Biomedical subjects

Kei Kurokawa

Publications and source records attributed to Kei Kurokawa.

4 recordsLinked to original sources

PubMed2006

Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase.

During development of the central and peripheral nervous systems, neurite extension mediated via glial-cell-line-derived neurotrophic factor (GDNF) and its receptor RET is critical for neuronal differentiation. In the present study, we investigated the role of the RET substrate Dok-4 in neurite outgrowth induced by the GDNF/RET signaling pathway. In TGW neuroblastoma cells, which endogenously express both RET and Dok-4, depletion of Dok-4 through treatment with small interfering RNA resulted in a marked decrease in GDNF-stimulated neurite outgrowth. By contrast, exogenous expression of wild-type Dok-4 induced sustained p44/42 mitogen-activated protein kinase (ERK1/2) activation and enhanced neurite outgrowth. Expression of Dok-4 mutants in which the tyrosine residues at codons 187, 220 and 270, conserved between Dok-4, -5, and -6, were each replaced with a phenylalanine inhibited sustained ERK1/2 activation and neurite outgrowth. We also found that Dok-4 induced a significant activation of the small G protein Rap1 and that expression of a dominant active Rap1 mutant restored neurite outgrowth in Dok-4-depleted cells. By contrast, expression of a dominant negative Rap1 mutant impaired GDNF-stimulated neurite outgrowth from TGW cells. Finally, we found that neurite formation in cultured rat hippocampal neurons was enhanced by the expression of Dok-4. Together, our results suggest that Dok-4, through activation of the Rap1-ERK1/2 pathway, regulates GDNF-mediated neurite outgrowth during neuronal development.

Adaptor Proteins, Signal Transducing↗

PubMed2004

A targeting mutation of tyrosine 1062 in Ret causes a marked decrease of enteric neurons and renal hypoplasia.

The Ret receptor tyrosine kinase plays a crucial role in the development of the enteric nervous system and the kidney. Tyrosine 1062 in Ret represents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins that are important for the activation of intracellular signaling pathways, such as the RAS/ERK, phosphatidylinositol 3-kinase/AKT, and Jun-associated N-terminal kinase pathways. To investigate the importance of tyrosine 1062 for organogenesis in vivo, knock-in mice in which tyrosine 1062 in Ret was replaced with phenylalanine were generated. Although homozygous knock-in mice were born normally, they died by day 27 after birth and showed growth retardation. The development of the enteric nervous system was severely impaired in homozygous mutant mice, about 40% of which lacked enteric neurons in the whole intestinal tract, as observed in Ret-deficient mice. The rest of the mutant mice developed enteric neurons in the intestine to various extents, although the size and number of ganglion cells were significantly reduced. Unlike Ret-deficient mice, a small kidney developed in all knock-in mice, accompanying a slight histological change. The reduction of kidney size was due to a decrease of ureteric bud branching during embryogenesis. Thus, these findings demonstrated that the signal via tyrosine 1062 plays an important role in histogenesis of the enteric nervous system and nephrogenesis.

Abnormalities, Multiple↗

PubMed2003

Establishment and characterization of mouse mammary carcinoma cell lines expressing RET with a multiple endocrine neoplasia 2A mutation.

We recently generated transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation (RET-MEN2A). Mammary tumors with frequent lung metastasis were developed in 22% of female transgenic mice in a stochastic fashion. In the current study, we established two cell lines (named MKK-f and MKK-s) from mammary tumors developed in RET-MEN2A transgenic mice. MKK-f and MKK-s were derived from well-differentiated ductal carcinoma and sarcomatous spindle cell carcinoma, respectively. MKK-f cells show epithelial-like morphology with a doubling time of 19 h, and MKK-s cells show spindle-shaped morphology with a doubling time of 15 h. When inoculated in immunodeficient mice, both cell lines were tumorigenic, metastasized to the lung and displayed histological features similar to those of the primary tumors. They maintained a high level of RET expression and activation of signaling molecules downstream of RET. Consistent with the histological phenotype, expression of E-cadherin was almost undetectable in MKK-s cells, whereas its expression was very high in MKK-f cells. When the difference of gene expression between the two cell lines was analyzed using cDNA microarrays including approximately 900 genes/ESTs, a total of 21 up- or down-regulated (> 2.0-fold) genes were identified. Differentially regulated genes included thymosin beta-10, fibroblast growth factor receptor 4, aldo-keto reductase and caspase 6 genes, which are known to be associated with tumor development and progression. These results may reflect the profiles of the transcriptional changes associated with dedifferentiation or progression of mammary carcinomas developed in genetically engineered mice.

Alcohol Oxidoreductases↗

PubMed2002

Role of Dok1 in cell signaling mediated by RET tyrosine kinase.

Using a yeast two-hybrid screen, we identified Dok1 as a docking protein for RET tyrosine kinase. Dok1 bound more strongly to RET with a multiple endocrine neoplasia (MEN) 2B mutation than RET with a MEN2A mutation and was highly phosphorylated in the cells expressing the former mutant protein. Analysis by site-directed mutagenesis revealed that tyrosine 361 in mouse Dok1 represents a binding site for the Nck adaptor protein and tyrosines 295, 314, 361, 376, 397, and 408 for the Ras-GTPase-activating protein. We replaced tyrosine 361 or these six tyrosines with phenylalanine (designated Y361F or 6F) in Dok1 and introduced the mutant Dok1 genes into the cells expressing the wild-type RET or RET-MEN2B protein. Overexpression of Dok1 or Dok1-Y361F, but not Dok1-6F, suppressed the Ras/Erk activation induced by glial cell line-derived neurotrophic factor or RET-MEN2B, implying that this inhibitory effect requires the Ras-GTPase-activating protein binding to Dok1. In contrast, overexpression of Dok1, but not Dok1-Y361F or Dok1-6F, enhanced the c-Jun amino-terminal kinase (JNK) and c-Jun activation. This suggested that the association of Nck to tyrosine 361 in Dok1 is necessary for the JNK and c-Jun activation by glial cell line-derived neurotrophic factor or RET-MEN2B. Because a high level of the JNK phosphorylation was observed in the cells expressing RET-MEN2B, its strong activation via Nck binding to Dok1 may be responsible for aggressive properties of medullary thyroid carcinoma developed in MEN 2B.

3T3 Cells↗