitor. These observed differences are likely caused by the constitutively open AKTE17K conformation. Since no differences in NR3C1 phosphorylation or nuclear shuttling were observed in our model, we hypothesize other mechanisms underlie the provoked steroid resistance by AKT wild type overexpression. To identify these novel mechanisms, we are currently performing an unbiased phospho-protein array to identify differentially phosphorylated AKT substrates. Disclosures No relevant conflicts of interest to declare.

The flavonoid baicalin improves glucose metabolism by targeting the PH domain of AKT and activating AKT/GSK3β phosphorylation.

Baicalin is one of the main flavonoids of the dried root of Scutellariabaicalensis Georgi and is reported to exert beneficial effects on the regulation of glucose/lipid metabolism. However, understanding its specific target and unique mechanism for improving glucose utilization is a challenge. In this paper, target fishing with a baicalin probe reveals that baicalin interacts with AKT. An immunofluorescence assay further demonstrates the colocalization of baicalin with AKT in the cytoplasm. A competitive test and virtual docking show that baicalin might bind to the pleckstrin homology domain of AKT. This specific binding hampers AKT membrane translocation, activates the phosphorylation of AKT on Ser473, induces the downstream glycogen synthase kinase 3β activation, and affects glycogen synthesis.

Chlorogenic Acid Targeting of the AKT PH Domain Activates AKT/GSK3β/FOXO1 Signaling and Improves Glucose Metabolism.

Chlorogenic acid (CGA), a bioactive component in the human diet, is reported to exert beneficial effects on the regulation of glucose metabolism. This study was designed to investigate the specific target of CGA, and explore its underlying mechanisms. Beneficial effects of CGA in glucose metabolism were confirmed in insulin-treated human hepatocarcinoma HepG2 cells. Protein fishing, via CGA-modified functionalized magnetic microspheres, demonstrated the binding of CGA with protein kinase B (AKT). Immunofluorescence using a CGA molecular probe further demonstrated the co-localization of CGA with AKT. A competitive combination test and hampering of AKT membrane translocation showed that CGA might bind to the pleckstrin homology (PH) domain of AKT. The specific binding did not lead to the membrane translocation to phosphatidylinositol (3,4,5)-trisphosphate (PIP3), but directly activated the phosphorylation of AKT on Ser-473, induced the phosphorylation of the downstream molecules, glycogen synthase kinase 3β (GSK3β) and forkhead box O1 (FOXO1), and improved glucose metabolism. Collectively, our data demonstrate that CGA exerts regulatory effects on glucose metabolism via direct targeting the PH domain of AKT. This study clarifies the mechanism of the potential benefits of nutrients containing CGA in the complementary therapy of glucose metabolism disorders.

Regulation of AKT Activity by Inhibition of the Pleckstrin Homology Domain-PtdIns(3,4,5)P3 Interaction Using Flavonoids.

The serine-threonine kinase AKT plays a pivotal role in tumor progression and is frequently overactivated in cancer cells; this protein is therefore a critical therapeutic target for cancer intervention. We aimed to identify small molecule inhibitors of the pleckstrin homology (PH) domain of AKT to disrupt binding of phosphatidylinositol-3,4,5-trisphosphate (PIP3), thereby downregulating AKT activity. Liposome pulldown assays coupled with fluorescence spectrometry were used to screen flavonoids for inhibition of the AKT PH-PIP3 interaction. Western blotting was used to determine the effects of the inhibitors on AKT activation in cancer cells, and in silico docking was used for structural analysis and optimization of inhibitor structure. Several flavonoids showing up to 50% inhibition of the AKT PH-PIP3 interaction decreased the level of AKT activation at the cellular level. In addition, the modified flavonoid showed increased inhibitory effects and the approach would be applied to develop anticancer drug candidates. In this study, we provide a rationale for targeting the lipid-binding domain of AKT, rather than the catalytic kinase domain, in anticancer drug development.

DICER1 hot-spot mutations in ovarian gynandroblastoma.

Gynandroblastoma is a rare ovarian sex cord-stromal tumour characterised by the presence of both male (Sertoli and/or Leydig cells) and female (granulosa cells) components. We investigated the mutational status of DICER1, FOXL2 and AKT1 genes at hot-spot regions that are known to be the key driving events in the development of Sertoli-Leydig cell tumour (SLCT), adult granulosa cell tumour (aGCT) and juvenile granulosa cell tumour (jGCT), respectively, to gain insights into the molecular pathogenesis of gynandroblastoma. Sixteen cases of gynandroblastoma were studied. All contained SLCT or Sertoli cell tumour components. aGCT and jGCT components were identified in seven and 10 cases, respectively, with one presenting both components. Heterozygous hot-spot mutations in the RNase IIIb domain of DICER1 were discovered in three cases, including one case with heterologous mucinous elements, all of which were composed of moderately or poorly differentiated SLCT and jGCT components, and harboured the mutations in both histological components. None of the 16 cases displayed mutations at the p.C134W (c.402C→G) of FOXL2 or within the pleckstrin-homology domain of AKT1. All cases showed FOXL2 immunostaining in both male and female components. DICER1 hot-spot mutation is the key-driving event in a subset of gynandroblastomas containing components of SLCT and jGCT. Gynandroblastomas composed of SLCT and jGCT may represent morphological variants of SLCT. The molecular basis of gynandroblastoma containing a component of aGCT is different from pure aGCT.

Inhibitory receptor FcγRIIb mediates the effects of IgG on a phagosome acidification and a sequential dephosphorylation system comprising SHIPs and Inpp4a.

The relative abundance of phosphoinositide (PI) species on the phagosome membrane fluctuates over the course of phagocytosis. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 rapidly increase in the forming of the phagocytic cup, following which they disappear after sealing of the cup. In the present study, we monitored the clearance of these PI species using the enhanced green fluorescent protein-fused pleckstrin homology domain of Akt, a fluorescence probe that binds both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in Raw 264.7 macrophages. The clearance of PIs was much faster when the phagocytosed particles were coated with IgG. The effect of IgG was not observed in the macrophages deficient in FcγRIIb, an inhibitory IgG receptor. To identify the lipid phosphatases responsible for the FcγRIIb-accelerated PI clearance, we prepared a panel of lipid phosphatase-deficient cells. The lack of a PI 5-phosphatase Src homology 2 domain-containing inositol-5-phosphatase (SHIP)1 or SHIP2 impaired the FcγRIIb-accelerated clearance of PIs. The lack of a PI 4-phosphatase Inpp4a also impaired the accelerated PIs clearance. In the FcγRIIb- and Inpp4a-deficient cells, acidification of the formed phagosome was slowed. These results suggested that FcγRIIb drives the sequential dephosphorylation system comprising SHIPs and Inpp4a, and accelerates phagosome acidification.

Angiomotin-like 2 interacts with and negatively regulates AKT.

Angiomotin-like 2 (AMOTL2) is a member of Angiomotin family proteins, which are potent inhibitors for the Hippo pathway effector YAP. However, additional cellular functions of AMOTL2 besides YAP inhibition are largely unknown. Here, we reported that AMOTL2 associates with and negatively regulates AKT. Mechanistically, AMOTL2 binds to AKT pleckstrin homology domain and interrupts AKT's membrane localization. Liver-specific depletion of AMOTL2 enlarged mouse liver and activated both YAP and AKT. Downregulation of AMOTL2 is found in human liver cancers, correlating with the concomitant activation of YAP and AKT. Together, our results provide novel insights into a dual tumor suppressive function of AMOTL2 by targeting both YAP and AKT in liver size control and cancer prevention.

Kinetic Analyses on the Binding of the RhoGEF P‐Rex1 by Gβγ and Potential Small Molecule Inhibitors

Phosphatidylinositol‐3,4,5‐triphosphate (PIP3)‐dependent Rac exchanger (P‐Rex1) is a Rho guanine nucleotide exchange factor (GEF) for Rac GTP‐binding proteins integral to cytoskeletal rearrangement and cell migration. In cases of some metastatic tumors, elevated expression of P‐Rex1 accompanies cancer cell migration. Cell‐membrane associated PIP3 acts to regulate P‐Rex1 by binding a cleft located in the pleckstrin homology (PH) domain. Additional activation and membrane recruitment is provided by the βγ subunits of the heterotrimeric G protein. The position of this activity is still unclear; however, and locating the residues of interest will identify a means by which to modify P‐Rex1 signaling. The second part of our investigation in P‐Rex1 activity is to develop and test a small molecule inhibitor for the established PIP3 activation site on the PH domain. By examining these synergistic paths of P‐Rex1 activation, we hope to arrest cancer metastasis in a clinical setting through regulation of a key cell motility protein. We quantified the interaction between Gβγ and P‐Rex1 using bio‐layer interferometry (BLI). We produced truncated P‐Rex1 constructs for the selective deletion of domains to isolate the regions necessary for Gβγ binding and activation. Results from the truncation of P‐Rex1 after the N‐terminal domains demonstrated a weaker affinity for Gβγ compared to the full‐length protein. This result supports the formation of a tertiary structure involving N‐terminally located domains and their C‐terminally located counterparts. In this conformation, multiple domains of P‐Rex1 contribute to the promotion of enzyme activation by Gβγ. In addition to identifying the site of Gβγ binding, targeting the PIP3 binding site through small molecule inhibition will provide alternate means of P‐Rex1 regulation. High‐throughput screening by differential scanning fluorimetry (DSF) produced candidates to test for P‐Rex1 inhibition. The small molecules that demonstrated thermostabilization of the P‐Rex1 PH domain in DSF were further tested using BLI to determine binding kinetics. Preliminary binding data for these compounds provides affinities in agreement with DSF results. Further analysis counter‐screening with the PH domain of Akt, which also binds PIP3, will allow us to confirm binding to the PIP3 site while developing inhibitors to interact with the P‐Rex1 activation site discriminately. This result supports the affinity of our potential inhibitors for PIP3 binding sites, but suggests that an alteration to increase P‐Rex1 specificity may be necessary. The synergistic activation of P‐Rex1 via Gβγ and PIP3 provides two alternate pathways to the regulation of this cell motility protein. Locating the site for Gβγ binding will provide us a target for future modulation of P‐Rex1 signaling. By developing a small molecule inhibitor for the well‐characterized activation site of PIP3, we hope to halt excessive enzyme activation. Investigating these processes will not only extend our understanding of P‐Rex regulation, but may provide a means by which to manage cancer metastasis.Support or Funding InformationThis work was supported by an American Cancer Society – Michigan Cancer Research Fund Postdoctoral Fellowship (PF‐14‐224‐01‐DMC) to J.C.

The LINK-A lncRNA interacts with PtdIns(3,4,5)P3 tohyperactivate AKTand confer resistance to AKTinhibitors.

Phosphatidylinositol-3,4,5-trisphosphate (PIP3) mediates signaling pathways as a second messenger in response to extracellular signals. Although primordial functions of phospholipids and RNAs have been hypothesized in the “RNA world”, physiological RNA-phospholipid interactions and their involvement in essential cellular processes has remained a mystery. We explicate the contribution of lipid-binding long non-coding RNAs (lncRNAs) in cancer cells. Among them, Long Intergenic Noncoding RNA for Kinase Activation (LINK-A) directly interacts with AKT pleckstrin homology domain and PIP3 at the single nucleotide level, facilitating AKT-PIP3 interaction and consequent enzymatic activation. LINK-A-dependent AKT hyperactivation leads to tumorigenesis and resistance to AKT inhibitors. Genomic deletions of the LINK-A PIP3-binding motif dramatically sensitized breast cancer cells to AKT inhibitors. Furthermore, meta-analysis showed the correlation between LINK-A expression and incidence of a SNP (rs12095274: A>G), AKT phosphorylation status, and poor outcomes for breast and lung cancer patients. PIP3-binding lncRNA modulates AKT activation with broad clinical implications.

The Interplay between Calmodulin and Membrane Interactions with the Pleckstrin Homology Domain of Akt

The Akt protein, a serine/threonine kinase, plays important roles in cell survival, apoptosis, and oncogenes. Akt is translocated to the plasma membrane for activation. Akt-membrane binding is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). It has been shown that Akt activation in breast cancer cells is modulated by calmodulin (CaM). However, the molecular mechanism of the interplay between CaM and membrane binding is not established. Here, we employed nuclear magnetic resonance (NMR) and biochemical and biophysical techniques to characterize how PI(3,4,5)P3, CaM, and membrane mimetics (nanodisc) bind to Akt(PHD). We show that PI(3,4,5)P3 binding to Akt(PHD) displaces the C-terminal lobe of CaM but not the weakly binding N-terminal lobe. However, binding of a PI(3,4,5)P3-embedded membrane nanodisc to Akt(PHD) with a 103-fold tighter affinity than PI(3,4,5)P3 is able to completely displace CaM. We also show that Akt(PHD) binds to both layers of the nanodisc, indicating proper incorporation of PI(3,4,5)P3 on the nanodisc surface. No detectable binding has been observed between Akt(PHD) and PI(3,4,5)P3-free nanodiscs, demonstrating that PI(3,4,5)P3 is required for membrane binding, CaM displacement, and Akt activation. Using pancreatic cancer cells, we demonstrate that inhibition of Akt-CaM binding attenuated Akt activation. Our findings support a model by which CaM binds to Akt to facilitate its translocation to the membrane. Elucidation of the molecular details of the interplay between membrane and CaM binding to Akt may help in the development of potential targets to control the pathophysiological processes of cell survival.

Abrus Agglutinin, a type II ribosome inactivating protein inhibits Akt/PH domain to induce endoplasmic reticulum stress mediated autophagy-dependent cell death.

Abrus agglutinin (AGG), a type II ribosome-inactivating protein has been found to induce mitochondrial apoptosis. In the present study, we documented that AGG-mediated Akt dephosphorylation led to ER stress resulting the induction of autophagy-dependent cell death through the canonical pathway in cervical cancer cells. Inhibition of autophagic death with 3-methyladenine (3-MA) and siRNA of Beclin-1 and ATG5 increased AGG-induced apoptosis. Further, inhibiting apoptosis by Z-DEVD-FMK and N-acetyl cysteine (NAC) increased autophagic cell death after AGG treatment, suggesting that AGG simultaneously induced autophagic and apoptotic death in HeLa cells. Additionally, it observed that AGG-induced autophagic cell death in Bax knock down (Bax-KD) and 5-FU resistant HeLa cells, confirming as an alternate cell killing pathway to apoptosis. At the molecular level, AGG-induced ER stress in PERK dependent pathway and inhibition of ER stress by salubrinal, eIF2α phosphatase inhibitor as well as siPERK reduced autophagic death in the presence of AGG. Further, our in silico and colocalization study showed that AGG interacted with pleckstrin homology (PH) domain of Akt to suppress its phosphorylation and consequent downstream mTOR dephosphorylation in HeLa cells. We showed that Akt overexpression could not augment GRP78 expression and reduced autophagic cell death by AGG as compared to pcDNA control, indicating Akt modulation was the upstream signal during AGG's ER stress mediated autophagic cell death. In conclusion, we established that AGG stimulated cell death by autophagy might be used as an alternative tumor suppressor mechanism in human cervical cancer. © 2016 Wiley Periodicals, Inc.