- Email: [email protected]
Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway
Biochemical and Biophysical Research Communications xxx (2017) 1e8
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway Chang Woo Han 1, Mi Suk Jeong 1, Se Bok Jang* Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 46241, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 June 2017 Accepted 21 July 2017 Available online xxx
Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. HREV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that HREV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. © 2017 Elsevier Inc. All rights reserved.
Keywords: Signaling pathway K-Ras H-REV107 Mutation
1. Introduction The Ras is a 21 kDa monomeric membrane-localized G protein that functions as a nucleotide-dependent switch for central growth signaling pathways [1]. This protein acts as a guanosine diphosphate (GDP)-inactive molecular switch in resting cells and becomes activated in response to extracellular receptors by binding guanosine triphosphate (GTP), as catalyzed by the guanine nucleotide exchange factors (GEFs) SOS1 and SOS2 [2,3]. In the GTP-bound active state, Ras interacts effectively with a series of cytoplasmic target or “effector” proteins that start clear intracellular signaling cascades that lead to deregulated cell growth, inhibition of cell death, invasiveness, and induction of angiogenesis [4]. By its essential GTPase activity, Ras hydrolyzes GTP to GDP to finish the Ras signaling. This reaction is increased by GTPase-activating proteins (GAPs), which stops the Ras signaling by switching Ras into an inactive GDP-bound signaling state [5]. Upon binding, two regions of RAS go through considerable structural changes depending on the type of bound nucleotide [6]. Two structurally dynamic loops,
* Corresponding author. E-mail address: [email protected] (S.B. Jang). 1 These authors contributed equally to this article.
“Switch I” (amino acids 34e40 in Ras) and “Switch II” (aa 59e77 in Ras), set up an interaction surface for effector molecules in a GTPdependent manner [7]. The Ras genes are proto-oncogenes that are mutated in human cancers, and the proteins are encoded by three expressed genes: K-, N, and H-Ras [8]. The amino-terminal catalytic domains (amino acids 1e165) of Ras are very homologous among the three Ras isoforms of K-Ras, N-Ras, and H-Ras [9]. The carboxyl-terminal sequences diverge notably and are collectively referred to as the hypervariable region (HVR) [9]. This region includes residues that define post-translational protein modifications, which are crucial for targeting Ras proteins to the cytosolic leaflet of cellular membranes [10]. Activating Ras mutations occur in about 30% of human cancers and at surprisingly higher frequencies in pancreatic (90%), lung (35%), thyroid gland (55%), colon (45%), and liver (30%) cancers [11]. More than 95% of Ras mutations are found in codons (amino acids) for glycine 12, glycine 13, or glutamine 61 [12]. These mutations make the Ras proteins insensitive to GTP-induced hydrolysis of GTP to GDP and immerse them in the activated state [13]. Activating mutations in Ras induce constitutive signaling to downstream targets, i.e., Ras effectors. The Raf-MEK-ERK cascade is the best characterized Ras effector pathway [14]. Factors known to be Ras effectors include PI3K, RalGDS, RIN1/2, PLCε, and TIAM1 [15].
http://dx.doi.org/10.1016/j.bbrc.2017.07.120 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
2
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Of the three Ras isoforms, K-Ras is most often mutated [10]. Although many studies of H-Ras have been conducted to date, aberrant K-Ras signaling is not yet understood. Oncogenic mutations such as Q61H are mainly observed in the K-Ras gene. Overall, 15e25% of lung adenocarcinomas harbor K-ras mutations. H-REV107, which has been identified as a K-Ras interacting protein [16] and is also known as H-REV107-1, H-REV107-3, and HRSL3, is a delegate of the H-REV107 type II tumor suppressor gene family. Members of the H-REV107 type II tumor suppressor gene family include H-REV107, retinoid-inducible gene 1 (RIG1), HRASLS (A-C1), HRASLS2 and iNAT, which are present in humans, rats, and mice [17]. Overexpression of H-REV107 protein exhibits activities that adjust cellular growth, differentiation, and apoptosis in tumor cell lines [18]. The H-REV107 protein is 18 kDa (aa 1e162) and contains the NC-motif (aa 111e123) at the N-terminus and a hydrophobic C-terminal membrane-anchoring domain (aa 126e162) that was found to be crucial for cellular localization, but not needful for enzyme activity [19]. The HREV107 protein family can inhibit the Ras signaling pathway, but its accurate molecular mechanism is unknown [20]. In this study, we investigated the interaction between K-Ras and H-REV107 in vitro using several biochemical and biophysical assays. In addition, we developed a molecular docking model of the Ras and H-REV107 complex based on the homology structures. These results provide an improved understanding that will enable design of new drugs against oncogenic K-Ras. 2. Materials and methods 2.1. Cloning, mutation, and expression The wild type human K-Ras (WT, residues 1e188) and H-REV107 (residues 1e125) were subcloned into the His-tagged fusion vector pET-28a. K-Ras and H-REV107 were amplified by PCR with oligonucleotides incorporating BamHI/XhoI sites on the 50 and 30 primers and the insert fragments were incorporated into the digested plasmid by T4 ligase. The His-tagged K-Ras, H-REV107, and GST-tagged H-REV107 were transformed into the expression host Escherichia coli BL21(DE3). Each colony was then inoculated in 5 ml of LuriaBertani (LB) medium enriched with 10 mg/ml kanamycin or 50 mg/ ml ampicillin, after which the bacteria were grown overnight at 37 C. These cells were added to 2 L of LB containing antibiotics and gown at 37 C until the OD600 reached 0.5e0.6. The expression of the proteins was induced overnight by 0.5 mM isopropyl-thio-b-Dgalactopyranoside (IPTG) at 25 C, and bacterial cells were harvested by centrifugation at 3830 g for 25 min at 4 C. The cell pellets were used directly or frozen at 70 C. For analysis, the cell pellets were resuspended with lysis buffer A [50 mM Tris-HCl (pH 7.5), and 200 mM NaCl] and sonicated for cell disruption. After sonication on ice, the cell suspensions were centrifuged at 20,017 g for 45 min to remove insoluble cellular debris. The GSTtagged H-REV107 cell pellets were then resuspended with lysis buffer [1 PBS; 4.3 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl (pH7.4)]. Point mutation of K-Ras Q61H was introduced by site-directed mutagenesis. PCR based site directed mutagenesis was performed using Dpn I enzyme digestion. 2.2. Purification For purification, the soluble supernatants of the His-tagged KRas (WT and Q61H) and H-REV107 proteins were applied to Ni-NTA columns and pre-equilibrated with buffer A. The column was then washed with buffer A, after which elution of the bound protein was accomplished by varying the imidazole amount (20e200 mM). The
clear GST-H-REV107 supernatant was loaded onto a Glutathionesepharose 4 Fast Flow at a flow rate of 2.5 ml/min, and washed broadly using 20 mL of 1 PBS. The bound protein was subsequently eluted in buffer A containing 5e30 mM glutathione. Gel filtration was performed by fast protein liquid chromatography (FPLC) using a Superdex 200 10/300 GL column. Each purification step was analyzed by SDS-PAGE using 15% polyacrylamaide gel and visualized using Coomassie blue staining. 2.3. Western blotting The purified K-Ras and H-REV107 proteins isolated from the 15% SDS-PAGE gel were transferred onto an immobilon-P membrane (Millipore) at 115 V for 1 h, after which the membrane was blocked with 5% skim milk in 1 PBS buffer containing 0.1% Tween 20 (PBST) for 45 min. Next, the membrane was incubated in primary antibody [His-probe (G-18) diluted to 1:5,000, Santa Cruz Biotechnology, Inc., and GST (B-14) diluted to 1:5,000, Santa Cruz Biotechnology, Inc.] for 1 h. After washing with 1 PBS-T for 30 min, the membrane was incubated for 1 h with goat anti-rabbit IgG-HRP for reaction with the His secondary antibody and with goat anti-mouse IgG-HRP for the GST secondary antibody diluted at a ratio of 1:10,000 in blocking buffer for 1 h. 2.4. GST pull-down assay The GST pull-down assay was performed by mixing 50 mg of purified His-K-Ras and 50 mg of purified GST/GST-H-REV107 proteins. These were incubated with glutathione-sepharose 4B beads and 1 PBS binding buffer containing 5 mM MgCl2 and 5 mM GDP/ GTP/GNP. After 3 h of reaction at 4 C, the bound proteins and beads were centrifuged at 600 g for 3 min and washed three times with 1 ml of 1 PBS binding buffer. The binding proteins were eluted with buffer [50 mM Tris-HCl (pH 7.5) and 30 mM glutathione] and analyzed by 15% SDS-PAGE. Finally, proteins were subsequently visualized by immunoblotting assay using anti-His and anti-GST (Santa Cruz Biotechnology, Inc.). 2.5. Structural modeling The models of K-Ras and H-REV107 complexes were constructed using the SWISS-MODEL program. The structures of human K-Ras (PDB ID: 3GFT) and human H-REV107 (PDB ID: 2KYT) were utilized as templates for homology protein modeling, after which the secondary structures of Ras (K-, N- and H-Ras) and H-REV107 were predicted. For the initial docking between K-Ras and H-REV107 proteins, the protein of H-REV107 was assigned as the receptor and K-Ras was assigned as the ligand molecule. 2.6. Biacore biosensor analysis Measurements of the apparent dissociation constants (KD) between K-Ras (WT and Q61H) and H-REV107 were followed up using a Biacore T100 biosensor (GE Healthcare, Sweden). By an amine coupling method, the H-REV107 (50 mg/mL in 10 mM sodium acetate with a pH of 5.0) was covalently bound to the carboxylated dextran matrix at a concentration corresponding to 640 response units (RU). A flow path including two cells was used to concurrently measure the kinetic parameters from one flow cell containing the H-REV107-immobilized sensor chip to another flow cell containing an underivatized chip. For kinetic measurements at room temperature, K-Ras with concentrations ranging from 31 to 500 nM were set up by dilution in HBS buffer (150 mM NaCl, 3 mM EDTA, 10 mM HEPES and 0.005% surfactant P20) with a pH of 7.4. Each sample was injected with K-Ras solution into the flow cells at a rate of
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
10 mL/min, after which the immobilized ligand was regenerated by injecting 50 mM NaOH at a rate 10 mL/min. 2.7. Fluorescence spectroscopic analysis Fluorescence emission spectra were obtained using a SCINCO FluoroMate FS-2 with 1 cm path length cuvettes containing excitation and emission slits with widths of 20 nm. The fluorescence emission spectra of K-Ras WT, GDP/GNP bound K-Ras and HREV107 were obtained to identify their characteristic chemical structures, including double bonds and aromatic groups. The emission intensity was recorded at 305e465 nm with an excitation wavelength of 295 nm. Wild-type K-Ras, GDP/GNP bound K-Ras and H-REV107 proteins at 5 mM were preincubated for 25 C. All spectra were obtained at a protein concentration of 50 mg/ml. 2.8. Cross-linking assay The cross-linking reaction was carried out in 20 mM HEPES (pH 8.0) containing 10 mg of K-Ras and H-REV107, and K-RaseH-REV107 mixed proteins at 37 C. The cross-linking reaction was started with the addition of varying concentrations of glutaraldehyde. The reaction was allowed to proceed for 5 min, then quenched by the addition of 1 M Tris-HCl (pH 7.0) for 10 min at room temperature. Finally, individual samples were resolved in 15% SDS/PAGE along with protein size markers, after which the gel was Coomassie blue stained. 3. Results and discussion The domain structures of full-length Ras (K-, H-, and N-Ras) and H-REV107 are shown in Fig. 1A. The full-length Ras structure has a conserved domain (aa 1e165), known as the G domain, and a less conserved C-terminal tail, which is known as the hypervariable region (aa 166e188/9). The full-length H-REV107 comprised the Nterminal phospholipase domain (aa 1e125) and the putative Cterminal transmembrane domain (aa 126e162), which were connected via a NC motif (aa 111e123). The domain structures and sequence alignments of the three different protein isoforms (K-, H-, and N-Ras) showed that the G domains shared significant sequence similarity (Fig. 1B). The Ras isoforms have six alpha helices and seven b-sheets. The amino acids sequence and secondary structure of H-REV107 (1e162) has five alpha helices and six b-sheets (Fig. 1C). The bands of His-K-Ras (WT and Q61H, 1e188) were located at 26 kDa, while His-H-REV107 (1e125) was at 18 kDa (Fig. 1D). We conducted various biochemical and biophysical experiments to estimate the interaction of K-Ras (WT and Q61H) with H-REV107. Their interactions were identified using a size exclusion column (Fig. 1DeF). The purified K-Ras (WT and Q61H) and H-REV107 were mixed in a 1:1 M ratio and incubated for 12 h at 4 C, after which the mixed protein was loaded onto a Superdex 200 10/300 GL column (Amersham Pharmacia Biotech). Bindings of K-Ras (WT and Q61H) with GDP/GTP/GNP and H-REV107 were detected using SDSPAGE. Each K-Ras and H-REV107 band appeared as a sharp peak and the complex bands came out earlier than each protein peak. In addition, GTP bound K-Ras (Q61H) formed an active larger sized complex with H-REV107 than GDP or GNP bound K-Ras-H-REV107 complexes, suggesting that it may make a stable large intermediate complex [21]. The interaction between K-Ras (WT and Q61H) and H-REV107 was investigated by a GST pull-down assay, which revealed that HREV107 interacts with GDP bound K-Ras WT and GTP/GNP bound K-Ras Q61H by Western blot analysis (Fig. 2AeC). GST-H-REV107 was co-purified with GDP/GTP/GNP bound His-K-Ras, after which
3
a point mutagenesis study was implemented on the five residues of K-Ras (Fig. 2A). As a result, four mutations of residues (T20A, Q25A, Y32A, and Y40A) in K-Ras disrupted the interaction with the HREV107 complex. Fluorescence analysis of K-Ras (WT and Q61H) and H-REV107 are shown in Fig. 2D and E. The spectra of both H-REV107-GDP/GNP bound K-Ras complexes were of lower intensity than those observed when only K-Ras and H-REV107 were combined. The simple combined intensity values were the sum of each measured value of K-Ras and H-REV107. A less rigid hydrophobic environment essential for the conformational changes related to GDP/GNP bound K-Ras and H-REV107 was observed as the fluorescence intensity decreased. The binding spectra between GTP bound K-Ras Q61H and H-REV107 were not obtained successfully. K-Ras and HREV107 proteins have few aromatic group residues; therefore, the fluorescence intensity of the complexes was slightly decreased compared to when they were simply combined. When combined with the fluorescence measurements of the K-Ras and H-REV107 complexes, the present study supports their interactions. Direct physical interaction between K-Ras (WT and Q61H) and H-REV107 was also verified by surface plasmon resonance spectroscopy (Fig. 2FeG). GDP bound K-Ras WT and GNP bound K-Ras (Q61H) were bound to H-REV107 with an apparent KD of 1 and 9 nM. Both GDP bound K-Ras WT and GNP bound Q61H showed higher affinity to the H-REV107. Data for GTP bound K-Ras Q61H was not obtained. To examine the formation of oligomer structures of K-Ras (WT and Q61H) and H-REV107 proteins, chemical cross-linking interaction analysis was conducted. SDS-PAGE analysis of the crosslinked product revealed bands of K-Ras protein as dimers or tetramers and the band of the H-REV107 protein as a dimer (Fig. 2H). The location of the generated cross-links indicates that the protein has a three dimensional structure or forms a protein complex [20]. The structures of the K-Ras (1e169) (WT and Q61H) and HREV107 (1e125) were modeled using the known structures of human K-Ras (PDB ID: 3GFT) and human H-REV107 (PDB ID: 2KYT). The interactions between the GDP bound K-Ras and H-REV107 are localized at the residues of K-Ras (T20, I21, I24, Q25, E31, Y32, I36, and Y40) on the a1, loop, and b2 regions and H-REV107 (F17, P19, F20, Y21 and R22) on the b1, loop and b2 regions (Fig. 3A). The complex structure of K-Ras-H-REV107 was shown as a ribbon representation and c-alpha trace. The interaction sites between the GDP bound K-Ras WT and H-REV107 were implemented with five residues, but the four mutations produced by the substitutions of Ala in K-Ras disrupted the interaction with H-REV107 (Fig. 2A). In addition, the interaction between GNP bound K-Ras (Q61H) and HREV107 is localized at the residues of K-Ras (E31, D33, and H61) on the loop regions and H-REV107 (G70, S71, and K107) on the a2 and loop regions (Fig. 3B). In both the K-Ras (WT and Q61H) and HREV107 interaction, E31 is an important binding residue, while E31A showed a weak interaction with H-REV107 after mutation. Superimposition of the c-alpha traces of K-Ras WT (GDP) and Q61H (GNP) with H-REV107 revealed that H-REV107 rotated dramatically by ~39 in the spatial arrangement to interact with K-Ras Q61H (GNP) from the binding position with K-Ras WT (GDP) (Fig. 3C). Conformational changes between the GDP- and GNP-bound states of K-Ras are confined to two distinct regions of the protein, switch I (aa 34e40) and switch II (aa 59e77). The detailed bindings between GDP/GNP bound K-Ras (WT and Q61H) and H-REV107 are shown (Fig. 4A and B). The large conformational changes are localized at the regions of Y32-E37 and A59-Y64. Among them, the residues of Y32, I36, Q61, and E63 showed large conformational changes. Comparison of GDP bound K-Ras-H-REV107 and GNP bound K-Ras (Q61H)-H-REV107 revealed that the opened switch loop of inactive GDP bound K-Ras-H-REV107 was closed with the
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
4
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Fig. 1. Domain and secondary structures of Ras and H-REV107 and SEC binding of K-Ras WT/Q61H and H-REV107 proteins. (A) Domain structures of Ras and H-REV107 are shown. (B) Sequence alignment and secondary structure of human Ras isoforms (K-, H- and N-Ras) are shown. The residues that are identically conserved in Ras isoforms are in the blue boxes. (C) The secondary structure of H-REV107 is shown. Alpha helices are shown as ellipses, beta sheets as arrows, and loops as black lines. (DeE) Size-exclusion chromatography of K-Ras WT/Q61H and H-REV107 proteins. After application of size exclusion chromatography, the interaction of K-Ras WT(GDP)/Q61H(GNP) (green) to H-REV107 (pink) is shown using SDS-PAGE. Elution profiles of the K-Ras WT(GDP)-H-REV107 (red) and K-Ras Q61H(GNP)-H-REV107 (blue) complexes from the Superdex 200 column are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
active form of GNP bound K-Ras (Q61H)-H-REV107 (Fig. 4C and D). The H-REV107 protein family negatively regulates the activity of Ras. Several studies have shown that H-REV107 down-regulates HRas via its PLA/AT activity, and inhibits the Ras signal. Moreover, HREV107 expression is reversibly down-regulated through the MAP/
ERK pathway in a branch of tumor cells [22]; however, its exact molecular mechanism is still unknown. This study showed that HREV107 interacts strongly with active GNP-bound K-Ras mutation. The active conformation of GNP-bound K-Ras shows interaction of the g-phosphate with two structurally dynamic loops (Switch I and
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
5
Fig. 2. Binding and cross-linking assay of K-Ras WT/Q61H and H-REV107 proteins. (A) GST-tag pull-down assay of GDP bound K-Ras WT/mutants (T20A, Q25A, E31A, Y32A, and Y40A) and H-REV107 interaction is shown by Western blot. (BeC) GST-tag pull-down assays of GTP/GNP bound K-Ras Q61H and H-REV107 interaction are shown. (DeE) Fluorescence spectra of the K-Ras WT(GDP)/Q61H(GNP)-H-REV107 complex and of each protein are shown. (FeG) Biacore biosensor analysis of K-Ras WT(GDP)/Q61H(GNP)-H-REV107 is shown. The sensorgrams for 31, 125, 250, and 500 nM K-Ras are shown. (H) Cross-linking reactions of K-Ras WT(GDP)/Q61H(GNP) and H-REV107 are shown.
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
6
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Fig. 3. Modeled structures of the K-Ras WT (GDP) (PDB ID: 4OBE, green)/Q61H (GNP) (PDB ID: 3GFT, blue) and H-REV107 (orange/purple) complexes. (AeB) Modeled structures of K-Ras WT(GDP)/Q61H(GNP) and H-REV107 complexes are shown as ribbon and c-alpha traces. The bound nucleotide is shown as a stick model, and Mg2þ is shown as a gray sphere. The Switch I and II regions of K-Ras WT(GDP)/Q61H(GNP) with H-REV107 are shown in yellow. (C) The modeled structures of K-Ras WT(GDP)/Q61H(GNP) with H-REV107 are superimposed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Switch II) that hold loops (Fig. 4D). In the active closed form of GNPbound K-Ras, the K-Ras mutation interacts with H-REV107 through the Switch I and II binding region. In contrast, the inactive conformation of GDP-bound K-Ras shows an open form and the wild-type K-Ras only interacts with H-REV107 through the switch I
region. In this study, the models of secondary and tertiary structures of K-Ras and H-REV107 were prepared and evaluated. GST-pull down, fluorescence spectroscopy, and BIAcore biosensor analysis were performed to evaluate the interaction between the K-Ras and H-
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
7
Fig. 4. Detailed binding and signaling of K-Ras WT/Q61H (GDP/GNP) and H-REV107 proteins. (A) Binding of the inactive open conformation of K-Ras WT (GDP) and H-REV107 is shown as ribbon and surface representations. (B) Binding of the active closed conformation of K-Ras Q61H (GNP) and H-REV107 is shown. (C) Cartoon model of the K-Ras in open (left: GDP bound) and closed (right: GNP bound) forms are shown. (D) Inactive open and active closed structures of K-Ras WT(GDP)/Q61H(GNP) with H-REV107 are superimposed.
REV107. The results suggest that H-REV107 interacts strongly with GNP-bound K-Ras mutation (Q61H). Hence, we suggest that HREV107 can be a stable drug target for oncogenic K-Ras mutation. Further elucidation of the molecular mechanisms of H-REV107 in K-Ras signaling is very important to understanding the biology of tumors and may eventually lead to novel anti-cancer therapy.
Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology
(2015R1D1A1A01059594) to S.B.J. and (2016R1D1A1B02011142) to J.M.S. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.07.120. References [1] T. Maurer, L.S. Garrenton, A. Oh, K. Pitts, D.J. Anderson, N.J. Skelton, B.P. Fauber, B. Pan, S. Malek, D. Stokoe, M.J. Ludlam, K.K. Bowman, J. Wu, A.M. Giannetti, M.A. Starovasnik, I. Mellman, P.K. Jackson, J. Rudolph,
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
8
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
[2] [3]
[4] [5]
[6] [7]
[8] [9] [10] [11]
[12]
W. Wang, G. Fang, Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 5299e5304. A.A. Adjei, Blocking oncogenic Ras signaling for cancer therapy, J. Natl. Cancer Inst. 93 (2001) 1062e1074. K. Scheffzek, M.R. Ahmadian, W. Kabsch, L. Wiesmüller, A. Lautwein, F. Schmitz, A. Wittinghofer, The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants, Science 277 (1997) 333e339. A.L. Feig, Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases, Nat. Cell Biol. 1 (1999) E25eE27. S.M. Lim, K.D. Westover, S.B. Ficarro, R.A. Harrison, H.G. Choi, M.E. Pacold, €nne, M. Meyerson, J.A. Marto, M. Carrasco, J. Hunter, N.D. Kim, T. Sim, P.A. Ja J.R. Engen, N.S. Gray, Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor, Angew. Chem. Int. Ed. Engl. 53 (2014) 199e204. K. Rajalingam, R. Schreck, U.R. Rapp, S. Albert, Ras oncogenes and their downstream targets, Biochim. Biophys. Acta 1773 (2007) 1177e1195. J.M. Ostrem, U. Peters, M.L. Sos, J.A. Wells, K.M. Shokat, K-Ras (G12C) inhibitors allosterically control GTP affinity and effector interactions, Nature 503 (2013) 548e551. I.A. Prior, P.D. Lewis, C. Mattos, A comprehensive survey of Ras mutations in cancer, Cancer Res. 72 (2012) 2457e2467. J.F. Hancock, Ras proteins: different signals from different locations, Nat. Rev. Mol. Cell Biol. 4 (2003) 373e385. S. Schubbert, K. Shannon, G. Bollag, Hyperactive Ras in developmental disorders and cancer, Nat. Rev. Cancer 7 (2007) 295e308. E.S. Leshchiner, A. Parkhitko, G.H. Bird, J. Luccarelli, J.A. Bellairs, S. Escudero, K. Opoku-Nsiah, M. Godes, N. Perrimon, L.D. Walensky, Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 1761e1766. S. Aviel-Ronen, F.H. Blackhall, F.A. Shepherd, M.S. Tsao, K-ras mutations in non-small-cell lung carcinoma: a review, Clin. Lung Cancer 8 (2006) 30e38.
[13] Y.S. Chang, K.T. Yeh, N.C. Hsu, S.H. Lin, T.J. Chang, J.G. Chang, Detection of N-, H-, and KRAS codons 12, 13, and 61 mutations with universal RAS primer multiplex PCR and N-, H-, and KRAS-specific primer extension, Clin. Biochem. 43 (2010) 296e301. [14] P.J. Roberts, C.J. Der, Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer, Oncogene 26 (2007) 3291e3310. [15] G.A. Repasky, E.J. Chenette, C.J. Der, Renewing the conspiracy theory debate: does Raf function alone to mediate Ras oncogenesis? Trends Cell Biol. 14 (2004) 639e647. [16] X. Ren, J. Lin, C. Jin, B. Xia, Solution structure of the N-terminal catalytic domain of human H-REV107eA novel circular permutated NlpC/P60 domain, FEBS Lett. 584 (2010) 4222e4226. [17] H. Wei, L. Wang, X. Ren, W. Yu, J. Lin, C. Jin, B. Xia, Structural and functional characterization of tumor suppressors TIG3 and H-REV107, FEBS Lett. 589 (2015) 1179e1186. [18] C.H. Wang, R.Y. Shyu, C.C. Wu, T.C. Tsai, L.K. Wang, M.L. Chen, S.Y. Jiang, F.M. Tsai, Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway, J. Biomed. Sci. 21 (2014) 1e9. [19] F.M. Tsai, M.L. Chen, L.K. Wang, M.C. Lee, H-rev107 regulates cytochrome P450 reductase activity and increases lipid accumulation, PLoS One 10 (2015) e0138586. [20] M.A. Trakselis, S.C. Alley, F.T. Ishmael, Identification and mapping of proteinprotein interactions by a combination of cross-linking, cleavage, and proteomics, Bioconjug Chem. 16 (2005) 741e750. [21] T.H. Jang, C. Zheng, J. Li, C. Richards, Y.S. Hsiao, T. Walz, H. Wu, H.H. Park, Structural study of the RIPoptosome core reveals a helical assembly for kinase recruitment, Biochemistry 53 (2014) 5424e5431. [22] I. Nazarenko, G. Kristiansen, S. Fonfara, R. Guenther, C. Gieseler, W. Kemmner, R. Schafer, I. Petersen, C. Sers, H-REV107-1 stimulates growth in non-small cell lung carcinomas via the activation of mitogenic signaling, Am. J. Pathol. 169 (2006) 1427e1439.
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway Chang Woo Han 1, Mi Suk Jeong 1, Se Bok Jang* Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 46241, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 June 2017 Accepted 21 July 2017 Available online xxx
Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. HREV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that HREV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. © 2017 Elsevier Inc. All rights reserved.
Keywords: Signaling pathway K-Ras H-REV107 Mutation
1. Introduction The Ras is a 21 kDa monomeric membrane-localized G protein that functions as a nucleotide-dependent switch for central growth signaling pathways [1]. This protein acts as a guanosine diphosphate (GDP)-inactive molecular switch in resting cells and becomes activated in response to extracellular receptors by binding guanosine triphosphate (GTP), as catalyzed by the guanine nucleotide exchange factors (GEFs) SOS1 and SOS2 [2,3]. In the GTP-bound active state, Ras interacts effectively with a series of cytoplasmic target or “effector” proteins that start clear intracellular signaling cascades that lead to deregulated cell growth, inhibition of cell death, invasiveness, and induction of angiogenesis [4]. By its essential GTPase activity, Ras hydrolyzes GTP to GDP to finish the Ras signaling. This reaction is increased by GTPase-activating proteins (GAPs), which stops the Ras signaling by switching Ras into an inactive GDP-bound signaling state [5]. Upon binding, two regions of RAS go through considerable structural changes depending on the type of bound nucleotide [6]. Two structurally dynamic loops,
* Corresponding author. E-mail address: [email protected] (S.B. Jang). 1 These authors contributed equally to this article.
“Switch I” (amino acids 34e40 in Ras) and “Switch II” (aa 59e77 in Ras), set up an interaction surface for effector molecules in a GTPdependent manner [7]. The Ras genes are proto-oncogenes that are mutated in human cancers, and the proteins are encoded by three expressed genes: K-, N, and H-Ras [8]. The amino-terminal catalytic domains (amino acids 1e165) of Ras are very homologous among the three Ras isoforms of K-Ras, N-Ras, and H-Ras [9]. The carboxyl-terminal sequences diverge notably and are collectively referred to as the hypervariable region (HVR) [9]. This region includes residues that define post-translational protein modifications, which are crucial for targeting Ras proteins to the cytosolic leaflet of cellular membranes [10]. Activating Ras mutations occur in about 30% of human cancers and at surprisingly higher frequencies in pancreatic (90%), lung (35%), thyroid gland (55%), colon (45%), and liver (30%) cancers [11]. More than 95% of Ras mutations are found in codons (amino acids) for glycine 12, glycine 13, or glutamine 61 [12]. These mutations make the Ras proteins insensitive to GTP-induced hydrolysis of GTP to GDP and immerse them in the activated state [13]. Activating mutations in Ras induce constitutive signaling to downstream targets, i.e., Ras effectors. The Raf-MEK-ERK cascade is the best characterized Ras effector pathway [14]. Factors known to be Ras effectors include PI3K, RalGDS, RIN1/2, PLCε, and TIAM1 [15].
http://dx.doi.org/10.1016/j.bbrc.2017.07.120 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
2
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Of the three Ras isoforms, K-Ras is most often mutated [10]. Although many studies of H-Ras have been conducted to date, aberrant K-Ras signaling is not yet understood. Oncogenic mutations such as Q61H are mainly observed in the K-Ras gene. Overall, 15e25% of lung adenocarcinomas harbor K-ras mutations. H-REV107, which has been identified as a K-Ras interacting protein [16] and is also known as H-REV107-1, H-REV107-3, and HRSL3, is a delegate of the H-REV107 type II tumor suppressor gene family. Members of the H-REV107 type II tumor suppressor gene family include H-REV107, retinoid-inducible gene 1 (RIG1), HRASLS (A-C1), HRASLS2 and iNAT, which are present in humans, rats, and mice [17]. Overexpression of H-REV107 protein exhibits activities that adjust cellular growth, differentiation, and apoptosis in tumor cell lines [18]. The H-REV107 protein is 18 kDa (aa 1e162) and contains the NC-motif (aa 111e123) at the N-terminus and a hydrophobic C-terminal membrane-anchoring domain (aa 126e162) that was found to be crucial for cellular localization, but not needful for enzyme activity [19]. The HREV107 protein family can inhibit the Ras signaling pathway, but its accurate molecular mechanism is unknown [20]. In this study, we investigated the interaction between K-Ras and H-REV107 in vitro using several biochemical and biophysical assays. In addition, we developed a molecular docking model of the Ras and H-REV107 complex based on the homology structures. These results provide an improved understanding that will enable design of new drugs against oncogenic K-Ras. 2. Materials and methods 2.1. Cloning, mutation, and expression The wild type human K-Ras (WT, residues 1e188) and H-REV107 (residues 1e125) were subcloned into the His-tagged fusion vector pET-28a. K-Ras and H-REV107 were amplified by PCR with oligonucleotides incorporating BamHI/XhoI sites on the 50 and 30 primers and the insert fragments were incorporated into the digested plasmid by T4 ligase. The His-tagged K-Ras, H-REV107, and GST-tagged H-REV107 were transformed into the expression host Escherichia coli BL21(DE3). Each colony was then inoculated in 5 ml of LuriaBertani (LB) medium enriched with 10 mg/ml kanamycin or 50 mg/ ml ampicillin, after which the bacteria were grown overnight at 37 C. These cells were added to 2 L of LB containing antibiotics and gown at 37 C until the OD600 reached 0.5e0.6. The expression of the proteins was induced overnight by 0.5 mM isopropyl-thio-b-Dgalactopyranoside (IPTG) at 25 C, and bacterial cells were harvested by centrifugation at 3830 g for 25 min at 4 C. The cell pellets were used directly or frozen at 70 C. For analysis, the cell pellets were resuspended with lysis buffer A [50 mM Tris-HCl (pH 7.5), and 200 mM NaCl] and sonicated for cell disruption. After sonication on ice, the cell suspensions were centrifuged at 20,017 g for 45 min to remove insoluble cellular debris. The GSTtagged H-REV107 cell pellets were then resuspended with lysis buffer [1 PBS; 4.3 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl (pH7.4)]. Point mutation of K-Ras Q61H was introduced by site-directed mutagenesis. PCR based site directed mutagenesis was performed using Dpn I enzyme digestion. 2.2. Purification For purification, the soluble supernatants of the His-tagged KRas (WT and Q61H) and H-REV107 proteins were applied to Ni-NTA columns and pre-equilibrated with buffer A. The column was then washed with buffer A, after which elution of the bound protein was accomplished by varying the imidazole amount (20e200 mM). The
clear GST-H-REV107 supernatant was loaded onto a Glutathionesepharose 4 Fast Flow at a flow rate of 2.5 ml/min, and washed broadly using 20 mL of 1 PBS. The bound protein was subsequently eluted in buffer A containing 5e30 mM glutathione. Gel filtration was performed by fast protein liquid chromatography (FPLC) using a Superdex 200 10/300 GL column. Each purification step was analyzed by SDS-PAGE using 15% polyacrylamaide gel and visualized using Coomassie blue staining. 2.3. Western blotting The purified K-Ras and H-REV107 proteins isolated from the 15% SDS-PAGE gel were transferred onto an immobilon-P membrane (Millipore) at 115 V for 1 h, after which the membrane was blocked with 5% skim milk in 1 PBS buffer containing 0.1% Tween 20 (PBST) for 45 min. Next, the membrane was incubated in primary antibody [His-probe (G-18) diluted to 1:5,000, Santa Cruz Biotechnology, Inc., and GST (B-14) diluted to 1:5,000, Santa Cruz Biotechnology, Inc.] for 1 h. After washing with 1 PBS-T for 30 min, the membrane was incubated for 1 h with goat anti-rabbit IgG-HRP for reaction with the His secondary antibody and with goat anti-mouse IgG-HRP for the GST secondary antibody diluted at a ratio of 1:10,000 in blocking buffer for 1 h. 2.4. GST pull-down assay The GST pull-down assay was performed by mixing 50 mg of purified His-K-Ras and 50 mg of purified GST/GST-H-REV107 proteins. These were incubated with glutathione-sepharose 4B beads and 1 PBS binding buffer containing 5 mM MgCl2 and 5 mM GDP/ GTP/GNP. After 3 h of reaction at 4 C, the bound proteins and beads were centrifuged at 600 g for 3 min and washed three times with 1 ml of 1 PBS binding buffer. The binding proteins were eluted with buffer [50 mM Tris-HCl (pH 7.5) and 30 mM glutathione] and analyzed by 15% SDS-PAGE. Finally, proteins were subsequently visualized by immunoblotting assay using anti-His and anti-GST (Santa Cruz Biotechnology, Inc.). 2.5. Structural modeling The models of K-Ras and H-REV107 complexes were constructed using the SWISS-MODEL program. The structures of human K-Ras (PDB ID: 3GFT) and human H-REV107 (PDB ID: 2KYT) were utilized as templates for homology protein modeling, after which the secondary structures of Ras (K-, N- and H-Ras) and H-REV107 were predicted. For the initial docking between K-Ras and H-REV107 proteins, the protein of H-REV107 was assigned as the receptor and K-Ras was assigned as the ligand molecule. 2.6. Biacore biosensor analysis Measurements of the apparent dissociation constants (KD) between K-Ras (WT and Q61H) and H-REV107 were followed up using a Biacore T100 biosensor (GE Healthcare, Sweden). By an amine coupling method, the H-REV107 (50 mg/mL in 10 mM sodium acetate with a pH of 5.0) was covalently bound to the carboxylated dextran matrix at a concentration corresponding to 640 response units (RU). A flow path including two cells was used to concurrently measure the kinetic parameters from one flow cell containing the H-REV107-immobilized sensor chip to another flow cell containing an underivatized chip. For kinetic measurements at room temperature, K-Ras with concentrations ranging from 31 to 500 nM were set up by dilution in HBS buffer (150 mM NaCl, 3 mM EDTA, 10 mM HEPES and 0.005% surfactant P20) with a pH of 7.4. Each sample was injected with K-Ras solution into the flow cells at a rate of
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
10 mL/min, after which the immobilized ligand was regenerated by injecting 50 mM NaOH at a rate 10 mL/min. 2.7. Fluorescence spectroscopic analysis Fluorescence emission spectra were obtained using a SCINCO FluoroMate FS-2 with 1 cm path length cuvettes containing excitation and emission slits with widths of 20 nm. The fluorescence emission spectra of K-Ras WT, GDP/GNP bound K-Ras and HREV107 were obtained to identify their characteristic chemical structures, including double bonds and aromatic groups. The emission intensity was recorded at 305e465 nm with an excitation wavelength of 295 nm. Wild-type K-Ras, GDP/GNP bound K-Ras and H-REV107 proteins at 5 mM were preincubated for 25 C. All spectra were obtained at a protein concentration of 50 mg/ml. 2.8. Cross-linking assay The cross-linking reaction was carried out in 20 mM HEPES (pH 8.0) containing 10 mg of K-Ras and H-REV107, and K-RaseH-REV107 mixed proteins at 37 C. The cross-linking reaction was started with the addition of varying concentrations of glutaraldehyde. The reaction was allowed to proceed for 5 min, then quenched by the addition of 1 M Tris-HCl (pH 7.0) for 10 min at room temperature. Finally, individual samples were resolved in 15% SDS/PAGE along with protein size markers, after which the gel was Coomassie blue stained. 3. Results and discussion The domain structures of full-length Ras (K-, H-, and N-Ras) and H-REV107 are shown in Fig. 1A. The full-length Ras structure has a conserved domain (aa 1e165), known as the G domain, and a less conserved C-terminal tail, which is known as the hypervariable region (aa 166e188/9). The full-length H-REV107 comprised the Nterminal phospholipase domain (aa 1e125) and the putative Cterminal transmembrane domain (aa 126e162), which were connected via a NC motif (aa 111e123). The domain structures and sequence alignments of the three different protein isoforms (K-, H-, and N-Ras) showed that the G domains shared significant sequence similarity (Fig. 1B). The Ras isoforms have six alpha helices and seven b-sheets. The amino acids sequence and secondary structure of H-REV107 (1e162) has five alpha helices and six b-sheets (Fig. 1C). The bands of His-K-Ras (WT and Q61H, 1e188) were located at 26 kDa, while His-H-REV107 (1e125) was at 18 kDa (Fig. 1D). We conducted various biochemical and biophysical experiments to estimate the interaction of K-Ras (WT and Q61H) with H-REV107. Their interactions were identified using a size exclusion column (Fig. 1DeF). The purified K-Ras (WT and Q61H) and H-REV107 were mixed in a 1:1 M ratio and incubated for 12 h at 4 C, after which the mixed protein was loaded onto a Superdex 200 10/300 GL column (Amersham Pharmacia Biotech). Bindings of K-Ras (WT and Q61H) with GDP/GTP/GNP and H-REV107 were detected using SDSPAGE. Each K-Ras and H-REV107 band appeared as a sharp peak and the complex bands came out earlier than each protein peak. In addition, GTP bound K-Ras (Q61H) formed an active larger sized complex with H-REV107 than GDP or GNP bound K-Ras-H-REV107 complexes, suggesting that it may make a stable large intermediate complex [21]. The interaction between K-Ras (WT and Q61H) and H-REV107 was investigated by a GST pull-down assay, which revealed that HREV107 interacts with GDP bound K-Ras WT and GTP/GNP bound K-Ras Q61H by Western blot analysis (Fig. 2AeC). GST-H-REV107 was co-purified with GDP/GTP/GNP bound His-K-Ras, after which
3
a point mutagenesis study was implemented on the five residues of K-Ras (Fig. 2A). As a result, four mutations of residues (T20A, Q25A, Y32A, and Y40A) in K-Ras disrupted the interaction with the HREV107 complex. Fluorescence analysis of K-Ras (WT and Q61H) and H-REV107 are shown in Fig. 2D and E. The spectra of both H-REV107-GDP/GNP bound K-Ras complexes were of lower intensity than those observed when only K-Ras and H-REV107 were combined. The simple combined intensity values were the sum of each measured value of K-Ras and H-REV107. A less rigid hydrophobic environment essential for the conformational changes related to GDP/GNP bound K-Ras and H-REV107 was observed as the fluorescence intensity decreased. The binding spectra between GTP bound K-Ras Q61H and H-REV107 were not obtained successfully. K-Ras and HREV107 proteins have few aromatic group residues; therefore, the fluorescence intensity of the complexes was slightly decreased compared to when they were simply combined. When combined with the fluorescence measurements of the K-Ras and H-REV107 complexes, the present study supports their interactions. Direct physical interaction between K-Ras (WT and Q61H) and H-REV107 was also verified by surface plasmon resonance spectroscopy (Fig. 2FeG). GDP bound K-Ras WT and GNP bound K-Ras (Q61H) were bound to H-REV107 with an apparent KD of 1 and 9 nM. Both GDP bound K-Ras WT and GNP bound Q61H showed higher affinity to the H-REV107. Data for GTP bound K-Ras Q61H was not obtained. To examine the formation of oligomer structures of K-Ras (WT and Q61H) and H-REV107 proteins, chemical cross-linking interaction analysis was conducted. SDS-PAGE analysis of the crosslinked product revealed bands of K-Ras protein as dimers or tetramers and the band of the H-REV107 protein as a dimer (Fig. 2H). The location of the generated cross-links indicates that the protein has a three dimensional structure or forms a protein complex [20]. The structures of the K-Ras (1e169) (WT and Q61H) and HREV107 (1e125) were modeled using the known structures of human K-Ras (PDB ID: 3GFT) and human H-REV107 (PDB ID: 2KYT). The interactions between the GDP bound K-Ras and H-REV107 are localized at the residues of K-Ras (T20, I21, I24, Q25, E31, Y32, I36, and Y40) on the a1, loop, and b2 regions and H-REV107 (F17, P19, F20, Y21 and R22) on the b1, loop and b2 regions (Fig. 3A). The complex structure of K-Ras-H-REV107 was shown as a ribbon representation and c-alpha trace. The interaction sites between the GDP bound K-Ras WT and H-REV107 were implemented with five residues, but the four mutations produced by the substitutions of Ala in K-Ras disrupted the interaction with H-REV107 (Fig. 2A). In addition, the interaction between GNP bound K-Ras (Q61H) and HREV107 is localized at the residues of K-Ras (E31, D33, and H61) on the loop regions and H-REV107 (G70, S71, and K107) on the a2 and loop regions (Fig. 3B). In both the K-Ras (WT and Q61H) and HREV107 interaction, E31 is an important binding residue, while E31A showed a weak interaction with H-REV107 after mutation. Superimposition of the c-alpha traces of K-Ras WT (GDP) and Q61H (GNP) with H-REV107 revealed that H-REV107 rotated dramatically by ~39 in the spatial arrangement to interact with K-Ras Q61H (GNP) from the binding position with K-Ras WT (GDP) (Fig. 3C). Conformational changes between the GDP- and GNP-bound states of K-Ras are confined to two distinct regions of the protein, switch I (aa 34e40) and switch II (aa 59e77). The detailed bindings between GDP/GNP bound K-Ras (WT and Q61H) and H-REV107 are shown (Fig. 4A and B). The large conformational changes are localized at the regions of Y32-E37 and A59-Y64. Among them, the residues of Y32, I36, Q61, and E63 showed large conformational changes. Comparison of GDP bound K-Ras-H-REV107 and GNP bound K-Ras (Q61H)-H-REV107 revealed that the opened switch loop of inactive GDP bound K-Ras-H-REV107 was closed with the
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
4
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Fig. 1. Domain and secondary structures of Ras and H-REV107 and SEC binding of K-Ras WT/Q61H and H-REV107 proteins. (A) Domain structures of Ras and H-REV107 are shown. (B) Sequence alignment and secondary structure of human Ras isoforms (K-, H- and N-Ras) are shown. The residues that are identically conserved in Ras isoforms are in the blue boxes. (C) The secondary structure of H-REV107 is shown. Alpha helices are shown as ellipses, beta sheets as arrows, and loops as black lines. (DeE) Size-exclusion chromatography of K-Ras WT/Q61H and H-REV107 proteins. After application of size exclusion chromatography, the interaction of K-Ras WT(GDP)/Q61H(GNP) (green) to H-REV107 (pink) is shown using SDS-PAGE. Elution profiles of the K-Ras WT(GDP)-H-REV107 (red) and K-Ras Q61H(GNP)-H-REV107 (blue) complexes from the Superdex 200 column are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
active form of GNP bound K-Ras (Q61H)-H-REV107 (Fig. 4C and D). The H-REV107 protein family negatively regulates the activity of Ras. Several studies have shown that H-REV107 down-regulates HRas via its PLA/AT activity, and inhibits the Ras signal. Moreover, HREV107 expression is reversibly down-regulated through the MAP/
ERK pathway in a branch of tumor cells [22]; however, its exact molecular mechanism is still unknown. This study showed that HREV107 interacts strongly with active GNP-bound K-Ras mutation. The active conformation of GNP-bound K-Ras shows interaction of the g-phosphate with two structurally dynamic loops (Switch I and
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
5
Fig. 2. Binding and cross-linking assay of K-Ras WT/Q61H and H-REV107 proteins. (A) GST-tag pull-down assay of GDP bound K-Ras WT/mutants (T20A, Q25A, E31A, Y32A, and Y40A) and H-REV107 interaction is shown by Western blot. (BeC) GST-tag pull-down assays of GTP/GNP bound K-Ras Q61H and H-REV107 interaction are shown. (DeE) Fluorescence spectra of the K-Ras WT(GDP)/Q61H(GNP)-H-REV107 complex and of each protein are shown. (FeG) Biacore biosensor analysis of K-Ras WT(GDP)/Q61H(GNP)-H-REV107 is shown. The sensorgrams for 31, 125, 250, and 500 nM K-Ras are shown. (H) Cross-linking reactions of K-Ras WT(GDP)/Q61H(GNP) and H-REV107 are shown.
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
6
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Fig. 3. Modeled structures of the K-Ras WT (GDP) (PDB ID: 4OBE, green)/Q61H (GNP) (PDB ID: 3GFT, blue) and H-REV107 (orange/purple) complexes. (AeB) Modeled structures of K-Ras WT(GDP)/Q61H(GNP) and H-REV107 complexes are shown as ribbon and c-alpha traces. The bound nucleotide is shown as a stick model, and Mg2þ is shown as a gray sphere. The Switch I and II regions of K-Ras WT(GDP)/Q61H(GNP) with H-REV107 are shown in yellow. (C) The modeled structures of K-Ras WT(GDP)/Q61H(GNP) with H-REV107 are superimposed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Switch II) that hold loops (Fig. 4D). In the active closed form of GNPbound K-Ras, the K-Ras mutation interacts with H-REV107 through the Switch I and II binding region. In contrast, the inactive conformation of GDP-bound K-Ras shows an open form and the wild-type K-Ras only interacts with H-REV107 through the switch I
region. In this study, the models of secondary and tertiary structures of K-Ras and H-REV107 were prepared and evaluated. GST-pull down, fluorescence spectroscopy, and BIAcore biosensor analysis were performed to evaluate the interaction between the K-Ras and H-
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
7
Fig. 4. Detailed binding and signaling of K-Ras WT/Q61H (GDP/GNP) and H-REV107 proteins. (A) Binding of the inactive open conformation of K-Ras WT (GDP) and H-REV107 is shown as ribbon and surface representations. (B) Binding of the active closed conformation of K-Ras Q61H (GNP) and H-REV107 is shown. (C) Cartoon model of the K-Ras in open (left: GDP bound) and closed (right: GNP bound) forms are shown. (D) Inactive open and active closed structures of K-Ras WT(GDP)/Q61H(GNP) with H-REV107 are superimposed.
REV107. The results suggest that H-REV107 interacts strongly with GNP-bound K-Ras mutation (Q61H). Hence, we suggest that HREV107 can be a stable drug target for oncogenic K-Ras mutation. Further elucidation of the molecular mechanisms of H-REV107 in K-Ras signaling is very important to understanding the biology of tumors and may eventually lead to novel anti-cancer therapy.
Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology
(2015R1D1A1A01059594) to S.B.J. and (2016R1D1A1B02011142) to J.M.S. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.07.120. References [1] T. Maurer, L.S. Garrenton, A. Oh, K. Pitts, D.J. Anderson, N.J. Skelton, B.P. Fauber, B. Pan, S. Malek, D. Stokoe, M.J. Ludlam, K.K. Bowman, J. Wu, A.M. Giannetti, M.A. Starovasnik, I. Mellman, P.K. Jackson, J. Rudolph,
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120
8
C.W. Han et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
[2] [3]
[4] [5]
[6] [7]
[8] [9] [10] [11]
[12]
W. Wang, G. Fang, Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 5299e5304. A.A. Adjei, Blocking oncogenic Ras signaling for cancer therapy, J. Natl. Cancer Inst. 93 (2001) 1062e1074. K. Scheffzek, M.R. Ahmadian, W. Kabsch, L. Wiesmüller, A. Lautwein, F. Schmitz, A. Wittinghofer, The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants, Science 277 (1997) 333e339. A.L. Feig, Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases, Nat. Cell Biol. 1 (1999) E25eE27. S.M. Lim, K.D. Westover, S.B. Ficarro, R.A. Harrison, H.G. Choi, M.E. Pacold, €nne, M. Meyerson, J.A. Marto, M. Carrasco, J. Hunter, N.D. Kim, T. Sim, P.A. Ja J.R. Engen, N.S. Gray, Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor, Angew. Chem. Int. Ed. Engl. 53 (2014) 199e204. K. Rajalingam, R. Schreck, U.R. Rapp, S. Albert, Ras oncogenes and their downstream targets, Biochim. Biophys. Acta 1773 (2007) 1177e1195. J.M. Ostrem, U. Peters, M.L. Sos, J.A. Wells, K.M. Shokat, K-Ras (G12C) inhibitors allosterically control GTP affinity and effector interactions, Nature 503 (2013) 548e551. I.A. Prior, P.D. Lewis, C. Mattos, A comprehensive survey of Ras mutations in cancer, Cancer Res. 72 (2012) 2457e2467. J.F. Hancock, Ras proteins: different signals from different locations, Nat. Rev. Mol. Cell Biol. 4 (2003) 373e385. S. Schubbert, K. Shannon, G. Bollag, Hyperactive Ras in developmental disorders and cancer, Nat. Rev. Cancer 7 (2007) 295e308. E.S. Leshchiner, A. Parkhitko, G.H. Bird, J. Luccarelli, J.A. Bellairs, S. Escudero, K. Opoku-Nsiah, M. Godes, N. Perrimon, L.D. Walensky, Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 1761e1766. S. Aviel-Ronen, F.H. Blackhall, F.A. Shepherd, M.S. Tsao, K-ras mutations in non-small-cell lung carcinoma: a review, Clin. Lung Cancer 8 (2006) 30e38.
[13] Y.S. Chang, K.T. Yeh, N.C. Hsu, S.H. Lin, T.J. Chang, J.G. Chang, Detection of N-, H-, and KRAS codons 12, 13, and 61 mutations with universal RAS primer multiplex PCR and N-, H-, and KRAS-specific primer extension, Clin. Biochem. 43 (2010) 296e301. [14] P.J. Roberts, C.J. Der, Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer, Oncogene 26 (2007) 3291e3310. [15] G.A. Repasky, E.J. Chenette, C.J. Der, Renewing the conspiracy theory debate: does Raf function alone to mediate Ras oncogenesis? Trends Cell Biol. 14 (2004) 639e647. [16] X. Ren, J. Lin, C. Jin, B. Xia, Solution structure of the N-terminal catalytic domain of human H-REV107eA novel circular permutated NlpC/P60 domain, FEBS Lett. 584 (2010) 4222e4226. [17] H. Wei, L. Wang, X. Ren, W. Yu, J. Lin, C. Jin, B. Xia, Structural and functional characterization of tumor suppressors TIG3 and H-REV107, FEBS Lett. 589 (2015) 1179e1186. [18] C.H. Wang, R.Y. Shyu, C.C. Wu, T.C. Tsai, L.K. Wang, M.L. Chen, S.Y. Jiang, F.M. Tsai, Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway, J. Biomed. Sci. 21 (2014) 1e9. [19] F.M. Tsai, M.L. Chen, L.K. Wang, M.C. Lee, H-rev107 regulates cytochrome P450 reductase activity and increases lipid accumulation, PLoS One 10 (2015) e0138586. [20] M.A. Trakselis, S.C. Alley, F.T. Ishmael, Identification and mapping of proteinprotein interactions by a combination of cross-linking, cleavage, and proteomics, Bioconjug Chem. 16 (2005) 741e750. [21] T.H. Jang, C. Zheng, J. Li, C. Richards, Y.S. Hsiao, T. Walz, H. Wu, H.H. Park, Structural study of the RIPoptosome core reveals a helical assembly for kinase recruitment, Biochemistry 53 (2014) 5424e5431. [22] I. Nazarenko, G. Kristiansen, S. Fonfara, R. Guenther, C. Gieseler, W. Kemmner, R. Schafer, I. Petersen, C. Sers, H-REV107-1 stimulates growth in non-small cell lung carcinomas via the activation of mitogenic signaling, Am. J. Pathol. 169 (2006) 1427e1439.
Please cite this article in press as: C.W. Han, et al., Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.120