Tsuyoshi Miyake

Epidermal Growth Factor Receptor Translocation to the Mitochondria: REGULATION AND EFFECT*

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.

c-Src modulates ErbB2 and ErbB3 heterocomplex formation and function

Overexpression and/or gene amplification of c-Src and members of the epidermal growth factor receptor (EGFR/ErbB) family have been implicated in the pathogenesis of breast cancer. Although members of the EGFR family are known to form heterocomplexes with one another, c-Src has also been shown to physically interact with members of this family in breast cancer cell lines and tumors. This paper investigates the role of c-Src in modulating the physical and functional interaction between ErbB2 and ErbB3, two family members that preferentially associate with one another and together exhibit high oncogenic potential. We show that overexpressed wild-type c-Src enhances heterocomplex formation of ErbB2 and ErbB3 that results in increased basal and/or heregulin-induced activation of receptors, and their downstream intracellular effectors. Expression of a kinase-inactive form of c-Src (K− c-Src) or pharmacological inhibition of c-Src by PP2 negatively affects these events. Furthermore, cellular motility and anchorage-independent growth promoted by the ErbB2/ErbB3 heterocomplex are dependent upon c-Src, as demonstrated by the effects of K− c-Src overexpression or treatment with PP2. In contrast to previous studies that defined a role for c-Src downstream of ErbB2/ErbB3, the current work suggests an upstream mechanism, whereby c-Src enhances ErbB2/ErbB3 signaling and biological functions by positively modulating the association between ErbB2 and ErbB3.

Comparison of ABF1 and RAP1 in Chromatin Opening and Transactivator Potentiation in the Budding Yeast Saccharomyces cerevisiae

ABSTRACT Autonomously replicating sequence binding factor 1 (ABF1) and repressor/activator protein 1 (RAP1) from budding yeast are multifunctional, site-specific DNA-binding proteins, with roles in gene activation and repression, replication, and telomere structure and function. Previously we have shown that RAP1 can prevent nucleosome positioning in the vicinity of its binding site and have provided evidence that this ability to create a local region of “open” chromatin contributes to RAP1 function at the HIS4 promoter by facilitating binding and activation by GCN4. Here we examine and directly compare to that of RAP1 the ability of ABF1 to create a region of open chromatin near its binding site and to contribute to activated transcription at the HIS4, ADE5,7, and HIS7 promoters. ABF1 behaves similarly to RAP1 in these assays, but it shows some subtle differences from RAP1 in the character of the open chromatin region near its binding site. Furthermore, although the two factors can similarly enhance activated transcription at the promoters tested, RAP1 binding is continuously required for this enhancement, but ABF1 binding is not. These results indicate that ABF1 and RAP1 achieve functional similarity in part via mechanistically distinct pathways.

Light effects on cell development and secondary metabolism in Monascus

In nature, light is one of most crucial environmental signals for developmental and physiological processes in various organisms, including filamentous fungi. We have found that both red light and blue light affect development in Monascus, influencing the processes of mycelium and spore formation, and the production of secondary metabolites such as γ-aminobutyric acid, red pigments, monacolin K and citrinin. Additionally, we observed that the wavelength of light affects these developmental and physiological processes in different ways. These findings suggest that Monascus possesses a system for differential light response and regulation.

Functional interactions between Choline kinase α, epidermal growth factor receptor and c-Src in breast cancer cell proliferation.

Epidermal growth factor receptor (EGFR) family members and c-Src are co-overexpressed in many cancers. The synergistic effect of EGFR and c-Src has been shown in the tumorigenesis of breast and other cancers. Reported mechanisms of synergy include transcriptional regulation by STAT5b and the regulation of cellular ATP production by mitochondrial protein COX II. Here, we report a new mechanism of EGFR-c-Src synergy through choline kinase α (CHKA). The first enzyme of the phosphatidyl choline production pathway, CHKA, is overexpressed in many cancers, and the product of the enzyme, phosphocholine, is also increased in tumor cells. In this report, we find that CHKA forms a complex with EGFR in a c-Src-dependent manner. Endogenous CHKA and EGFR co-immunoprecipitated from a variety of breast cancer cell lines and immortalized mammary epithelial cells. CHKA interacted with the EGFR kinase domain upon c-Src co-overexpression and was phosphorylated in a c-Src-dependent manner on Y197 and Y333. Overexpression of EGFR and c-Src increased total cellular activity and protein levels of CHKA. Mutation of CHKA Y197 and Y333 reduced complex formation, EGFR-dependent activation of CHKA enzyme activity and epidermal growth factor (EGF)-dependent DNA synthesis. Furthermore, small interfering RNA-mediated knockdown of CHKA in MCF-7 and MCF-10A cells reduced EGF-dependent cell proliferation. Together, these results strongly implicate a new c-Src-dependent link between CHKA and EGFR, which contributes to the regulation of cell proliferation and tumorigenesis.

Effects of the Principal Nutrients on Lovastatin Production by Monascus pilosus

Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose–glycerol–peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.

Characterization of a Novel Trans-Activation Domain of BRCA1 That Functions in Concert with the BRCA1 C-terminal (BRCT) Domain

Mutations in the breast cancer susceptibility gene, BRCA1, account for a significant proportion of hereditary breast and ovarian cancers. The BRCA1 C-terminal (BRCT) domain, which can activate transcription when fused to a heterologous DNA binding domain, is required for BRCA1 function in suppression of tumorigenesis. Here, we provide evidence for a new activation domain in BRCA1 that lies adjacent to the BRCT domain. We name the two domains AD1 and AD2, respectively. Like AD2, the newly discovered AD1 can act independently as an activation domain in both yeast and human cells. However, unlike AD2, AD1 activity in mammalian cells is cell type context-dependent. Furthermore, combination of these two domains in mammalian cells can result in a robust synergy in transcriptional activation. A highly conserved coiled-coil motif in AD1 is required for the cooperative transcription activation. Interestingly, the functional cooperativity between AD1 and AD2 is absent in certain breast and ovarian cancer cell lines, although each domain can still activate transcription. Therefore, the differential and cooperative actions of the two activation modules may contribute to the heterogeneous risk of BRCA1 mutations in different tissues.

Impaired Core Promoter Recognition Caused by Novel Yeast TAF145 Mutations Can Be Restored by Creating a Canonical TATA Element within the Promoter Region of the TUB2 Gene

ABSTRACT The general transcription factor TFIID, which is composed of TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs), has been shown to play a crucial role in recognition of the core promoters of eukaryotic genes. We isolated Saccharomyces cerevisiae yeast TAF145 (yTAF145) temperature-sensitive mutants in which transcription of a specific subset of genes was impaired at restrictive temperatures. The set of genes affected in these mutants overlapped with but was not identical to the set of genes affected by a previously reportedyTAF145 mutant (W.-C. Shen and M. R. Green, Cell 90:615–624, 1997). To identify sequences which rendered transcription yTAF145 dependent, we conducted deletion analysis of theTUB2 promoter using a novel mini-CLN2 hybrid gene reporter system. The results showed that the yTAF145mutations we isolated impaired core promoter recognition but did not affect activation by any of the transcriptional activators we tested. These observations are consistent with the reported yTAF145 dependence of the CLN2 core promoter in the mutant isolated by Shen and Green, although the CLN2 core promoter functioned normally in the mutants we report here. These results suggest that different promoters require different yTAF145 functions for efficient transcription. Interestingly, insertion of a canonical TATA element into the TATA-less TUB2 promoter rescued impaired transcription in the yTAF145 mutants we studied. It therefore appears that strong binding of TBP to the core promoter can alleviate the requirement for at least one yTAF145 function.

Identification of a Multifunctional Domain in Autonomously Replicating Sequence-Binding Factor 1 Required for Transcriptional Activation, DNA Replication, and Gene Silencing

ABSTRACT Autonomously replicating sequence-binding factor 1 (ABF1) is a multifunctional, site-specific DNA binding protein that is essential for cell viability in Saccharomyces cerevisiae. ABF1 plays a direct role in transcriptional activation, stimulation of DNA replication, and gene silencing at the mating-type loci. Here we demonstrate that all three activities of ABF1 are conferred by the C terminus of the protein (amino acids [aa] 604 to 731). Furthermore, a detailed mutational analysis has revealed two important clusters of amino acid residues in the C terminus (C-terminal sequence 1 [CS1], aa 624 to 628; and CS2, aa 639 to 662). While both regions play a pivotal role in supporting cell viability, they make distinct contributions to ABF1 functions in various nuclear processes. CS1 specifically participates in transcriptional silencing and/or repression in a context-dependent manner, whereas CS2 is universally required for all three functions of ABF1. When tethered to specific regions of the genome, a 30-aa fragment that contains CS2 alone is sufficient for activation of transcription and chromosomal replication. In addition, CS2 is responsible for ABF1-mediated chromatin remodeling. Based on these results, we suggest that ABF1 may function as a chromatin-reorganizing factor to increase accessibility of the local chromatin structure, which in turn facilitates the action of additional factors to establish either an active or repressed chromatin state.

Cep57, a multidomain protein with unique microtubule and centrosomal localization domains.

The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to gamma-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.