Toshiyoshi Yamamoto

Localization of a novel v-erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line.

The c-erbB-2 gene is a v-erbB-related proto-oncogene which is distinct from the gene encoding the epidermal growth factor receptor. By using two independent methods, hybridization of both sorted chromosomes and metaphase spreads with cloned c-erbB-2 DNA, we mapped the c-erbB-2 locus on human chromosome 17 at q21, a specific breakpoint observed in a translocation associated with acute promyelocytic leukemia. Furthermore, we observed amplification and elevated expression of the c-erbB-2 gene in the MKN-7 gastric cancer cell line. These data suggest possible involvement of the c-erbB-2 gene in human cancer.

Heterodimeric Phosphoinositide 3-Kinase Consisting of p85 and p110β Is Synergistically Activated by the βγ Subunits of G Proteins and Phosphotyrosyl Peptide

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses. Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the βγ subunits of G protein (Gβγ) from rat liver. The Gβγ-sensitive PI 3-kinase appeared to be a heterodimer consisting of p110β and p85 (or their related subunits). The stimulation by Gβγ was inhibited by the GDP-bound α subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of Gβγ was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110β/p85α expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110α and p85 in the same rat liver, together with a recombinant p110α/p85α, was not activated by Gβγ, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110β/p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.

Phosphorylation sites of protein kinase C δ in H2O2-treated cells and its activation by tyrosine kinase in vitro

Protein kinase C δ (PKC δ) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H2O2, the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC δ in the H2O2-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC δ in response to H2O2.

The yes-related cellular gene lyn encodes a possible tyrosine kinase similar to p56lck.

With v-yes DNA as the probe, a human cDNA library made from placental RNA was screened under relaxed conditions, and DNA clones derived from a novel genetic locus, termed lyn, were obtained. Nucleotide sequencing revealed that lyn could encode a novel tyrosine kinase that was very similar to mouse T-lymphocyte-specific tyrosine kinase p56lck and the v-yes protein as well as to the gene products of v-fgr and v-src. Northern hybridization analysis revealed that a 3.2-kilobase lyn mRNA was expressed in a variety of tissues of the human fetus. The pattern of lyn mRNA expression was different from those of related genes, such as yes and syn. Hybridization analysis of DNA from sorted chromosomes showed that the lyn gene is located on human chromosome 8 q13-qter.

Ceramide-induced Apoptosis by Translocation, Phosphorylation, and Activation of Protein Kinase Cδ in the Golgi Complex

Protein kinase C (PKC), a Ca2+/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCδ in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCδ-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCδ expression using small interfering RNA. Ceramide induced the translocation of PKCδ to the Golgi complex and the concomitant activation of PKCδ via phosphorylation of Tyr311 and Tyr332 in the hinge region of the enzyme. Unphosphorylatable PKCδ (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCδ lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCδ to the Golgi complex and that PKCδ is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCδ by small interfering RNA to study the significance of phosphorylation of Tyr311 and Tyr332 in PKCδ for ceramide-induced apoptosis and found that phosphorylation of Tyr311 and Tyr332 is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCδ, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.

Specific expression of human c-fgr in natural immunity effector cells

The c-fgr gene product was shown by immune complex protein kinase assay with specific antibodies to be a 58-kilodalton protein (p58c-fgr) with tyrosine-specific autophosphorylating activity. On examination of peripheral blood cells by immunoblotting with anti-c-fgr antibodies, p58c-fgr was found only in the fractions of monocytes, granulocytes, and natural killer cells. On the other hand, histochemical studies of hybridization demonstrated accumulation of c-fgr transcripts on most monocytes and large lymphocytes. In hematopoietic cell lines, p58c-fgr was detected in differentiated granulocytic cells as well as in differentiated monocytic cells of HL-60-cell origin. These data suggest a specific role for p58c-fgr in natural immunity effector cells.

Search for aflatoxin and trichothecene production inhibitors and analysis of their modes of action

Mycotoxin contamination of crops is a serious problem throughout the world because of its impact on human and animal health as well as economy. Inhibitors of mycotoxin production are useful not only for developing effective methods to prevent mycotoxin contamination, but also for investigating the molecular mechanisms of secondary metabolite production by fungi. We have been searching for mycotoxin production inhibitors among natural products and investigating their modes of action. In this article, we review aflatoxin and trichothecene production inhibitors, including our works on blasticidin S, methyl syringate, cyclo(l-Ala-l-Pro), respiration inhibitors, and precocene II.

Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor

Significance Several in situ click chemistry studies have been reported. To date, there is evidence to indicate that proteins act as mold between azide and alkyne fragments by X-ray analysis of protein–ligand complexes. However, only “postclick” structural evidence has been available. We succeeded in obtaining crystal structures of a chitinase complexed with an azide inhibitor and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating triazole formation in chitinase. This is an example to express the “preclick” state of in situ click chemistry and a demonstration to show that the in situ click chemistry approach will benefit from this analysis for future plans. We also performed density functional theory calculations to explore the chitinase-contributed Huisgen cycloaddition. The Huisgen cycloaddition of azides and alkynes, accelerated by target biomolecules, termed “in situ click chemistry,” has been successfully exploited to discover highly potent enzyme inhibitors. We have previously reported a specific Serratia marcescens chitinase B (SmChiB)-templated syn-triazole inhibitor generated in situ from an azide-bearing inhibitor and an alkyne fragment. Several in situ click chemistry studies have been reported. Although some mechanistic evidence has been obtained, such as X-ray analysis of [protein]–[“click ligand”] complexes, indicating that proteins act as both mold and template between unique pairs of azide and alkyne fragments, to date, observations have been based solely on “postclick” structural information. Here, we describe crystal structures of SmChiB complexed with an azide ligand and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating syn-triazole formation, which allows generation of its own distinct inhibitor. We have also performed density functional theory calculations based on the X-ray structure to explore the acceleration of the Huisgen cycloaddition by SmChiB. The density functional theory calculations reasonably support that SmChiB plays a role by the cage effect during the pretranslation and posttranslation states of selective syn-triazole click formation.

Biochemical assays for multiple activation states of protein kinase C

This protocol describes biochemical procedures to monitor the activation of the protein kinase C (PKC) family using PKCδ as the representative. The PKC family is composed of ten isoforms divided into cPKC, nPKC and aPKC groups, and their catalytic activity is regulated by multiple mechanisms. For example, PKCδ in the nPKC group is activated by diacylglycerol as a second messenger in the receptor-coupled manner, through tyrosine phosphorylation and protein complex formation in stress-stimulated cells, and by the caspase-catalyzed cleavage during apoptosis. The isoform is immunoprecipitated from cultured cells, the protein kinase activity is measured by in vitro kinase assay and the tyrosine phosphorylation and protein complex formation are characterized by immunoblot, whereas the generation of the catalytic fragment is detected by immunoblot in the cell extract. The combination of these procedures is useful to evaluate the activation states of the PKC family in cells. This protocol can be completed in 3–5 d.

Creation of Customized Bioactivity within a 14-Membered Macrolide Scaffold: Design, Synthesis, and Biological Evaluation Using a Family-18 Chitinase.

Argifin, a 17-membered pentapeptide, inhibits chitinase. As argifin has properties that render it unsuitable as a drug development candidate, we devised a mechanism to create the structural component of argifin that bestows the chitinase inhibition and introduce it into a 14-membered macrolide scaffold. Here we describe (1) the designed macrolide, which exhibits ∼200-fold more potent chitinase inhibition than argifin, (2) the binding modes of the macrolide with Serratia marcescens chitinase B, and (3) the computed analysis explaining the reason for derivatives displaying increased inhibition compared to argifin, the macrolide aglycone displaying inhibition in a nanomolar range. This promises a class of chitinase inhibitors with novel skeletons, providing innovative insight for drug design and the use of macrolides as adaptable, flexible templates for use in drug discovery research and development.