Shigeyuki Yokoyama

Oncogenic Ras triggers cell suicide through the activation of a caspase-independent cell death program in human cancer cells

To prevent neoplasia, cells of multicellular organisms activate cellular disposal programs such as apoptosis in response to deregulated oncogene expression, making the suppression of such programs an essential step for potentially neoplastic cells to become established as clinically relevant tumors. Since the mutation of ras proto-oncogenes, the most frequently mutated proto-oncogenes in human tumors, is very rare in some tumor types such as glioblastomas and gastric cancers, we hypothesized that mutated ras genes might activate a cell death program that cannot be overcome by these tumor types. Here we show that the expression of oncogenically mutated ras gene induces cellular degeneration accompanied by cytoplasmic vacuoles in human glioma and gastric cancer cell lines. Cells dying as a result of oncogenic Ras expression had relatively well-preserved nuclei that were negative for TUNEL staining. An immunocytochemical analysis demonstrated that the cytoplasmic vacuoles are derived mainly from lysosomes. This oncogenic Ras-induced cell death occurred in the absence of caspase activation, and was not inhibited by the overexpression of anti-apoptotic Bcl-2 protein. These observations suggested that oncogenic Ras-induced cell death is most consistent with a type of programmed cell death designated `type 2 physiological cell death or `autophagic degeneration, and that this cell death is regulated by a molecular mechanism distinct from that of apoptosis. Our findings suggest a possible role for this non-apoptotic cell death in the prevention of neoplasia, and the activation of the non-apoptotic cell death program may become a potential cancer therapy complementing apoptosis-based therapies. In addition, the approach used in this study may be a valuable way to find genetically-regulated cell suicide programs that cannot be overcome by particular tumor types.

Raf/MAPK and rapamycin-sensitive pathways mediate the anti-apoptotic function of p21Ras in IL-3-dependent hematopoietic cells

The Ras signal transduction pathway is activated by a number of hematopoietic cytokines and is implicated in the prevention of apoptotic death in hematopoietic cells. Recent studies have provided evidence that the downstream of Ras is highly divergent and several independent pathways appear to mediate distinct biological functions of Ras. In the present study, we investigated the downstream pathway(s) of Ras responsible for the maintenance of hematopoietic cell survival by using various mutants of signaling molecules. Activation of the Raf/MAPK pathway in interleukin (IL) 3-dependent cells by expression of an oncogenic Raf or a Ras mutant (G12V/T35S) prevented apoptosis following IL-3 deprivation. In contrast, another Ras mutant (G12V/V45E), which is apparently incapable of activating MAPK, efficiently blocked apoptosis as well. It is therefore likely that the activation of the Raf/MAPK pathway is not an absolute requirement for the prevention of apoptosis, and there appears to be a Raf/MAPK-independent pathway that contributes to hematopoietic cell survival. Since Ras(G12V/V45E) was able to cause the phosphorylation of p70/S6 kinase, we inhibited the S6 kinase pathway by rapamycin and by wortmannin, and found that the anti-apoptotic function of Ras(G12V/V45E), but not of Ras(G12V), was critically influenced by both inhibitors. These results indicate that the Raf/MAPK and a rapamycin/wortmannin-sensitive pathways mediate Ras function to prevent apoptotic death in hematopoietic cells.

Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis

We developed two methods for stable-isotope labeling of proteins by cell-free synthesis. Firstly, we applied cell-free synthesis to the dual amino acid-selective 13C-15N labeling method, originally developed for in vivo systems by Kainosho and co-workers. For this purpose, we took one of the advantages of a cell-free protein synthesis system; the amino acid-selective stable-isotope labeling is free of the isotope scrambling problem. The targets of selective observation were Thr35 and Ser39 in the ‘effector region’ (residues 32–40) of the Ras protein complexed with the Ras-binding domain of c-Raf-1 (Raf RBD) (the total molecular mass is about 30 kDa). Using a 15-mL Escherichia coli cell-free system, which was optimized to produce about 0.4 mg of Ras protein per 1-mL reaction, with 2 mg each of DL-[13C′]proline and L-[15N]threonine, we obtained about 6 mg of Ras protein. As the Pro–Thr sequence is unique in the Ras protein, the Thr35 cross peak of the Ras•Raf RBD complex was unambiguously identified by the 2D 1H–15N HNCO experiment. The Ser39 cross peak was similarly identified with the [13C′]Asp/[15N]Ser-selectively labeled Ras protein. There were no isotope scrambling problems in this study. Secondly, we have established a method for producing a milligram quantity of site-specifically stable-isotope labeled protein by a cell-free system involving amber suppression. The E. coli amber suppressor tRNATyr_CUA (25 mg) was prepared by in vitro transcription with T7 RNA polymerase. We aminoacylated the tRNATyr_CUA transcript with purified E. coli tyrosyl-tRNA synthetase, using 2 mg of l-[15N]tyrosine. In the gene encoding the Ras protein, the codon for Tyr32 was changed to an amber codon (TAG). This template DNA and the [15N]Tyr-tRNATyr_CUA were reacted for 30 min in 30 mL of E. coli cell-free system. The subsequent purification yielded 2.2 mg of [15N]Tyr32-Ras protein. In the 1H–15N HSQC spectrum of the labeled Ras protein, only one cross peak was observed, which was unambiguously assigned to Tyr32.

Differential Structural Requirements for Interaction of Ras Protein with Its Distinct Downstream Effectors

Ras proteins have multiple effectors of distinct structures that do not share significant structural homology at their Ras interaction sites. To prove possible differences in their recognition mechanisms of Ras, we screened 44 human Ha-Ras proteins carrying mutations in the effector region and its flanking sequences for interaction with human Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenylyl cyclase. The Ras binding specificities were largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their affinities for Byr2 selectively reduced. The only exception was Ras(D38N), which lost the ability to bind Raf-1 while retaining the activity to bind Byr2 and complement the Byr2 phenotype of S. pombe. On the other hand, adenylyl cyclase had quite distinct requirements for Ras residues; mutations P34G and T58A selectively abolished the ability to bind and activate it without considerably affecting the interaction with Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In addition, V45E mutation was found to impair the ability of Ras to activate both Raf-1 and adenylyl cyclase without significantly affecting the binding affinities for them. These results demonstrate that significant differences exist in the recognition mechanisms by which the three effector molecules associate with Ras and suggest that a region of Ras required for activation of the effectors in general may exist separately from that for binding the effectors.

An approach to global fold determination using limited NMR data from larger proteins selectively protonated at specific residue types

SummaryA combination of calculation and experiment is used to demonstrate that the global fold of larger proteins can be rapidly determined using limited NMR data. The approach involves a combination of heteronuclear triple resonance NMR experiments with protonation of selected residue types in an otherwise completely deuterated protein. This method of labelling produces proteins with α-specific deuteration in the protonated residues, and the results suggest that this will improve the sensitivity of experiments involving correlation of side-chain (1H and 13C) and backbone (1H and 15N) amide resonances. It will allow the rapid assignment of backbone resonances with high sensitivity and the determination of a reasonable structural model of a protein based on limited NOE restraints, an application that is of increasing importance as data from the large number of genome sequencing projects accumulates. The method that we propose should also be of utility in extending the use of NMR spectroscopy to determine the structures of larger proteins.

A helix-turn-helix structure unit in human centromere protein B (CENP-B).

CENP‐B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes. The N‐terminal portion of CENP‐B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically binds to the CENP‐B box sequence (17 bp) in centromere DNA. We determined the solution structure of human CENP‐B DBD RP1 by multi‐dimensional 1H, 13C and 15N NMR methods. The CENP‐B DBD RP1 structure consists of four helices and has a helix–turn–helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found. The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP‐B DBD RP1. We studied the interaction between CENP‐B DBD RP1 and the CENP‐B box by the use of NMR chemical shift perturbation. The results suggest that CENP‐B DBD RP1 interacts with one of the essential regions of the CENP‐B box DNA, mainly at the N‐terminal basic region, the N‐terminal portion of helix 2 and helix 3.

Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis

Nonnatural amino acids with photofunctional groups were incorporated site‐specifically into a polypeptide by using in vitro protein synthesizing system. The nonnatural amino acids were attached to tRNACCU through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon. l‐p‐Phenylazophenylalanine, l‐2‐anthrylalanine, l‐1‐naphthylalanine, l‐2‐naphthylalanine and l‐p‐biphenylalanine were successfully incorporated into a polypeptide, but l‐1‐pyrenylalanine was not. The polypeptides containing the nonnatural amino acids showed photofunctionalities.

Solution structure of the Ras‐binding domain of RGL

The RGL protein, a homolog of the Ral GDP dissociation stimulator (RalGDS), has been identified as a downstream effector of Ras. In the present study, the solution structure of the Ras‐binding domain of RGL (RGL‐RBD) was determined by NMR spectroscopy. The overall fold of RGL‐RBD consists of a five‐stranded β‐sheet and two α‐helices, which is the same topology as that of RalGDS‐RBD. The backbone chemical shift perturbation of RGL‐RBD upon interaction with the GTP analog‐bound Ras was also examined. The solution structure of RGL‐RBD, especially around some of the Ras‐interacting residues, is appreciably different from that of RalGDS‐RBD.

RNA aptamers that specifically bind to the Ras-binding domain of Raf-1

RNA aptamers that bind to the Ras‐binding domain (RBD) of a proto‐oncogene product, Raf‐1, were isolated from a pool of random sequences using a glutathione S‐transferase‐fused RBD (GST‐RBD). The RNA molecules bind to the GST‐RBD, but not to GST, with dissociation constants of about 300 nM. In contrast, these RNA aptamers do not bind to the Ras‐binding domain of the RGL protein, which is also known to be activated by Ras. The aptamers actually compete with Ras for binding to the Raf‐1 RBD. The anti‐Raf‐1 aptamers may be used to specifically inhibit the Ras‐Raf interaction in the complicated signaling network in mammalian cells.

Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-sevenless 1 (mSos1).

A fragment consisting of residues 584–1071 of the mouse Son-of-sevenlessu20091 (mSos1) protein was found to be sufficient for stimulation of the guanine nucleotide exchange of Ras in vitro, which defines the CDC25 homology (CDC25H) domain of mSos1. Furthermore, we found that the CDC25H-domain fragment activated the extracellular signal-regulated protein kinases (ERKs), and was mainly membrane localized, when expressed in unstimulated human embryonic kidney 293 cells. Then, we examined the roles of other mSos1 domains in autoinhibition of the CDC25H-domain functions in unstimulated cellular environments. First, longer fragments that have the CDC25H domain and the following proline-rich Grb2-binding domain exhibited negligible membrane localization, and accordingly much lower ERK-activation activities, under serum-starved conditions. On the other hand, the preceding Pleckstrin–homology (PH) domain affects neither the ERK-activation activity nor the membrane-localization activity of the CDC25H domain. By contrast, the cells expressing a fragment containing the Dbl homology (DH) domain in addition to the PH and CDC25H domains exhibited remarkably low ERK activities under serum-starved conditions. This autoinhibitory effect of the DH domain on the CDC25H-domain function was shown to be relieved when cells were stimulated with epidermal growth factor. The DH-domain extention affected neither the in vitro guanine nucleotide exchange activity nor the membrane-localization activity of the CDC25H domain. Therefore, one of the roles of the DH domain is to exert an autoinhibition over the CDC25H-domain function on the cell membrane, in the absence, but not in the presence, of extracellular stimuli.