Biomedical subjects

Kazuya Machida

Publications and source records attributed to Kazuya Machida.

8 recordsLinked to original sources

PubMed2006

Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains.

Deciphering global signaling networks is of great importance for the detailed understanding of cellular signaling processes controlling many important biological functions. Among signaling processes, tyrosine phosphorylation has a central role. At present, adequate techniques for the global characterization of the tyrosine phosphoproteome are lacking, particularly for the analysis of small amounts of protein. By combining the power of PCR amplification with the unique properties of Src homology region 2 (SH2) domains to specifically recognize tyrosine-phosphorylated proteins, we developed a new proteomic approach, termed oligonucleotide-tagged multiplex assay (OTM). For OTM, multiple SH2 domains are labeled by domain-specific oligonucleotide tags, applied as probes to complex protein mixtures in a multiplex reaction and phosphotyrosine-specific interactions are quantified by PCR. Using OTM we reproducibly quantified differential states of tyrosine phosphorylation with high sensitivity and specificity in small amounts of whole cellular extracts as demonstrated for various tumor cell lines and human leukemia samples.

3T3 Cells↗

PubMed2005

Exploring the mechanism for the synthesis of silsesquioxanes. 4. The synthesis of T8.

In an attempt to explore the mechanism for the synthesis of silsesquioxanes (POSS), the entire reaction processes from HSi(OH)3 to T8, one of the most common and stable POSS was investigated with electronic structure theory calculations, including electron correlation effects. In addition, the effect of some controlling factors, which play important roles in the reaction mechanism, is discussed.

Journal Article↗

PubMed2004

The SH2 domain: versatile signaling module and pharmaceutical target.

The Src homology 2 (SH2) domain is the most prevalent protein binding module that recognizes phosphotyrosine. This approximately 100-amino-acid domain is highly conserved structurally despite being found in a wide variety proteins. Depending on the nature of neighboring protein module(s), such as catalytic domains and other protein binding domains, SH2-containing proteins play many different roles in cellular protein tyrosine kinase (PTK) signaling pathways. Accumulating evidence indicates SH2 domains are highly versatile and exhibit considerable flexibility in how they bind to their ligands. To illustrate this functional versatility, we present three specific examples: the SAP, Cbl and SOCS families of SH2-containing proteins, which play key roles in immune responses, termination of PTK signaling, and cytokine responses. In addition, we highlight current progress in the development of SH2 domain inhibitors designed to antagonize or modulate PTK signaling in human disease. Inhibitors of the Grb2 and Src SH2 domains have been extensively studied, with the aim of targeting the Ras pathway and osteoclastic bone resorption, respectively. Despite formidable difficulties in drug design due to the lability and poor cell permeability of negatively charged phosphorylated SH2 ligands, a variety of structure-based strategies have been used to reduce the size, charge and peptide character of such ligands, leading to the development of high-affinity lead compounds with potent cellular activities. These studies have also led to new insights into molecular recognition by the SH2 domain.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Tyrosine phosphorylation of NOS3 in a breast cancer cell line and Src-transformed cells.

We investigated the tyrosine phosphorylation of NOS3 by active Src. In a cell line derived from human breast cancer, BT474, we found activation of c-Src and tyrosine phosphorylation of NOS3. Phosphorylation of NOS3 was suppressed by treatment of BT474 with PP1, an Src kinase inhibitor, in a dose-dependent manner, suggesting that phosphorylation of NOS3 is catalyzed by active c-Src. Phosphorylation of NOS3 was further examined by a series of Src mutants. In cells expressing v-Src, substantial phosphorylation of NOS3 was observed, whereas NOS3 phosphorylation was not evident in cells expressing c-Src. Similarly, NOS1 was also phosphorylated in cells expressing v-Src. Consistently, in cells expressing a temperature-sensitive mutant of v-Src, NOS3 phosphorylation was temperature-dependent. Moreover, transforming mutant of c-Src, Y527Fc-Src, could activate NOS3 phosphorylation. In contrast, non-myristoylated form of v-Src, G2Av-Src and a kinase-inactive mutant of v-Src, K295Mv-Src, could not activate NOS3 phosphorylation. Taken together, our results suggest that active, membrane-bound form of Src can induce constitutive phosphorylation of NOS3.

Animals↗

PubMed2003

Profiling the global tyrosine phosphorylation state.

Protein tyrosine kinases and protein tyrosine phosphatases play a key role in cell signaling, and the recent success of specific tyrosine kinase inhibitors in cancer treatment strongly validates the clinical relevance of basic research on tyrosine phosphorylation. Functional profiling of the tyrosine phosphoproteome is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel molecular diagnostic approaches. The ultimate aim of current mass spectrometry-based phosphoproteomic approaches is the comprehensive characterization of the phosphoproteome. However, current methods are not yet sensitive enough for routine detection of a large percentage of tyrosine-phosphorylated proteins, which are generally of low abundance. In this article, we discuss alternative methods that exploit Src homology 2 (SH2) domains for profiling the tyrosine phosphoproteome. SH2 domains are small protein modules that bind specifically to tyrosine-phosphorylated peptides; there are more than 100 SH2 domains in the human genome, and different SH2 domains bind to different classes of tyrosine-phosphorylated ligands. These domains play a critical role in the propagation of signals in the cell, mediating the relocalization and complex formation of proteins in response to changes in tyrosine phosphorylation. We have developed an SH2 profiling method based on far-Western blotting, in which a battery of SH2 domains is used to probe the global state of tyrosine phosphorylation. Application to the classification of human malignancies suggests that this approach has potential as a molecular diagnostic tool. We also describe ongoing efforts to modify and improve SH2 profiling, including the development of a multiplexed assay system that will allow high-throughput functional profiling of the tyrosine phosphoproteome.

Blotting, Far-Western↗

PubMed2002

A role for SHPS-1/SIRPalpha1 in IL-1beta- and TNFalpha-dependent signaling.

We investigated the role of SHPS-1/SIRPalpha1 in IL-1beta- and TNFalpha-dependent signaling that leads to the activation of Erk 1/2 and Akt. Treatment of Balb3T3 cells with IL-1beta or TNFalpha activated tyrosine phosphorylation of SHPS-1, its association with SHP-2 and the phosphorylation of Erk 1/2 and Akt. PP1, a specific inhibitor for the Src family protein tyrosine kinases, strongly inhibited tyrosine phosphorylation of SHPS-1 and complex formation of SHPS-1 with SHP-2 by IL-1beta. In addition, PP1 substantially inhibited the IL-2beta- and TNFalpha-dependent activation of Erk 1/2 and Akt. Exogenous expression of either SHPS-1 mutants that lack SHP-2 binding function or a dominant negative mutant of SHP-2 markedly inhibited the activation of Erk 1/2 and Akt by IL-1beta, whereas wild type SHPS-1 did not. Moreover, IL-1beta-stimulation induced association of SHPS-1 with IL-1RAcP, a second subunit of IL-1 receptor, whereas expression of SHPS-1 mutant that lack SHP-2 binding function clearly blocked the association and tyrosine phosphorylation of endogenous SHPS-1. Taken together, our results strongly suggest that activation of Erk 1/2 and Akt by proinflammatory cytokines requires tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2.

3T3 Cells↗

PubMed2002

SHPS-1: a budding molecule against cancer dissemination.

The expression of SHPS-1 is down-regulated by several oncogene products such as v-Src in fibroblasts. In addition, the down-regulation of SHPS-1 is also found inhuman breast cancer tissues compared with the matched normal tissues. On the other hand, forced expression of SHPS-1 suppresses anchorage-independent cell growth of v-Src-transformed cells in vitro as well as peritoneal dissemination of the cells in nude mice. Surprisingly, the extracellular region (EC) of SHPS-1 seems to take part on the inhibitory effect. Because the EC domain interacts with fibronectin and DTT abrogates the inhibitory effects of the EC, we speculate that super-fibronectin may function with the EC as a suppressor of cancer cells. Here we show that SHPS-1 expression provides a unique potential that links suppression of anchorage-independent cell growth and cancer dissemination.

3T3 Cells↗

PubMed2002

Hyaluronan-CD44s signaling regulates matrix metalloproteinase-2 secretion in a human lung carcinoma cell line QG90.

We investigated the production of matrix metalloproteinase (MMP) by hyaluronan(HA) stimulation in a human cancer cell line, QG90, that expresses a large amount of CD44s, a HA receptor. Treatment of QG90 with HA strongly activated MMP-2 secretion in a time- and dose-dependent manner. We found that expression of antisense CD44s in QG90 cells substantially inhibited the HA-dependent secretion of MMP-2, whereas overexpression of full-length CD44s augmented the HA-dependent secretion of MMP-2. In addition, pretreatment of cells with the neutralizing anti-CD44 antibody significantly inhibited both the HA-dependent MMP-2 secretion and the HA-dependent activation of mitogen-activated protein kinase in a dose-dependent manner. Similarly, treatment of cells with a Ras farnesyltransferase inhibitor, manumycin A, strongly inhibited the HA-dependent MMP-2 secretion. Moreover, in vitro invasiveness of QG90 and its activation by HA were clearly suppressed by the expression of antisense CD44s. In addition, treatment of cells with anti-CD44, a mitogen-activated protein/extracellular signal-regulated kinase kinase 1 inhibitor, PD98059, or phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, effectively blocked the HA-dependent activation of the invasiveness. In contrast, overexpression of full-length CD44 substantially activated the invasiveness of QG90. Taken together, HA-CD44s signaling plays a key role in the HA-dependent secretion of MMP-2 and, hence, in the invasiveness of QG90 cells.

Carcinoma, Small Cell↗