KR20120112196A - Use of hades as a negative regulator of akt - Google Patents

Abstract

PURPOSE: An anticancer agent containing Hades as a Akt negative regulator is provided to suppress proliferation and presence of cancer cells. CONSTITUTION: An Akt(protein kinase B) negative regulator contains Hades protein with an amino acid of sequence number 2 or a gene encoding the same as an active ingredient. The gene encoding Hades contains a base of sequence number 1. The gene is inserted into a recombinant expression vector. Hades contains RING finger-domain and has E3-ubiquitin ligase activity. An anticancer agent contains Hades protein or the gene encoding the same as an active ingredient. The anticancer agent functions as an Akt(protein kinase B) negative regulator.

Description

Use of Hades as a negative regulator of Akt

The present invention relates to the use of Hades as an Akt negative regulator, and more particularly, to an Akt (protein kinase B) negative regulator or anticancer agent containing a Hades protein or a gene encoding the same as an active ingredient.

Akt (protein kinase B), a serine / threonine kinase, is involved in response to essential cellular stimuli such as growth factors and insulin [1, 2]. Akt activation regulates a variety of biological responses, including cell proliferation, protein synthesis, cell growth, cell cycle progression, and inhibition of apoptosis [3]. Akt is activated through platelet-derived growth factor, insulin, epidermal growth factor, basic fibroblast growth factor, and insulin-like growth factor I [2, 4, 5]. Downstream substrates of Akt have been identified at the cellular and molecular level. Glycogen synthase kinase 3 (GSK3), the first identified substrate of Akt, is negatively regulated through direct phosphorylation by Akt [6]. When Akt inhibits GSK3 (especially GSK3β) activity, it induces β-catenin stabilization, nuclear localization and gene activation [7-9].

Several modulators of Akt activation were identified and provided important information on the Akt regulatory signaling pathway. Phosphatidilinositol 3-kinase (PI3K) and phosphatase and tensine homologs (PTEN) are regulators of Akt [10]. PI3K induces Akt phosphorylation / activation whereas PTEN induces Akt phosphorylation through phosphorylation of phosphatidylinositol-3,4,5-triphosphate produced from phosphatidylinositol-4,5-bisphosphate by PI3K , And [2, 4, 11]. Negative regulators such as C1 domain-containing phosphatase and tensin homologs, carboxy-terminal regulatory proteins, Trb3, and Keratin K10 have also been reported to inactivate Akt [12-15].

The proteolytic activity of the ubiquitin-proteasome system (UPS) is important in many cellular processes [16]. The formation of polyubiquitin-protein conjugates recognized and destroyed by the 26S proteasome involves three components that participate in the cascade of the ubiquitin transfer reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) And specificity factor (E3) called ubiquitin ligase. E3 ligase controls the specificity of target protein selection and controls the abundance of individual target proteins [16]. Although the molecular pathways and physiological roles of Akt ubiquitination have been recently studied [17-22], much of Akt ubiquitination remains unclear.

Here we identified Hades as a new E3 ligase of Akt. That is, the inventors have identified Hades as a new negative regulator of Akt that functions in a variety of cellular processes, including cell proliferation, survival and tumor development, and completed the present invention.

It is therefore a primary object of the present invention to provide the use of Hades as an Akt negative modulator.

Another object of the present invention is to provide an Akt negative regulator containing a Hades protein or a gene encoding the same as an active ingredient.

Another object of the present invention is to provide an anticancer agent containing a Hades protein or a gene encoding the same as an active ingredient.

Another object of the present invention is to provide a method for screening an Akt negative or positive modulator using the interaction of Akt and Hades.

According to an aspect of the present invention, the present invention provides an Akt (protein kinase B) negative modulator containing a Hades protein having the amino acid sequence of SEQ ID NO: 2 or a gene encoding the same as an active ingredient.

The Hades protein was identified among cDNA library proteins capable of interacting with Akt and named after Greek subordinates. Hades protein has been deposited with UniProtKB accession number Q969V5 and has the amino acid sequence of SEQ ID NO: 2. The Akt (protein kinase B) is a serine / threonine kinase known to be involved in various biological processes including cell proliferation, cell growth, cell cycle progression and inhibition of apoptosis. In particular, Akt1 is known to be involved in cell survival pathways by inhibiting apoptosis, and Akt2 is known as an important signaling molecule in the insulin signaling pathway. Hades of the present invention have been found to effectively interact with both Akt1 and Akt2. Here, a negative regulator refers to a regulator that inhibits the expression of Akt or inhibits downstream signaling pathways of Akt.

In the present invention, the gene encoding Hades may have any nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 2, preferably Akt (protein kinase) having a nucleotide sequence of SEQ ID NO: 1 B) providing negative modulators.

As used herein, the term “gene” refers to a nucleic acid (eg, DNA, RNA) sequence that includes a coding sequence necessary to produce a protein or polypeptide. A protein or polypeptide may be encoded by a full length coding sequence or by a portion of the coding sequence as long as the desired active or functional properties are maintained. The term also encompasses coding regions of structural genes and sequences located adjacent to coding regions of the 5 'and 3' ends at distances of at least about 1 kb from the ends such that the gene corresponds to the length of a full-length mRNA. . Sequences located 5 ′ of the coding region and present in the mRNA are referred to as 5′-nontranslated sequences. Sequences located 3 'or below the coding region and present in the mRNA are referred to as 3'-untranslated sequences. The term "gene" includes both cDNA and genomic forms of a gene. Genomic forms or clones of genes include coding regions that are interrupted by non-coding sequences called "introns" or "insert regions" or "insert sequences". Introns are segments of genes that are transcribed into nuclear RNA (hnRNA); Introns can include regulatory elements, eg, enhancers. Introns are removed or “spliced” from the nucleus or initial transcript; Thus, introns are not present in mRN transcripts. The mRNA functions during translation to specify the sequence or sequence of amino acids of the initial polypeptide.

Gene expression converts genetic information encoded in a gene into RNA (e.g., mRNA rRNA, tRNA or snRNA) through transcription of the gene (e.g., by enzymatic action of RNA polymerase) and In this regard, the process of converting into protein via "detoxification" of mRNA is mentioned. Gene expression can be regulated at many stages of the process; "Up-regulation" or "activation refers to regulation that increases production of gene expression products, and" down-regulation "or" inhibition "refers to regulation that reduces product. The resulting molecule (eg, transcription factor) is often referred to as an “activator” and an “inhibitor,” respectively. In addition to including an intron, the genomic form of the gene is also the 5 'and 3' ends of the sequence present in the RNA transcript. May be referred to as “flanking” sequences or regions (these flanking sequences may be referred to as 5 ′ or 3 ′ for non-translated sequences present in RNA transcripts). The 5 'flanking region may comprise regulatory sequences, for example, promoters and enhancers that regulate or influence the transcription of the gene The 3' flanking region may terminate transcription, post-transcriptional cleavage, and polya Contains sequences that direct denylation Can be included.

In the present invention, the Hades gene provides an Akt (protein kinase B) negative regulator, characterized in that inserted in the recombinant expression vector. In this case, the Hades gene is operably linked with the regulatory elements of the recombinant expression vector.

As used herein, the term "vector" is used for nucleic acid molecules that deliver DNA fragments from one cell to another. Vectors are often derived from plasmids, bacteriophages or plant or animal viruses.

As used herein, the term “expression vector” refers to a recombinant DNA comprising a suitable nucleic acid sequence necessary for the expression of the coding sequence of interest and the coding sequence operably linked in a particular host organism. Nucleic acid sequences required for expression in prokaryotic cells generally, along with other sequences, include promoters, operators (optional), and ribosomal binding sites. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

As used herein, the term "recombinant expression vector" is a vector capable of expressing a protein of interest in the appropriate host cell, in the present invention, a Hades protein, a genetic construct comprising an essential regulatory element operably linked to express a gene insert. Say the offering.

As used herein, the term “operably linked” refers to a functional link between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence. Operational linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.

Suitable expression vectors include promoters, initiation codons, termination codons, expression control elements such as polyadenylation signals and enhancers, and the like, and can be prepared in various ways depending on the purpose. The start codon and the stop codon must be functional in the subject when the gene construct is administered and must be in frame with the coding sequence.

In the present invention, Hades provides an Akt (protein kinase B) negative modulator, characterized in that it has a RING finger-domain and has E3-ubiquitin ligase activity. In the examples of the present invention, it was demonstrated that Hades has RING finger-domain and has E3-ubiquitin ligase activity.

In the present invention, the Akt provides an Akt (protein kinase B) negative modulator, characterized in that it directly interacts with Hades and is ubiquitinated by Hades. Embodiments of the present invention demonstrated that Akt interacts directly with Hades and is ubiquitinate by Hades.

In the present invention, it provides an Akt (protein kinase B) negative modulator, characterized in that the kinase domain (KD) of the Akt interacts mainly in association with Hades. In the examples of the present invention it was demonstrated that the kinase domain (KD) of the Akt interacts mainly in association with Hades.

In the present invention, Akt (protein kinase B) negative regulator is characterized in that Akt is decomposed by ubiquitination by Hades to inhibit Akt expression. In the examples of the present invention, it was demonstrated that Akt was decomposed by ubiquitination by Hades to inhibit Akt expression.

In the present invention, Hades provides an Akt (protein kinase B) negative modulator, characterized in that it specifically ubiquitizes only Akt activated by phosphorylation. In the embodiment of the present invention, it was demonstrated that Hades specifically ubiquitizes only Akt activated by phosphorylation.

In the present invention, Akt (protein kinase B) negative modulator, characterized in that the lysine 284 of Akt is a major site of ubiquitination by Hades. Example of the present invention demonstrated that the lysine 284 of Akt is a major site of ubiquitination by Hades.

In the present invention, the Akt is an Akt (protein kinase B) negative regulator, characterized by inducing cell proliferation, inhibition of apoptosis and cell migration by downstream signaling. To provide. It is well known in the prior art that Akt induces cell proliferation, inhibition of apoptosis and cell migration by downstream signaling.

In the present invention, Hades provides an Akt (protein kinase B) negative modulator, characterized in that it inhibits the proliferation, survival and cell migration of cancer cells. Examples of the present invention demonstrated that Hades inhibits the proliferation, survival and cell migration of cancer cells by negatively regulating downstream signaling of Akt. In the examples of the present invention, the effects of the present invention were tested on HeLa cells as cervical cancer cells and HCT116 as colon cancer cells as typical examples of cancer cells.

According to another aspect of the present invention, the present invention is characterized in that it functions as an Akt (protein kinase B) negative regulator, containing a Hades protein having the amino acid sequence of SEQ ID NO: 2 or a gene encoding the same as an active ingredient. To provide an anticancer agent. Examples of the present invention demonstrated that Hades inhibits the proliferation, survival and cell migration of cancer cells by negatively regulating downstream signaling of Akt. In the examples of the present invention, the effects of the present invention were tested on HeLa cells as cervical cancer cells and HCT116 as colon cancer cells as typical examples of cancer cells.

In addition, preferably, the anticancer agent of the present invention may further include one or more pharmaceutically acceptable carriers or excipients (see Remington's Pharmaceutical Sciences and US Pharmacopoeia. 1984, Mack Publishing Company, Easton, PA, USA).

As used herein, "pharmaceutically acceptable" generally means suitable for administration to a mammal, including a human, from a toxicity or stability standpoint. Carriers include diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, surfactants, absorbent carriers, brighteners commonly used in the pharmaceutical art. If necessary, flavors, sweeteners and the like may be added. The carrier may also contain other pharmaceutically acceptable excipients to modify other conditions such as pH, osmotic pressure, viscosity, sterility, lipidity, solubility and the like. Pharmaceutically acceptable excipients which allow sustained or delayed release after administration may also be included.

Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like. In addition, if desired, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, triethanolamine sodium acetate, ≪ / RTI >

The anticancer agent compositions of the present invention may be infused via a route of administration, eg, by systemic or oral route. Preferred forms of systemic administration include injection, generally intravenous injection. Other injection routes may be used, for example, subcutaneously, intramuscularly or intraperitoneally. Alternative means of systemic administration include transmucosal and transdermal administration using penetrants such as bile acids or fusidic acids or other surfactants. Moreover, the anticancer composition of this invention can also be formulated with an enteric agent, a capsolization agent, or an oral administration agent. Administration of these anticancer agents may be local and / or partial in the form of ointments, pastes, gels and the like.

In embodiments in which the anticancer agent composition of the present invention comprises a nucleic acid, the nucleic acid may be included in a vector. For example, the vector can be a viral vector. In certain embodiments, the viral vector is an adenovirus vector. In some embodiments, the adenovirus is formulated with protamine. Any number of viral particles can be administered to the patient. In certain embodiments, about 10 ⁸ to about 10 ¹⁴ viral particles are administered to the patient per administration.

In some embodiments of the invention in which the nucleic acid composition is administered, the nucleic acid composition may comprise one or more lipids. Any of the lipids described above can be included in such lipid-nucleic acid compositions. Examples of such lipids include DOTAP and cholesterol, or derivatives thereof.

As used herein, the term "therapeutically effective amount" means the amount of active agent, such as the Hades protein of the present invention, that when administered according to a desired therapeutic regimen, produces a desired therapeutic effect or response or provides a desired benefit. The amount of composition actually administered may be determined by the physician, based on the severity of the condition, the composition administered, age, weight, and the response of each patient, the chosen route of administration, to the physician, but may be determined by the physician as a Hades protein. In this case, the pharmaceutically or prophylactically effective amount to be administered to the cancer patient is preferably about 0.01 to 10 mg / kg / day.

According to another aspect of the present invention, the present invention provides a method for screening an Akt (protein kinase B) negative modulator comprising the following steps: (a) Cotransfection of Akt and Hades to an animal cell; ); (b) treating any of the agents on the transfected animal cells; And (c) determining whether the interaction of Akt and Hades is promoted in the animal cell.

According to another aspect of the present invention, the present invention provides a method for screening Akt (protein kinase B) positive modulators comprising the following steps: (a) Cotransfection of Akt and Hades into animal cells; ); (b) treating the transformed animal cell with any agent; And (c) determining whether the interaction of Akt and Hades in the animal cell is inhibited.

Hereinafter, the present invention will be described more specifically.

Akt, a serine / threonine kinase, functions in a variety of cellular processes, including cell proliferation, cell survival, and tumor development. In the meantime, the study of the mechanisms that negatively regulate Akt has focused on dephosphorylation-mediated inactivation. In the present invention, we have identified Hades, which is a negative regulator of Akt, having a RING finger domain and E3 ubiquitin ligase activity. Akt interacts directly with Hades, in vitro and by Hades. It has been found to be ubiquitinated in vivo. Other molecular assays have demonstrated that phosphorylated Akt is a practical target for both interaction and ubiquitination by Hades. The results of these functional studies suggest that the degradation of Akt by Hades can inhibit the proliferation and survival of cancer cells. These data provide a clarification for the Akt-ubiquitination signaling network.

The present invention demonstrates the function of Hades in the negative regulation of Akt cell signaling. Hades binds directly to Akt in vivo and in vitro, and it binds to Hades through the kinase domain of Akt. Although there are three isoforms in Akt, and the kinase domains are 90-95% identical in amino acid sequence among these isoforms, Hades binds to Akt1 and Akt2, but not to Akt3. And the same SGK1 in Akt and most amino acid sequence was neither bound by Hades nor ubiquitized by Hades (FIG. 2D). In addition, Hades confirmed that it was not phosphorylated by Akt kinase.

In the present invention, the correlation between Hades and Akt was found to be highly dependent on the phosphorylation status of Akt, thereby finding that Akt protein stability is strictly regulated. Serum stimulation to induce phosphorylation of Akt increased the binding between Akt and Hades, thus increasing Akt ubiquitination and Akt degradation. This phosphorylation-dependent Akt proteolysis was re-validated by constructing and using Akt (Myc / His-Akt-myr), which is artificially genetically engineered and can always remain activated. By immunoprecipitation, Hades bound to Akt-myr but not to unphosphorylated dominant mutant Akt (Akt-DN). In addition, treatment of the cells with LY294002 and wortmannin, which inhibits Akt phosphorylation, showed that the degradation of Akt through Hades was inhibited. Conversely, low expression of Hades via siRNA increased Akt protein stability and increased p-Akt levels. Taken together, the data suggest that Hades prefers to bind p-Akt, thereby promoting the degradation of p-Akt.

Structurally, Akt is phosphorylated at the 473 th serine at the C-terminus, which is a phosphorylated and non-enzymatic domain at the amino acid 308 th thronine in the T-loop present in its enzyme activity domain. Complete activity [40]. In the inactivated state of Akt, a bond is formed between the PH domain and the kinase domain in Akt, thereby inhibiting the in-loop phosphorylation of Akt by higher phosphate-inositide-dependent protein kinase-1 [37, 41]. Although the exact structural mechanism is not fully known, it can be concluded that the phosphorylation of Akt exposes a portion capable of binding Hades in the kinase domain of Akt. First, the pull-down analysis data shows that a bond is formed between the kinase domain of Akt and Hades. Second, as a result of competing binding assay by synthesizing the phosphorylated moiety of Akt into small peptides, there was no difference in Hades' binding capacity between phosphorylated and non-phosphorylated peptides (Mishiddata). Third, mimicking phosphorylation of Akt (T308D / S473D, genetically engineered to always activate) also had binding capacity with Hades. Therefore, these results indicate that the phosphorylation residues of Akt are not important for binding with Hades, but the structural deformation that appears to be phosphorylated is important.

Proteolysis by the ubiquitin-proteome system (UPS) can modulate various intracellular responses [16]. First, several papers have reported that the degradation of Akt by UPS is not a special phenomenon but a wide range of events in many different cells [42]. Akt protein is stabilized in the presence of Hsp90, and therefore Hsp90 inhibitors induce Akt degradation [18]. However, in the present invention, it was found that the degradation of Akt by Hades was not related to the degradation of Hsp inhibitor-induced Akt. Riesterer et al. Found that inhibition of VEGF signaling by VEGF inhibitor PTK787 / ZK222584 induced degradation of Akt by UPS [20, 42, 43]. In addition, treatment with rapamycin, an inhibitor of protein mTOR discovered as a target for the drug rapamycin, has been reported to eliminate the protective mechanism of VEGF against Akt degradation by UPS-. Yan et al reported that the degradation of dendritic Akt by the UPS in the hippocampal nerve occurs selectively [43]. They found that Akt destabilization of the dendritic portion was necessary to form neuropolarity. Although the breakdown of Akt by UPS has been reported under different cellular conditions, it is still unclear about the specific E3 ligase that causes Akt degradation by UPS. Recently, tetratricopeptide repeat domain 3 is an Akt-specific E3 ligase, which has been reported to induce ubiquitination and degradation of Akt in the nucleus [20]. However, since activated Akt can exist not only in the nucleus but also in the cytoplasm and cell membrane, the meaning of the present invention is that Hades [23] in the mitochondria negatively regulates p-Akt in the cytoplasm.

In a previous report, Hades' intracellular location was mitochondria and Hades activated apoptosis signaling [45]. In addition, another study group reported that degradation of Akt occurs caspase dependent during apoptosis [46]. Although the present invention did not address the possibility that caspase activation by Hades affects the Akt signaling system, the degradation of Hades-dependent Akt did not seem to be associated with caspase cell signaling. This is because Akt continues to be decomposed by Hades even when cells were treated with Z-VAD, a caspase inhibitor (FIG. 17).

To date, no amino acid residues in Akt corresponding to polyubiquitin-dependent proteolysis have been reported. Although Akt-KD and Hades bind, they do not rule out the possibility of ubiquitination in the Akt-PH and Akt-HM regions. As shown in FIG. 5A, ubiquitination occurred by Hades in the kinase domain of Akt but not Akt-PH and Akt-HM. In order to find out the residues in Akt that ubiquitin is bound by Hades, eleven lysine residues in Akt-KD were used for the experiment. As a result, the point mutation of the 284th lysine residue in Akt was sufficient to inhibit Akt ubiquitination. However, our experiments do not rule out the possibility that the involvement of Akt protein stability may be other than the 284 residue.

Akt enzymes play an important role in promoting various intracellular processes involved in cell growth and cell migration through phosphorylation of several downstream targets [36]. In the present invention, Hades cancer cell overexpression demonstrated that the cell growth and growth capacity dependently inhibit Hades' E3 ligase activity. GSK-3β and TSC2 are well known sub-targets of Akt and are known to regulate protein synthesis, cell proliferation, differentiation, migration and apoptosis [47,48]. Several reports have shown that GSK-3β's ability to promote apoptosis is inhibited by phosphorylation of serine, the ninth amino acid in GSK-3β, by Akt [6]. When the 1462 threonine residue in TSC2 is phosphorylated by Akt, it has been reported to promote cell growth signals for insulin signaling [49]. Therefore, it was proved that inhibition of cell death and survival by Hades resulted from degradation of Akt and thus inhibition of phosphorylation level of ASK phosphorylation target proteins GSK-3β and TSC2. In addition, inhibition of Akt signaling by Hades resulted in that Hades can regulate this cellular process by negatively regulating Akt signals through pTOPFLASH reporter and wound healing assay. In addition, we revealed that NF-κB activation via Hades is independent of Hades's E3 ligase activity, and that NF-κB activation via Akt is dependent on Hades' E3 ligase activity. Recently, Zhang et al. Reported that Hades / GIDE and Hades without RING domains slightly increased the activity of NF-κB. Overexpression of IKKβ-KA and IκBα (SS / AA) reduced the activity of NF-κB by Hades. Inhibition, but also did not inhibit the effect of inhibiting cell growth through Hades / GIDE. Thus, although the mechanism of activation of NF-κB by Hades is still unclear, the activation of NF-κB does not seem to be related to Hades' ability to inhibit cell growth. Therefore, although Hades increases the activity of NF-κB, this phenomenon can be understood to be different from the NF-κB mechanism by Akt.

The results of the present invention demonstrate that Hades can negatively regulate Akt, thereby regulating many related cellular processes. The results of the present invention may also offer the possibility of having clinical significance as a future new target as a modulator of Akt.

1 shows Akt interacts with Hades E3 ubiquitin ligase in vitro and in vivo.
FIG. 2 shows that the ubiquitination and degradation of Akt is mediated by Hades.
FIG. 3 shows that pAkt is a preferred substrate of ubiquitination by Hades.
4 shows that activated Akt is the preferential target of the ubiquitin E3 ligase Hades.
FIG. 5 shows that lysine 284 of Akt is a site for Hades-mediated polyubiquitination.
FIG. 6 shows that the cell growth function of Akt is inhibited by Hades.
FIG. 7 shows that siRNA-mediated Hades expression removal stabilizes endogenous Akt1 and Akt2 proteins.
8 shows that Hades induces Akt1 degradation and co-locates with Akt1 in the mitochondria.
9 shows that serum reduces Akt protein stability.
FIG. 10 shows that geldanamycin induces Akt degradation in both control cells and Hades-deficient cells.
FIG. 11 shows that Hades-mediated Akt proteolysis is restored by wortmannin.
12 shows that mutation of lysine 284 prevents proteolytic degradation of isotopically expressed Akt by Hades.
FIG. 13 shows that the depletion of Hades in HeLa cells by shRNA increases cell growth.
FIG. 14 shows that Akt-mediated cell survival increase is reduced by Hades in a RING domain-dependent manner.
FIG. 15 shows that endogenous Hades deficiency inhibits cell migration.
FIG. 16 shows that Hades-induced NF-κB activation is independent of its E3 ligase activity, and Akt-induced NF-κB activation is reduced by Hades in an E3 ligase activity-dependent manner.
FIG. 17 shows that isotopically expressed Akt is degraded by Hades in HeLa cells in the presence of a caspase inhibitor Z-VAD.
FIG. 18 shows that Hades reduces colony formation in HCT116 colon cancer cell line.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  1: plasmid ( plasmids )

The coding sequences of Akt and Hades were amplified and isolated by PCR of cDNA derived from cervical cancer cell line HeLa. Amplified human Akt1 cDNA was cloned into pcDNA3.1-Myc / His (Invitrogen, Carlsbad, Calif.), PGEX6p (Promega, Madison, Wis.) And pET28 (Novagen, EMD Chemicals Inc. Merck, Darmstat, Germany) vectors. HadescDNA was cloned into pEGFP-C (Clontech, Mountain View, Calif.) And pGEX6p vectors. The functional divisions of the Akt gene were cloned into the pcDNA3.1 (Invitrogen) vector. The dominant negative mutated Akt genes (DN-Akt, T308A and S473A), mutants of lysine residues in the Akt amino acid sequence, and Hades' RING domain-mutations (Hades MT-C302S and C305S) were QuikChange site-directed Using the products of the mutagenesis kit (Stratagene, Santa Clara, CA), the product instructions were followed. HA-ubiquitin (HA-Ub) plasmid pMT123 was provided by Dr. Dirk Bohmann (University of Rochester, Rochester, NY). HA-Ub mutant genes (HA-Ub-K48R and HA-Ub-K63R) were provided by Dr. Zhijian Chen (University of Texas Southwestern Medical Center, Dallas, TX). Primer sequences used for PCR are listed in Table 1 below.

TABLE 1 Primer sequences used to generate constructs of Akt and Hades

Example  2: recombinant protein ( recombinant proteins )

GST- and GST-labeled Hades (GST-Hades) proteins were expressed for 1 hour by treatment with 0.1 mM IPTG at 37 ° C using Escherichia coli BL21 strain. The strain cells were reproduced using NP-40 lysis buffer (50 mMTris-HCl, pH 7.5, 150 mMNaCl, 1% NP-40, 1 mM DTT, 1 mM EDTA, lysozyme and protease inhibitors). It was cloudy. The recombinant proteins were extracted using glutathione-sepharose 4B bead following the instructions of the product, and the extracted product was dialyzed using the product of Slide-A-Lyzer dialysis cassette (Pierce, Rockford, IL). Concentrated using Amicon Ultra-15 device (Millipore, Billerica, Mass.). His-Akt was purchased from Millipore.

Example  3: antibodies and reagents ( Antibodies and reagents )

The antibodies used for this study are as follows. Antibodies of EFGP, Myc-tag, phosphorylated Akt (pAkt) (S308), pAkt (T473), Akt, pGSK-3β, TSC2, pTSC2 (T1462) were purchased from Cell Signaling Technology (Danvers, MA) and HA- The antibody of tag, GSK-3β was purchased from Santa Cruz Biotechnology, (Santa Cruz, CA), and purchased from Millipore under SGK1. Hades polyclonal antibody was injected into rabbits using peptides consisting of amino acid sequences N-SGERPKGIQETEEM-C and N-SRAKPEDRESLKSAC-C and immunized after extraction with antibodies. Transfection of artificially injecting plasmid into cancer cells was performed using Lipofectamine 2000 reagent (Invitrogen). Z-VAD and MG132 were purchased from Sigma-Aldrich (St. Louis, Mo.) and used. It was purchased from LY294002 and wortmannin Cell Signaling Technology. 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt (MTS) assay kit was purchased from Promega.

Example  4: In vitro  Protein binding Potency  Measurement experiment ( in vitro binding assay )

35S-labeled proteins and protein assemblies were obtained by in vitro transcription / translation reactions, with 0.5% NP-40 buffer containing 3 μg of GST or GST-Akt recombinant protein attached to glutathione-Sepharose 4B beads. The mixture was reacted for 3 hours at 37 degrees, and after washing three times, the sample was boiled into the SDS sample buffer. Analysis of bound proteins was done by developing using autoradiography via SDS-PAGE.

Example  5: Immunoprecipitation  ( Immunoprecipitation )

Immunoprecipitation experiments showed that the cell lysate suspended in CHAPS buffer (0.5% CHAPS, 10 mMTris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 5 mM β-mercaptoethanol and 10% glycerol) was shown in Figure 4 Reaction was carried out as follows. Afterwards Protein A / G PLUS-Agarose beads (Santa Cruz Biotechnology) was added, followed by further reaction for 2 hours and washed with water.

Example  6: In Vitro Ubiquitination Experiment  ( in vitro ubiquitination assay )

150ng recombinant E1 (Boston Biochem, Cambridge, MA) enzyme, 250 treated with His-Akt or λ-protein dephosphatase (λ-PPase, New England Biolabs, Ipswich, Mass.) Purified from insect cells ubiquitination buffer (50 mMTris-HCl, pH 7.4, 5 mM MgCl2, 15 μM ZnCl2, 0.3 mM) with ng recombinant UbcH5C (Boston Biochem), 500 ng GST-Hades and 5 ug His-ubiquitin (Boston Biochem) DTT, 0.006% DTT, 2 mM ATP, 10 U creatine phosphokinase and 10 mM phosphocreatine) were reacted for 90 minutes at 30 ° C. In the ubiquitination reaction treated with λ-protein dekinase, His-Akt and λ-PPase were reacted in ubiquitination buffer for 1 hour at room temperature at 30 ° C. After the reaction, 2-fold concentrated dephosphatase inhibitor was added. The reaction mixture was then reacted with E1, E2, GST-Hades and ubiquitin. The reaction product was analyzed by immunoblotting using anti-Akt antibody via 8% SDS-PAGE.

Example  7: In vivo  Ubiquitination test in vivo ubiquitination assay )

After transfection, MG132 treated cells were washed with PBS and vortexed by adding 200 μl of a denaturing lysis buffer (50 mMTris-HCl, pH 7.4, 0.5% SDS and 70 mM β-mercaptoethanol). Cells were completely lysed by boiling at room temperature for 95 minutes. The lysed cell solution was diluted with 800 μl of CHAPS buffer. The diluted cell solution was immunoprecipitated by adding Akt antibody and protein agarose A / G PLUS-Agarose beads. After the reaction, the beads were washed with CHAPS lysis buffer 5 times and then boiled. Ubiquitinated Akt was developed through immunoblotting with anti-HA antibodies. His-pull down assay was performed by diluting the denatured cell solution with 800 μl of buffer A (50 mM NaH 2 PO 4, 300 mM NaCland 10 mM imidazole, pH 8.0) and then adding Ni-NTA bead. It was reacted overnight at room temperature 4 degrees. The beads were washed five times using buffer B (50 mM NaH 2 PO 4, 300 mM NaCl and 20 mM imidazole, pH 8.0), and the proteins bound to the beads were eluted by boiling with SDS-PAGE sample buffer. The eluted proteins were immunoblotted using anti-HA antibodies.

Example  8: RNA  Interference ( RNA interference )

Sides targeting Hades were purchased from Ambion. First, Hades siRNA (100 pmol) was transduced into HEK293 cells (5 × 10 5 cells) in a serum-free medium via LipofectamineRNAiMAX (Invitrogen), and then exchanged into normal medium for a further 48 hours to finish.

Example  9: Cell viability test cell viability assay )

Cell viability was observed through a general MTS experiment. The results are presented graphically and compared in percentages with the control.

Example  10: Colony formation  ( Clonogenic survival assay )

First, heLa cells, cervical cancer cell lines, were transduced with a control plasmid encoded with green fluorescent protein (EGFP), Hades labeled with EGFP, or Hades MT gene. After 2 weeks, the resulting colonies (colonies) were fixed and stained with 0.1% crystal violet to analyze the results. Anchorage-independent soft-agar assay was performed on 3 ml of DMEM medium containing .45% low-melting agarose and 10% fetal bovine serum. After addition, 2 ml of DMEM containing 0.9% agarose and 10% fetal bovine serum was added to cover the surface, and colonies were observed by culturing for 2 weeks.

Example  11: Luciferase reporter gene analysis luciferase reporter gene assay )

This experiment used pTOPFLASH (upstate), a TCF-reacting luciferase plasmid. There are six TCF-response sites in the pTOPFLASH vector. After transducing this vector and reacting for 48 hours, it was analyzed how many times the luciferase activity was increased with β-galactosidase as a control. The result is the average of three independent experiments.

Example  12: Battlefield ( full - length ) human cDNA  Library building

To construct a full-length human cDNA library, total RNA was first extracted from cervical cancer HeLa cells and liver Chang cells using TRIzol reagent (Invitrogen, Carlsbad, Calif.) Based on the manufacturer's instructions. Normalized full-length cDNA libraries were constructed using SMART cDNA library construction kit (Clontech, Mountain View, CA) and TRIMMER-cDNA normalization kit (Evrogen, Russia) based on the manufacturer's instructions. Normalized cDNA was fractionated by size using CHROMA SPIN-400 column (Clontech), and modified to insert SfiI restriction enzyme reaction site for all cDNAs over 500 bp by genetic method. It was then cloned into pcDNA3.1 (+) vector (Invitrogen) and transformed into TOP10 Escherichia coli.

Example  13: new Akt -Can be combined Protein  Screening experiment

If in vitro binding assays revealed binding positive protein aggregates, the cDNA aggregates were further subdivided and continued until one positive cDNA clone was isolated. Positive clones bound to Akt were identified through NCBI gene database for information from sequencing.

Example  14: subcellular fraction ( subcellular fractionation )

Cervical cancer HeLa cells are grown to approximately 70% population on a 100-mm plate, and then 5 μg of EGFP-MULAN-WT or EGFP-MULAN-MT with Myc / His-Akt1-myr (2 μg) Lipofectamine 2000 (Invitrogen) was used to transduce cells into cells based on the manufacturer's instructions. The cells were then incubated for 24 hours. The cultured cells were harvested and resuspended in NKM buffer (1 mM Tris-HCl, pH 7.4), 130 mM NaCl, 5 mM KCl, 7.5 mM MgCl 2). The cells were lysed using a Dounce homogenizer and centrifuged for 5 minutes at 2 ° C. (final concentration: 0.25M) at 12,000 xg intensity at room temperature. After centrifugation, the supernatant was transferred to a new tube and again centrifuged for 15 min at 4 ° C. at an intensity of 7,000 × g. Mitochondrial pellets were resuspended in suspension buffer (10 mM Tris-HCl (pH 6.7), 0.15 mM MgCl 2, 0.25 mM sucrose, protease inhibitor cocktail) and centrifuged again at 4 ° C. for 10 minutes at an intensity of 9,500 × g. After centrifugation the pellet is the mitochondrial fraction and the supernatant is the cytoplasmic fraction. The cytoplasm and mitochondrial proteins were analyzed via immunoblotting using 10-15% SDS-PAGE and corresponding antibodies.

Example  15: immunofluorescence assay ( immunofluorescence assay )

The transduced cells were incubated in glass coverslips, immobilized by reaction with 4% formaldehyde for 5 minutes at room temperature, washed twice with PBS to remove the remaining solution, and then permeabilization buffer (0.5% Triton X). -100 in PBS) for 3 minutes. Immobilized cells were blocked by reaction with PBS containing 5% BSA and 2% goat serum for 30 minutes, and the anti-myc antibody (1: 200 in PBS) was reacted at 4 ° C. overnight. After washing with PBS three times, the remaining ones were removed and reacted for an additional hour using a secondary antibody (1: 500 in PBS) labeled with fluorescein Texas red. The coverslips were turned upside down and firmly immobilized in Vectashield (Vector Laboratories, USA) for fluorescence analysis using a confocal laser-scanning microscope (FV-1000 Spectral, Olympus, Japan).

Example  16: Lentivirus  Synthesis and Infection

First, the injection of the Hades shRNA-coded pLKO vector (Sigma) into a microorganism was purchased, and the vector was purely separated from the microorganism. LKO shRNA lentiviral vectors were produced by transducing pLKO-MULAN shRNA, packaging plasmid (pCMV-Δ8.2) and VSVG plasmid into 293T cells via Lipofectamine 2000. 48 hours after transfection, the virus-containing medium was collected and concentrated through a filter having a 0.45-μm pore. After culturing cervical cancer HeLa cells in a 6-well plate, the virus was treated with polybrene, and after 6 hours, the medium was changed anew, and finally, 72 hours with 1 μg / ml puromycin added medium. Incubated. Finally, puromycin-resistant HeLa cells expressing the control shRNA and Hades shRNA were selected and used in the experiment.

Example  17: Statistical Analysis

Statistical analysis was performed using the χ2 test, Fisher's exact test and Spearman's phase correlation analysis. P <0.05 can be said to be statistically significant.

[Experiment result]

1 shows Akt interacts with Hades E3 ubiquitin ligase in vitro and in vivo. (A) In vitro interactions between Akt and Hades. The 35S-methionine-labeled Akt was tested for interaction with GST-tagged Hades (GST-Hades) using a full-union assay. (B) In vivo association between Akt and Hades. After transfection with plasmid as indicated, HEK293 cells were treated with MG132, followed by immunoprecipitation with anti-Akt antibody and immunoblotting with anti-EGFP. Immunoglobulin G was used as a negative control. (C) Endogenous interactions between Hades and Akt isoforms. MG132-pretreated HeLa cells were immunoprecipitated with Akt isoform-specific antibodies and immunoblotting using anti-Hades antibodies. (D) Functional domain structure and guidance of plasmids of Akt deletion mutants. (E) Akt's KD interacts with Hades. The 35S-methionine-labeled Akt deletion mutants were tested for interaction with GST-Hades using a pull-down assay.

FIG. 2 shows that the ubiquitination and degradation of Akt is mediated by Hades. (A) Akt protein levels in cells were reduced by Hades via proteasome degradation pathways in a RING-dependent manner. After transfection with plasmid as indicated, HEK293 cells were treated with MG132 and then immunoblotted with the indicated antibodies. Isotopic expression levels of GST were normalized to the transfection control. (B) Akt was ubiquitized by Hades in vitro. In vitro ubiquitination assays were performed as described in the Examples. (C) Akt was ubiquitized by Hades in vivo. After transfection with the plasmid as indicated, HEK293 cells were treated with MG132, followed by an in vivo ubiquitination assay as described in the Examples. (D) Ubiquitination of Akt was eliminated by Hades siRNA transfection. After transfection with siRNAs and HA-Ub plasmids as indicated, HEK293 cells were treated with 1 μM MG132 for 12 hours followed by an in vivo ubiquitination assay. (E) K48-linked polyubiquitination is responsible for Hades-dependent Akt ubiquitination. HeLa cells were transfected with plasmids as indicated, followed by in vivo ubiquitination.

FIG. 3 shows that pAkt is a preferred substrate of ubiquitination by Hades. (A) The interaction between Hades and Akt is dependent on phosphorylated Akt. Serum-deficient HeLa cells were treated with 10% serum or insulin and MG132 for 6 hours. Following immunoprecipitation assay with Akt antibody, immunoblotting with anti-Hades antibody. (B) pAkt levels in cells were reduced by Hades. HeLa cells were transfected with plasmids as described in the silencing example. After 24 hours incubation, the medium was replaced with serum free medium and the cells were then stimulated with 10% serum for 2 hours. Cells were lysed and immunoblotted with antibodies as indicated. (C) Cell pAkt protein was ubiquitized by Hades. After transfection with plasmid as indicated, HeLa cells were incubated in serum free medium for 18 hours. Cells were treated with 10 μΜ LY294002 and then stimulated with 10% serum in the presence of 10 μΜ MG132. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (D) Dephosphorylation of pAkt reduced Hades-mediated Akt ubiquitination in vitro. In vitro ubiquitination assays were performed with purified human Akt protein derived from Sf9 cells and λ-PPase. Ubiquitinated Akt and pAkt were detected by immunoblotting with the indicated antibodies. (E) Hades siRNA treatment increased Akt protein stability. HeLa cells were transfected with control and Hades siRNA and treated with 40 μg / ml cycloheximide for the indicated times, after which Akt protein turnover was investigated via immunoblotting.

4 shows that activated Akt is the preferential target of the ubiquitin E3 ligase Hades. (A) Hades interacted with constitutively active Akt in vivo. After transfection with plasmid as indicated, HEK293 cells were treated with MG132, followed by immunoprecipitation with anti-myc antibody and immunoblotting with the indicated antibodies. (B) Activated Akt was efficiently ubiquitized by Hades in vivo. After transfection with the plasmid as indicated, HEK293 cells were treated with MG132, followed by an in vivo ubiquitination assay as described in the Examples. (C) Proteolysis of Hades-mediated Akt was inhibited by LY294002. HEK293 cells were cotransfected with plasmids as indicated. After 24 hours incubation, cells were treated with 10 μM LY29402 for 4 hours. Cells were lysed and immunoblotted with the indicated antibodies. Isotopic expression levels of GST were normalized to transfection control. The graph shows the mean ± SD of the relative intensities of Akt from three experiments.

FIG. 5 shows that lysine 284 of Akt is a site for Hades-mediated polyubiquitination. (A) KD of Akt is ubiquitized by Hades. A series of Akt deletion constructs were transcribed and translated in the presence of 35S-methionine. The 35S-labeled Akt mutations were ubiquitized by Hades via an in vitro ubiquitination assay. (B) Lysine 284 of Akt is a polyubiquitination site. HEK293 cells were cotransfected with plasmids as indicated. 24 hours after transfection, cells were treated with 10 μM MG132 and subjected to in vivo ubiquitination assay as described in the Examples. Total cell lysates were analyzed by immunoblotting with the indicated antibodies. (C) K284R-Akt was more stable than WT Akt. HeLa cells were cotransfected with the indicated plasmids and then treated with 40 μg / ml cycloheximide. Cells were immunoblotted with indicated antibodies.

FIG. 6 shows that the cell growth function of Akt is inhibited by Hades. (A) Cell growth was alleviated by Hades. After transfection with plasmids as indicated in HeLa cells, the number of viable cells was counted every 24 hours using a hemocytometer. The result is representative of three independent experiments. (B) Hades inhibits cell survival in a dose-dependent manner. HeLa cells were transfected with plasmids as indicated. After 24 hours incubation, cell viability was determined by MTS assay. (C) Hades inhibits clonal growth. HeLa cells were transfected with plasmids as indicated, then incubated in G418 (500 μg / ml) containing medium for 2 weeks and stained with crystal violet. (D) Hades inhibits anchorage-independent growth. Soft-agar assays were performed with HeLa cells transfected with the indicated plasmids. Colonies were counted two weeks after seeding. The result is representative of three independent experiments (mean ± SD plot). Student's t-test was used for statistical significance (* P <0.05). (E, F, G) Akt downstream signaling is regulated by Hades. HeLa cells were transfected with the indicated Hades plasmid (E) and Hades siRNA (F). After 24 hours incubation, cells were lysed and immunoblotted with the indicated antibodies. HeLa cells were cotransfected with TCF / LEF1 reporter plasmid, pTOPFLASH and the other plasmids indicated. After 24 hours of incubation, cells were lysed and reporter luciferase activity driven by the TCF response element was determined (G). (H, I) Hades inhibited Akt-induced cell migration (H), but did not inhibit K284R-Akt-induced cell migration (I). HeLa cells were cotransfected with plasmids as indicated. After 48 hours transfection, 100% confluent cells were scratched to form wounds. 72 hours after the induction of the wound, the migration of cells was visualized on a phase contrast microscope. (J) Cell growth of K284R-Akt-expressing cells is not inhibited by Hades. After HeLa cells were transfected with the plasmid as indicated, the number of viable cells was counted every 24 hours using a hemocytometer.

FIG. 7 shows that siRNA-mediated Hades expression removal stabilizes endogenous Akt1 and Akt2 proteins. HeLa serotransfected with Hades specific siRNA. The effect of Hades clearance on Akt isoforms was determined by immunoblotting with Akt isoform-specific antibodies.

8 shows that Hades induces Akt1 degradation and co-locates with Akt1 in the mitochondria. (A) Hades induced Akt1 degradation in both the cytosol and mitochondria. HeLa cells were cotransfected with the indicated plasmids and the cytoplasmic and mitochondrial fractions were isolated. Immunoblotting with the indicated antibodies determined the effect of Akt1 degradation by Hades in the cytoplasm and mitochondrial fraction. Tubulin and Bcl-xL were used as cytoplasmic and mitochondrial markers, respectively. (B) Colocation of Hades with Akt1-WT (top) and Akt1-myr (bottom) was investigated in HeLa cells. HeLa cells were cotransfected with the indicated plasmids and the cells were incubated in the presence of MG132 (10 μM) for 4 hours, immobilized and visualized by confocal microscopy. DAPI staining was used to make nuclei visible. Abbreviations: cyto, cytoplasm; mito, mitochondria

9 shows that serum reduces Akt protein stability. HeLa cells were serum-deficient for 20 hours and a set of cells were stimulated with 10% fetal bovine serum. Cells were then treated with 40 μg / ml cycloheximide for 0, 3, 6, 12, and 24 hours and endogenous Akt protein stability was investigated using immunoblotting.

FIG. 10 shows that geldanamycin induces Akt degradation in both control cells and Hades-deficient cells. HeLa cells were transfected with negative control siRNA or Hades siRNA and then treated with geldanamycin (50 nM) overnight. Protein levels of Hades and Akt were detected by immunoblotting.

FIG. 11 shows that Hades-mediated Akt proteolysis is restored by wortmannin. HEK293 cells were cotransfected with plasmids as indicated. After 24 hours of incubation, cells were treated with 10 μM Wortmannin for 4 hours. Cells were lysed and immunoblotted with the indicated antibodies.

12 shows that mutation of lysine 284 prevents proteolytic degradation of isotopically expressed Akt by Hades. HEK293 cells were cotransfected with plasmids for wild type or lysine mutations of Akt in combination with plasmids for EGFP-Hades or control EGFP. 24 hours after transfection, cells were lysed and immunoblotted with the indicated antibodies.

FIG. 13 shows that the depletion of Hades in HeLa cells by shRNA increases cell growth. HeLa cells were infected with lentivirus expressing negative control shRNA or Hades shRNA. Cells were selected with 5 μg / ml puromycin for 2 weeks. Cell growth was determined by counting using a hemocytometer every 24 hours. Data is shown as mean of 3 independent experiments (mean ± SD plot).

FIG. 14 shows that Akt-mediated cell survival increase is reduced by Hades in a RING domain-dependent manner. HeLa cells were transfected with plasmids for control EGFP, EGFP-tagged wild type Hades (EGFPHADES-WT) or RING domain mutant Hades (EGFP-HADES-MT) in combination with Myc / His-Akt-myr. Twenty four hours after transfection, cell viability was determined using MTS assay. Three independent experiments were conducted. The results represent three independent experiments (mean ± SD plot). Student's t-test was used to determine statistical significance (* P <0.05).

FIG. 15 shows that endogenous Hades deficiency inhibits cell migration. Lentivirus-mediated Hades-deficient cells and control cells were incubated and scratched to scratch 100% confluent cells. Cell migration was visualized by phase contrast microscopy 72 hours after scratching to form a wound.

FIG. 16 shows that Hades-induced NF-κB activation is independent of its E3 ligase activity, and Akt-induced NF-κB activation is reduced by Hades in an E3 ligase activity-dependent manner. HeLa cells are transfected with EGFP control, EGFP-HADES-WT, or EGFP-HADES-MT, or for Myc / His-Akt-WT with NF-κB luciferase reporter plasmid and β-galactosidase plasmid Cotransfected with plasmids. After 24 hours incubation, cells were lysed and reporter luciferase activity was determined.

FIG. 17 shows that isotopically expressed Akt is degraded by Hades in HeLa cells in the presence of a caspase inhibitor Z-VAD. HEK293 cells were transfected with a plasmid for EGFP-HADES-WT or EGFP-HADES-MT with a combination of Myc / His-Akt-WT. 24 hours after transfection, cells were treated with Z-VAD (10 μM) and incubated for another 4 hours. Cell extracts were immunoblotted with the indicated antibodies.

FIG. 18 shows that Hades reduces colony formation in HCT116 colon cancer cell line. (a) Cancer cells are characterized by growing morphologically to form colonies. In order to confirm how much colonies were generated, the EGFP vector, EGFP-Hades WT vector, and EGFP-Hades MT vector were first transfected into colon cancer cell HCT116 through Lipofectamin 2000 according to the manufacturer's instructions. . Thereafter, the cultures were cultured in a medium containing the antibiotic puromycin for 2 weeks to generate colonies. After that, the cells were immobilized and finally stained with 0.1% crystal violet. (B) The graph shows the colony numbers shown in (a).

Result 1: Akt  end Hades E3 Ubiquitin Ligase and  Interact

In order to identify proteins that interact with Akt, human full-length cDNA libraries derived from HeLa and Chang cells were prepared (Supplementary Materials and Methods). In vitro transcription and translated protein pools from the cDNA library were screened for human Akt1-binding proteins with a modified SMART technique. In the present invention, all experiments were conducted using human Akt1, which is denoted as 'Akt' unless otherwise specified. From this library, several positive cDNA clones were isolated and one of the clones was identified as the main Akt-binding partner (data not shown). This clone contained several putative functional domains: a transmembrane (TM) domain or signal peptide at the N terminus, a second TM domain in the middle of the protein, and a RING finger domain at the C terminus (ie, a label of the E3 ligase domain). . While the present invention focuses on functional communication between this clone and Akt, Li et al. [23] named this protein MULAN, meaning mitochondrial ubiquitin ligase activator of NF-κB.

In vitro pull-down studies have shown that Akt physically interacts with Hades (FIG. 1A). Intermolecular associations between endogenous Akt and Hades were investigated using coimmunoprecipitation assays (FIG. 1B). Human Akt has three isoforms: Akt1, Akt2 and Akt3. Akt isoform-specific immunoprecipitation assay showed that Hades interacts with Akt1 and Akt2 but not with Akt3 (FIG. 1C). In addition, Hades deficiency increased the protein levels of Akt1 and Akt2 but not Akt3 (FIG. 7). It has been reported that Akt2 is translocated to mitochondria but Akt3 is not [24]. Mitochondrial translocation of Akt1 is controversial [24-26]. However, experiments of the present invention revealed that Akt1 can be translocated to mitochondria (FIG. 8A). Confocal microscopy also revealed that Akt1 colocalized with Hades (FIG. 8B). In vitro binding assays using a series of Akt lacking mutants revealed that the kinase domain of Akt is primarily associated with Hades (FIGS. 1D and 1E).

Result 2: Akt Ubiquitination  And disassembly Hades Directly controlled by

To determine the functional role of the interaction between Akt and Hades, we investigated whether Hades functions as an E3 ligase for Akt. Hades expression resulted in a decrease in Akt protein levels in an E3 ligase activity-dependent manner. In addition, the proteasome inhibitor MG132 completely recovered this decrease in intracellular Akt protein levels (FIG. 2A, lane 5). Next, in vitro and in vivo ubiquitination assays demonstrated that recombinant and endogenous Akt proteins were ubiquitinated by Hades in an E3 ligase activity-dependent manner (FIGS. 2B and 2C). The opposite was observed in Hades siRNA-induced knockdown cells. Hades siRNA transfection resulted in inhibition of Akt ubiquitination in HEK293 cells (FIG. 2D, left panel). Interestingly, serum / glucocorticoid regulatory kinase 1 (SGK1) [27] with high homology with Akt was not affected by the endogenous Hades deficiency (FIG. 2D, right panel).

The ability to generate diverse substrate-ubiquitin structures is important for targeting proteins with different fates [28]. To this end, in vivo ubiquitination assays were performed in HeLa cells expressing HA-tagged ubiquitin (HA-Ub WT, HA-Ub K48R, and HA-Ub K63R) in which lysine 48 or 63 was mutated to arginine. As shown in FIG. 2E, Ub K48R completely abolished Hades-mediated Akt ubiquitination, while Ub WT and Ub K63R did not. This indicates that K48-linked ubiquitination chains are formed during Hades-mediated ubiquitination of Akt. These results show that Hades E3 ligase specifically targets Akt to induce its ubiquitination and subsequent degradation.

Result 3: pAkt The Hades E3 Ubiquitin Ligase  Preferential ( preferential Target

Since the upregulation of Akt kinase activity is tightly controlled by the phosphorylation of serine 308 and threonine 473, it was tested whether the active / inactive state of Akt affected Akt degradation by Hades. To test this hypothesis, we first tested the interaction between endogenous Hades and Akt under the stimulus of growth factors. Interestingly, the interaction between endogenous Hades and Akt was detected in the presence of serum and insulin in HeLa cells (FIG. 3A). Similarly, Hades-induced Akt degradation was preferentially induced in serum-stimulated HEK293 cells (FIG. 3B). In vivo ubiquitination assays also demonstrated that serum stimulation induced endogenous Akt ubiquitination by Hades (FIG. 3C). In addition, LY294002, a PI3K inhibitor that inhibits phosphorylation of Akt, inhibited Hades-induced Akt ubiquitination in serum-stimulated HEK293 cells (FIG. 3C, lanes 5-8). These observations suggest an association between Akt activation status and Hades-mediated degradation. To demonstrate this hypothesis, an in vitro ubiquitination assay was performed in the presence of λ-PPase [30, 31] having activity with phosphorylated serine, threonine and tyrosine residues. Treatment with λ-PPase effectively eliminated phosphorylation of Akt residues, inhibiting Akt ubiquitination (FIG. 3D).

The results led to testing whether serum or growth factors reduce the stability of endogenous Akt. Interestingly, it was found that Akt protein levels were slightly decreased in serum-deficient cells and significantly increased in serum-stimulated cells, indicating that phosphorylated Akt degrades more efficiently than non-phosphorylated Akt (FIG. 9). . It was also found that serum-induced Akt degradation was inhibited by Hades deficiency (FIG. 3E) and that siRNA-induced Hades deficiency increased the protein levels of Akt and pAkt in a dose-dependent manner (FIG. 6F). . In addition, geldanamycin induced Akt degradation in both control and Hades-deficient cells, suggesting that Hades-induced Akt degradation is independent of Hsp90 inhibitor-induced Akt degradation (FIG. 10). . Overall, these results indicate that Hades preferentially targets pAkt for ubiquitination.

Result 4: enabled Akt The Hades of Polyubiquitination  Target

Akt activation is induced by cross-domain morphologically-mediated phosphorylation [32, 33]. We then tested the intracellular regulation of permanently active Akt by Hades by coimmunoprecipitation assay. Myristolation signal-attached Akt (Akt-myr) is a constitutively active form of Akt [34]. Coimmunoprecipitation experiments interacted with functionally active Akts (Myc / His-Akt-WT and Myc / His-Akt-myr) in which Hades were ectopically expressed, but dominant-negative Akt (Myc) in HEK293 cells. / His-Akt-DN) did not interact with (Fig. 4A). We confirmed using ubiquitination experiments in vivo that ubiquitination of Akt was dependent on Akt activation in transfected HEK293 cells (FIG. 4B). Similarly, LY294002 completely inhibited Akt phosphorylation and mostly blocked Hades-mediated Akt degradation (Figure 4C, lanes 4, 5, and 6). Another PI3K / Akt inhibitor, wortmannin [35] also blocked Hades-mediated Akt degradation (FIG. 11). These results indicate that Hades preferentially binds to pAkt and promotes its degradation, which is consistent with our results that Hades negatively regulates active pAkt.

Result 5: Akt  Lysine in 284 is Hades On by Ubiquitination  It is a major part

Akt consists of the N-terminal flextrin homology (PH) domain, the central catalytic kinase domain (KD) and the C-terminal short regulatory hydrophobic motif (HM) [36]. Phosphorylation of Akt induces a structural change, separating the PH and HM domains from the KD domain [37]. In vitro binding assays demonstrated that Hades specifically ubiquitizes Akt WT as well as Akt-KD (FIG. 5A). We then determined which lysine residues of Akt-KD are required for protein stabilization and ubiquitination. To assess the association between Akt protein stabilization and mutation of lysine residues, putative ubiquitin-conjugation residues (K158, K163, K170, K183, K189, K214, K276, K284, K289, by site-directed mutation) Mutations from Akt lysine (K) to arginine (R) of K297 and K307) were generated. As shown in FIG. 12, degradation of Akt K284R was inhibited compared to Akt WT and other Akt mutations. In addition, we confirmed in vivo ubiquitination assay that Akt K284R showed significantly reduced ubiquitination (FIG. 5B). In addition, cycloheximide chase assay showed that Hades did not reduce the stability of Akt K284R compared to Akt WT (FIG. 5C). The results indicate that lysine 284 in Akt is a specific residue of Hades-mediated Akt ubiquitination.

Result 6: Hades On by Akt Signaling  Voice control

Akt plays an important role in regulating cell cycle progression, cell survival and cell growth [38]. We found that the growth of cells expressing EGFP-Hades-WT was significantly inhibited compared to the growth of cells expressing EGFP-Hades-MT or control EGFP (FIG. 6A). Hades-deficient cells grew faster than control cells (FIG. 13). Hades inhibited proliferation of HeLa cells in a concentration-dependent manner (FIG. 6B). MTS assay also showed that the increase in Akt-mediated cell viability is reduced by Hades overexpression in HeLa cells (FIG. 14). Inhibition of cell growth by Hades was confirmed by clonality and anchorage-independent soft agar assays (FIG. 6D). HeLa cells expressing EGFP-Hades-WT showed a 50% reduction in colony formation compared to colony formation of cells expressing EGFP-Hades-MT or control EGFP (FIG. 6C). These results indicate that Hades modulates the Akt downstream signaling pathway. GSK-3 / β-catenin / TCF signaling is an important downstream pathway of PI3K / Akt-mediated cell survival and proliferation [6, 39]. In FIG. 6E, we showed that both phosphorylation of Akt downstream target proteins GSK-3β and TSC2 is inhibited by Hades in a RING ligase activity-dependent manner (FIG. 6E). In addition, the opposite phenomenon was observed in a Hades siRNA-dependent manner (FIG. 6F). In the TCF / LEF1 response element-driven luciferase system, pTOPFLASH reporter activity was measured to confirm inhibition of Akt downstream signaling by Hades (FIG. 6G). Taken together, these results indicate that Hades inhibits cell growth by regulating the Akt signaling pathway. We assessed the effect of Hades on Akt-induced cell migration using the following in vivo wound healing assay. Isotopically expressed WT Hades inhibited HeLa cell migration better than RING ligase MT Hades (FIG. 6H) and Hades siRNA transfection increased HeLa cell migration (FIG. 15). This indicates that Hades E3 ligase efficiently inhibits Akt and its downstream signaling.

In addition, we tested the effect of Akt K284R on cell growth and cell migration, which is regulated by Hades. Hades inhibited Akt-induced cell growth more efficiently than it inhibited Akt-K284R-induced cell growth (FIG. 6J). Similarly, Akt-K284R-induced cell migration was not inhibited by Hades in contrast to Akt WT-induced cell migration (FIG. 6I). These results indicate that Hades E3 ligase efficiently inhibits Akt function by ubiquitination of Akt in lysine 284.

Akt is known to activate NF-κB, and Hades has been reported as an NF-κB activator [23,45]. Therefore, it is unclear whether Hades induces NF-κB activity or inhibits NF-κB activity by inhibiting Akt. To address this problem, we tested NF-κB activity using a reporter plasmid containing an NF-κB-binding site (NF-κB luciferase reporter). HeLa cells were transfected with EGFP control, EGFP-Hades-WT and EGFP-Hades-MT or cotransfected with plasmids for Myc / His-Akt WT. In these transfected HeLa cells, Hades WT and Hades MT increased NF-κB activity (up to 3 fold), but upregulation of NF-κB activity by Akt WT (up to 7 fold) was Hades E3 ligase-dependent Reduced in a manner (FIG. 16). These results indicate that Hades can upregulate NF-κB activity, but the effect is independent of Akt-mediated NF-κB pathway.

We identified Hades, a negative regulator of Akt, with a RING finger domain and E3 ubiquitin ligase activity. According to the present invention, Akt interacts directly with Hades and is ubiquitinated by Hades in vitro and in vivo. Therefore, Hades according to the present invention can inhibit the proliferation and survival of cancer cells by decomposing Akt and negatively controlling its downstream signaling, and thus can be usefully used as an anticancer agent.

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<110> Konkuk University Industrial Cooperation Corp <120> Use of Hades as a negative regulator of Akt <130> PN120202 <150> US 61 / 469,814 <151> 2011-03-31 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1059 <212> DNA <213> Homo sapience <400> 1 atggagagcg gagggcggcc ctcgctgtgc cagttcatcc tcctgggcac cacctctgtg 60 gtcaccgccg ccctgtactc cgtgtaccgg cagaaggccc gggtctccca agagctcaag 120 ggagctaaaa aagttcattt gggtgaagat ttaaagagta ttctttcaga agctccagga 180 aaatgcgtgc cttatgctgt tatagaagga gctgtgcggt ctgttaaaga aacgcttaac 240 agccagtttg tggaaaactg caagggggta attcagcggc tgacacttca ggagcacaag 300 atggtgtgga atcgaaccac ccacctttgg aatgattgct caaagatcat tcatcagagg 360 accaacacag tgccctttga cctggtgccc cacgaggatg gcgtggatgt ggctgtgcga 420 gtgctgaagc ccctggactc agtggatctg ggtctagaga ctgtgtatga gaagttccac 480 ccctcgattc agtccttcac cgatgtcatc ggccactaca tcagcggtga gcggcccaaa 540 ggcatccaag agaccgagga gatgctgaag gtgggggcca ccctcacagg ggttggcgaa 600 ctggtcctgg acaacaactc tgtccgcctg cagccgccca aacaaggcat gcagtactat 660 ctaagcagcc aggacttcga cagcctgctg cagaggcagg agtcgagcgt caggctctgg 720 aaggtgctgg cgctggtttt tggctttgcc acatgtgcca ccctcttctt cattctccgg 780 aagcagtatc tgcagcggca ggagcgcctg cgcctcaagc agatgcagga ggagttccag 840 gagcatgagg cccagctgct gagccgagcc aagcctgagg acagggagag tctgaagagc 900 gcctgtgtag tgtgtctgag cagcttcaag tcctgcgtct ttctggagtg tgggcacgtt 960 tgttcctgca ccgagtgcta ccgcgccttg ccagagccca agaagtgccc tatctgcaga 1020 caggcgatca cccgggtgat acccctgtac aacagctaa 1059 <210> 2 <211> 352 <212> PRT <213> Homo sapience <400> 2 Met Glu Ser Gly Gly Arg Pro Ser Leu Cys Gln Phe Ile Leu Leu Gly   1 5 10 15 Thr Thr Ser Val Val Thr Ala Ala Leu Tyr Ser Val Tyr Arg Gln Lys              20 25 30 Ala Arg Val Ser Gln Glu Leu Lys Gly Ala Lys Lys Val His Leu Gly          35 40 45 Glu Asp Leu Lys Ser Ile Leu Ser Glu Ala Pro Gly Lys Cys Val Pro      50 55 60 Tyr Ala Val Ile Glu Gly Ala Val Arg Ser Val Lys Glu Thr Leu Asn  65 70 75 80 Ser Gln Phe Val Glu Asn Cys Lys Gly Val Ile Gln Arg Leu Thr Leu                  85 90 95 Gln Glu His Lys Met Val Trp Asn Arg Thr Thr His Leu Trp Asn Asp             100 105 110 Cys Ser Lys Ile Ile His Gln Arg Thr Asn Thr Val Pro Phe Asp Leu         115 120 125 Val Pro His Glu Asp Gly Val Asp Val Ala Val Arg Val Leu Lys Pro     130 135 140 Leu Asp Ser Val Asp Leu Gly Leu Glu Thr Val Tyr Glu Lys Phe His 145 150 155 160 Pro Ser Ile Gln Ser Phe Thr Asp Val Ile Gly His Tyr Ile Ser Gly                 165 170 175 Glu Arg Pro Lys Gly Ile Gln Glu Thr Glu Glu Met Leu Lys Val Gly             180 185 190 Ala Thr Leu Thr Gly Val Gly Glu Leu Val Leu Asp Asn Asn Ser Val         195 200 205 Arg Leu Gln Pro Pro Lys Gln Gly Met Gln Tyr Tyr Leu Ser Ser Gln     210 215 220 Asp Phe Asp Ser Leu Leu Gln Arg Gln Glu Ser Ser Val Arg Leu Trp 225 230 235 240 Lys Val Leu Ala Leu Val Phe Gly Phe Ala Thr Cys Ala Thr Leu Phe                 245 250 255 Phe Ile Leu Arg Lys Gln Tyr Leu Gln Arg Gln Glu Arg Leu Arg Leu             260 265 270 Lys Gln Met Gln Glu Glu Phe Gln Glu His Glu Ala Gln Leu Leu Ser         275 280 285 Arg Ala Lys Pro Glu Asp Arg Glu Ser Leu Lys Ser Ala Cys Val Val     290 295 300 Cys Leu Ser Ser Phe Lys Ser Cys Val Phe Leu Glu Cys Gly His Val 305 310 315 320 Cys Ser Cys Thr Glu Cys Tyr Arg Ala Leu Pro Glu Pro Lys Lys Cys                 325 330 335 Pro Ile Cys Arg Gln Ala Ile Thr Arg Val Ile Pro Leu Tyr Asn Ser             340 345 350

Claims (14)

An Akt (protein kinase B) negative modulator containing a Hades protein having the amino acid sequence of SEQ ID NO: 2 or a gene encoding the same as an active ingredient. The Akt (protein kinase B) negative modulator according to claim 1, wherein the Hades-encoding gene has a nucleotide sequence of SEQ ID NO: 1. The Akt (protein kinase B) negative modulator according to claim 2, wherein the Hades-encoding gene is inserted into a recombinant expression vector. The Akt (protein kinase B) negative modulator of claim 1, wherein the Hades has a RING finger-domain and has E3-ubiquitin ligase activity. 2. Akt (protein kinase B) negative modulator according to claim 1, wherein the Akt interacts directly with Hades and is ubiquitinated by Hades. 6. Akt (protein kinase B) negative modulator according to claim 5, characterized in that the kinase domain (KD) of Akt interacts mainly in association with Hades. The Akt (protein kinase B) negative modulator according to claim 5, wherein Akt is decomposed by ubiquitination by Hades to inhibit Akt expression. 6. The Akt (protein kinase B) negative modulator according to claim 5, wherein Hades specifically ubiquitizes only Akt activated by phosphorylation. 6. Akt (protein kinase B) negative modulator according to claim 5, characterized in that lysine 284 of Akt is the major site of ubiquitination by Hades. The Akt (protein kinase B) negative of claim 1, wherein the Akt induces cell proliferation, inhibition of apoptosis, and cell migration by downstream signaling. Modulator. The Akt (protein kinase B) negative modulator according to claim 1, wherein the Hades inhibits the proliferation, survival and cell migration of cancer cells. An anticancer agent comprising a Hades protein having an amino acid sequence of SEQ ID NO: 2 or a gene encoding the same as an active ingredient, which functions as an Akt (protein kinase B) negative regulator. Screening method for Akt (protein kinase B) negative modulator comprising the following steps:
(a) cotransfection of Akt and Hades to animal cells;
(b) treating any of the agents on the transfected animal cells; And
(c) determining whether the interaction of Akt and Hades is promoted in said animal cell. Screening method for Akt (protein kinase B) positive modulator comprising the following steps:
(a) cotransfection of Akt and Hades to animal cells;
(b) treating the transformed animal cell with any agent; And
(c) determining whether the interaction of Akt and Hades is inhibited in said animal cell.

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