Masayuki Noguchi

Identification of Akt Association and Oligomerization Domains of the Akt Kinase Coactivator TCL1

ABSTRACT Serine/threonine kinase Akt/protein kinase B, the cellular homologue of the transforming viral oncogene v-Akt, plays a central role in the regulation of cell survival and proliferation. We have previously demonstrated that the proto-oncogene TCL1 is an Akt kinase coactivator. TCL1 binds to Akt and mediates the formation of oligomeric TCL1-Akt high-molecular-weight protein complexes in vivo. Within these protein complexes, Akt is preferentially phosphorylated and activated. The MTCP1/TCL1/TCL1b oncogene activation is the hallmark of human T-cell prolymphocytic leukemia (T-PLL), a form of adult leukemia. In the present study, using a PCR-generated random TCL1 library combined with a yeast two-hybrid screening detecting loss of interaction, we identified D16 and I74 as amino acid residues mediating the association of TCL1 with Akt. Based on molecular modeling, we determined that the βC-sheet of TCL1 is essential for TCL1 homodimerization. Studies with mammalian overexpression systems demonstrated that both Akt association and oligomerization domains of TCL1 are distinct functional domains. In vitro kinase assays and overexpression experiments in mammalian cells demonstrated that both TCL1-Akt interaction and oligomerization of TCL1 were required for TCL1-induced Akt activation and substrate phosphorylation. Assays for mitochondrial permeability transition, nuclear translocation, and cell recovery demonstrated that both Akt association and homodimerization of TCL1 are similarly needed for the full function of TCL1 as an Akt kinase coactivator in vivo. The results demonstrate the structural basis of TCL1-induced activation of Akt, which causes human T-PLL.

Inhibition of Akt Kinase Activity by a Peptide Spanning the βA Strand of the Proto-oncogene TCL1

Akt plays a central role in the regulation of cellular anti-apoptosis underlying various human neoplastic diseases. We have demonstrated previously that TCL1 (a proto-oncogene underlying human T cell prolymphocytic leukemia) interacts with Akt and functions as an Akt kinase co-activator. With the aim to develop an Akt kinase inhibitor, we hypothesized that a peptide, which spans the Akt-binding site, binds to Akt and modulates Akt kinase activity and its downstream biological responses. Indeed, we demonstrated that a peptide, named “Akt-in” (Akt inhibitor, NH2-AVTDHPDRLWAWEKF-COOH, encompassing the βA strand of human TCL1), interacted with Akt and specifically inhibited its kinase activity. Nuclear magnetic resonance studies suggested that interaction of Akt-in with the pleckstrin homology domain (PH) of Akt caused conformational changes on the variable loop 1 of Akt, the locus mediating phosphoinositide binding. Consistently, interaction of Akt-in with the Akt PH domain prevented phosphoinositide binding and hence inhibited membrane translocation and activation of Akt. Moreover, Akt-in inhibited not only cellular proliferation and anti-apoptosis in vitro but also in vivo tumor growth without any adverse effect. The roles of Akt, which possesses a PH domain, in intracellular signaling were well established. Hence, Akt inhibitors create an attractive target for anticancer therapy. However, no effective inhibitors specific for Akt have been developed. Akt-in, which inhibits association of phosphatidylinositol with Akt, is the first molecule to demonstrate specific Akt kinase inhibition potency. This observation will facilitate the design of specific inhibitors for Akt, a core intracellular survival factor underlying various human neoplastic diseases.

The E3 Ligase TTC3 Facilitates Ubiquitination and Degradation of Phosphorylated Akt

The serine threonine kinase Akt is a core survival factor that underlies a variety of human diseases. Although regulatory phosphorylation and dephosphorylation have been well documented, the other posttranslational mechanisms that modulate Akt activity remain unclear. We show here that tetratricopeptide repeat domain 3 (TTC3) is an E3 ligase that interacts with Akt. TTC3 contains a canonical RING finger motif, a pair of tetratricopeptide motifs, a putative Akt phosphorylation site, and nuclear localization signals, and is encoded by a gene within the Down syndrome (DS) critical region on chromosome 21. TTC3 is an Akt-specific E3 ligase that binds to phosphorylated Akt and facilitates its ubiquitination and degradation within the nucleus. Moreover, DS cells exhibit elevated TTC3 expression, reduced phosphorylated Akt, and accumulation in the G(2)M phase, which can be reversed by TTC3 siRNA or Myr-Akt. Thus, interaction between TTC3 and Akt may contribute to the clinical symptoms of DS.

Proto-oncogene TCL1: more than just a coactivator for Akt

Serine threonine kinase Akt, also called PKB (protein kinase B), plays a central role in regulating intracellular survival. Deregulation of this Akt signaling pathway underlies various human neoplastic diseases. Recently, the proto‐oncogene TCL1 (T cell leukemia 1), with a previously unknown physiological function, was shown to interact with the Akt pleckstrin homology domain, enhancing Akt kinase activity; hence, it functions as an Akt kinase coactivator. In contrast to pathological conditions in which the TCL1 gene is highly activated in various human neoplasmic diseases, the physiological expression of TCL1 is tightly limited to early developmental cells as well as various developmental stages of immune cells. The NBRE (nerve growth factor‐responsive element) of the proximal TCL1 promoter sequences can regulate the restricted physiological expression of TCL1 in a negative feedback mechanism. Further, based on the NMR structural studies of Akt‐TCL1 protein complexes, an inhibitory peptide, “Akt‐in,” consisting of the βA strand of TCL1, has been identified and has therapeutic potential. This review article summarizes and discusses recent advances in the understanding of TCL1‐Akt functional interaction in order to clarify the biological action of the proto‐oncogene TCL1 family and the development avenues for a suppressive drug specific for Akt, a core intracellular survival regulator.—Noguchi, M., Ropars, V., Roumestand, C., Suizu, F. Proto‐oncogene TCL1: more than just a coactivator for Akt. FASEB J. 21, 2273–2284 (2007)

Structural Basis for the Co-activation of Protein Kinase B by T-cell Leukemia-1 (TCL1) Family Proto-oncoproteins

Chromosomal translocations leading to overexpression of p14TCL1 and its homologue p13MTCP1 are hallmarks of several human T-cell malignancies (1). p14TCL1/p13MTCP1 co-activate protein kinase B (PKB, also named Akt) by binding to its pleckstrin homology (PH) domain, suggesting that p14TCL1/p13MTCP1 induce T-cell leukemia by promoting anti-apoptotic signals via PKB (2, 3). Here we combined fluorescence anisotropy, NMR, and small angle x-ray-scattering measurements to determine the affinities, molecular interfaces, and low resolution structure of the complex formed between PKBβ-PH and p14TCL1/p13MTCP1. We show that p14TCL1/p13MTCP1 target PKB-PH at a site that has not yet been observed in PH-protein interactions. Located opposite the phospholipid binding pocket and distal from known protein-protein interaction sites on PH domains, the binding of dimeric TCL1 proteins to this site would allow the crosslinking of two PKB molecules at the cellular membrane in a preactivated conformation without disrupting certain PH-ligand interactions. Thus this interaction could serve to strengthen membrane association, promote trans-phosphorylation, hinder deactivation of PKB, and involve PKB in a multi-protein complex, explaining the array of known effects of TCL1. The binding sites on both proteins present attractive drug targets against leukemia caused by TCL1 proteins.

Identification of nerve growth factor-responsive element of the TCL1 promoter as a novel negative regulatory element

The serine/threonine kinase, Akt (protein kinase B) plays a central role in the regulation of intracellular cell survival. Recently, we demonstrated that the proto-oncogene TCL1, overexpressed in human T-cell prolymphocytic leukemia, is an Akt kinase co-activator. Tightly restricted TCL1 gene expression in early developmental cells suggested that the TCL1 gene is regulated at a transcriptional level. To characterize how TCL1 gene expression is regulated, we cloned the 5′-promoter of the TCL1 gene located at human chromosome 14q32. The 5′-TCL1 promoter region contains a TATA box with cis-regulatory elements for Nur77/NGFI-B (nerve growth factor-responsive element (NBRE), CCAAGGTCA), NFκB, and fork head transcription factor. Nur77/NGFI-B, an orphan receptor superfamily transcription factor implicated in T-cell apoptosis, is a substrate for Akt. We hypothesized that TCL1 transactivity is regulated through Akt-induced phosphorylation of Nur77/NGFI-B in vivo. In an electrophoretic mobility shift assay with chromosomal immunoprecipitation assays, wild-type Nur77, but not S350A mutant Nur77, could specifically bind to TCL1-NBRE. A luciferase assay demonstrated that TCL1-NBRE is required for inhibition of TCL1 transactivity upon nerve growth factor/platelet-derived growth factor stimulation, which activates Akt and phosphorylates Nur77. Using a chromosomal immunoprecipitation assay with reverse transcription-PCR, nerve growth factor stimulation inhibited binding of endogenous Nur77 to TCL1-NBRE, in turn, suppressing TCL1 gene expression. The results together establish that TCL1-NBRE is a novel negative regulatory element of Nur77 (NGFI-B). To the best of our knowledge, TCL1-NBRE is the first direct target of Nur77 involving the regulation of intracellular cell death survival. This Akt-induced inhibitory mechanism of TCL1 should play an important role in immunological and/or neuronal development in vivo.

The links between AKT and two intracellular proteolytic cascades: Ubiquitination and autophagy

The serine threonine kinase AKT plays a central role in the regulation of cell survival in a variety of human neoplastic diseases. A series of studies have revealed a connection between AKT signaling and two important protein degradation pathways in mammalian cells: the ubiquitin-proteasome system and autophagy. Two distinct ubiquitination systems have been reported to regulate AKT signaling: K63-linked ubiquitination, which promotes the oncogenic activation of AKT, and K48-linked ubiquitination, which triggers the proteasomal degradation of phosphorylated AKT. Autophagy is an evolutionarily conserved mechanism for the gross disposal and recycling of intracellular proteins in mammalian cells. AKT signaling may play a regulatory role in autophagy; however, the underlying mechanisms have not been fully clarified. Recently, AKT was shown to phosphorylate key molecules involved in the regulation of autophagy. Furthermore, lysosomal co-localization of the AKT-Phafin2 complex is reportedly critical for the induction of autophagy. In this review, we will discuss the connection between AKT, a core intracellular survival regulator, and two major intracellular proteolytic signaling pathways in mammalian cells.

The impact of microRNA-mediated PI3K/AKT signaling on epithelial-mesenchymal transition and cancer stemness in endometrial cancer

Activation of the PI3K/AKT pathway, a common mechanism in all subtypes of endometrial cancers (endometrioid and non-endometrioid tumors), has important roles in contributing to epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) features. MicroRNAs (miRNAs) are small non-coding RNA molecules that concurrently affect multiple target genes, and regulate a wide range of genes involved in modulating EMT and CSC properties. Here we overview the recent advances revealing the impact of miRNAs on EMT and CSC phenotypes in tumors including endometrial cancer via regulating PI3K/AKT pathway. MiRNAs are crucial mediators of EMT and CSC through targeting PTEN-PI3K-AKT-mTOR axis. In endometrial cancer cells, miRNAs can activate or attenuate EMT and CSC by targeting PTEN and other EMT-associated genes, such as Twist1, ZEB1 and BMI-1. More detailed studies of miRNAs will deepen our understanding of the molecular basis underlying PI3K/AKT-induced endometrial cancer initiation and progression. Targeting key signaling components of PI3K/AKT pathway by restoring or inhibiting miRNA function holds promise as a potential therapeutic approach to suppress EMT and CSC in endometrial cancer.

Characterization of the interaction of influenza virus NS1 with Akt.

Avian influenza viruses belong to the genus influenza A virus of the family Orthomyxoviridae. The influenza virus consists of eight segmented minus stranded RNA that encode 11 known proteins. Among the 11 viral proteins, NS1 (non-structural protein 1, encoded on segment 8) has been implicated in the regulation of several important intra-cellular functions. In this report, we investigated the functional interaction of NS1 with serine threonine kinase Akt, a core intra-cellular survival regulator. In co-immunoprecipitation assays and GST pull-down assays, NS1 directly interacted with Akt. The interaction was mediated primarily through the Akt-PH (Pleckstrin Homology) domain and the RNA-binding domain of NS1. NS1 preferentially interacted with phosphorylated Akt, but not with non-phosphorylated Akt. Functionally, the NS1-Akt interaction enhanced Akt activity both in the intra-cellular context and in in vitro Akt kinase assays. Confocal microscopic analysis revealed that phosphorylated Akt interacted with NS1 during the interphase of the cell cycle predominantly within the nucleus. Finally, mass spectrometric analysis demonstrated the position at Thr215 of NS1 protein is primary phosphorylation target site through Akt activation. The results together supported the functional importance of influenza virus NS1 with Akt, a core intra-cellular survival regulator.

Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3)P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.