Kei Kurokawa

Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor.

Glial cell line derived neurotrophic factor (GDNF) signals through a multicomponent receptor complex consisting of RET receptor tyrosine kinase and a member of GDNF family receptor α (GFRα). Recently, it was shown that tyrosine 1062 in RET represents a binding site for SHC adaptor proteins and is crucial for both RAS/mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways. In the present study, we characterized how these two pathways diverge from tyrosine 1062, using human neuroblastoma and primitive neuroectodermal tumor cell lines expressing RET at high levels. In response to GDNF stimulation, SHC bound to GAB1 and GRB2 adaptor proteins as well as RET, and SHC and GAB1 were highly phosphorylated on tyrosine. The complex formation consisting of SHC, GAB1 and GRB2 was almost abolished by replacement of tyrosine 1062 in RET with phenylalanine. Tyrosine-phosphorylated GAB1 was also associated with p85 subunit of PI3-K, resulting in PI3-K and AKT activation, whereas SHC-GRB2-SOS complex was responsible for the RAS/ERK signaling pathway. These results suggested that the RAS and PI3-K pathways activated by GDNF bifurcate mainly through SHC bound to tyrosine 1062 in RET. Furthermore, using luciferase reporter-gene assays, we found that the RAS/ERK and PI3-K signaling pathways are important for activation of CREB and NF-κB in GDNF-treated cells, respectively.

Biological and biochemical properties of Ret with kinase domain mutations identified in multiple endocrine neoplasia type 2B and familial medullary thyroid carcinoma.

Several mutations were identified in the kinase domain of the RET proto-oncogene in patients with multiple endocrine neoplasia (MEN) 2B, familial medullary thyroid carcinoma (FMTC) or sporadic medullary thyroid carcinoma. We introduced seven mutations (glutamic acid 768→aspartic acid (E768D), valine 804→leucine (V804L), alanine 883→phenylalanine (A883F), serine 891→alanine (S891A), methionine 918 →threonine (M918T), alanine 919→proline (A919P) and E768D/A919P) into the short and long isoforms of RET cDNA and transfected the mutant cDNAs into NIH3T3 cells. The transforming activity of the long isoform of Ret with each mutation was much higher that that of its short isoform. Based on the levels of the transforming activity, these mutant RET genes were classified into two groups; a group with high transforming activity (A883F, M918T and E768D/A919P) and a group with low transforming activity (E768D, V804L, S891A and A919P) (designated high group and low group). Interestingly, the level of transforming activity correlated with clinical phenotypes; high group Ret with the A883F or M918T mutation and low group Ret with the E768D, V804L or S891A mutation were associated with the development of MEN 2B and FMTC, respectively. In addition, we found that substitution of phenylalanine for tyrosine 905 present in the kinase domain abolished both transforming and autophosphorylation activities of low group Ret whereas it did not affect the activities of high group Ret.

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

ABSTRACT 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.

Identification of SNT/FRS2 docking site on RET receptor tyrosine kinase and its role for signal transduction.

SNT/FRS2 is a lipid anchored docking protein that contains an amino-terminal myristylation signal, followed by a phosphotyrosine-binding (PTB) domain and a carboxy-terminal region with multiple tyrosine residues. Here we show that the SNT/FRS2 PTB domain binds to RET receptor tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF) or multiple endocrine neoplasia (MEN) 2 mutations. Analyses by site directed-mutagenesis revealed that it binds to tyrosine 1062 in RET that is also known to be a binding site for the SHC adaptor protein. Whereas SHC bound to RET was associated with GRB2 and GAB1 proteins, SNT/FRS2 was associated with GRB2 only, suggesting that SNT/FRS2 is involved mainly in the activation of the RAS/mitogen activated protein kinase (MAPK) pathway but not the phosphatidylinositol 3-kinase (PI3-K)/AKT pathway. In addition, phosphorylated SNT/FRS2 appeared to directly complex with SHP-2 tyrosine phosphatase. These results suggest that tyrosine 1062 in RET provides a site for the interaction of multiple signaling molecules and that the balance of SHC and SNT/FRS2 binding may affect the nature of the intracellular signaling for cell proliferation, differentiation and survival induced by activated RET.

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) inDok1 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.

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.

Implication of expression of GDNF/Ret signalling components in differentiation of bone marrow haemopoietic cells.

Glial cell line‐derived neurotrophic factor (GDNF) and neurturin (NTN) mediate their actions through a unique multicomponent receptor system composed of Ret receptor tyrosine kinase and glycosyl‐phosphatidylinositol‐linked cell surface proteins (designated GFRα‐1 and GFRα‐2). In the present study, expression of these signalling components in the process of differentiation of haemopoietic cells was investigated. Ret was expressed at variable levels in normal and malignant cells of the myelomonocyte lineage. Immunohistochemical analysis of human and mouse tissues revealed that Ret expression was increased in intermediate mature myeloid cells such as promyelocytes and myelocytes and decreased in mature granulocytes and monocytes. Consistent with this observation, when THP‐1 monocytic and HL‐60 promyelocytic leukaemia cells expressing Ret were differentiated toward macrophages or granulocytes by treatment of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or all‐trans retinoic acid (RA), Ret expression strikingly decreased during differentiation. Expression of GDNF, NTN, GFRα‐1 and GFRα‐2 was undetectable in THP‐1 and HL‐60 cells as well as in bone marrow haemopoietic cells. In contrast, bone marrow stromal cells appeared to express GDNF, GFRα‐1 and GFRα‐2 but not Ret. These findings suggested that the interaction between stromal cells and Ret‐expressing haemopoietic cells in the bone marrow microenvironment may play a role in the differentiation of myelomonocyte‐lineage cells through activation of the GDNF/Ret signalling pathway.

Analysis of DOK‐6 function in downstream signaling of RET in human neuroblastoma cells

Point mutations and structural alterations of the RET tyrosine kinase gene cause multiple endocrine neoplasia type 2 (MEN 2) and papillary thyroid carcinoma, respectively. RET activation by glial cell line‐derived neurotrophic factor (GDNF) is essential for the development of the enteric nervous system and the kidney. The signal through RET tyrosine kinase requires several adaptor proteins including the DOK (downstream of kinase) family of proteins. Of the seven members of the DOK protein family, DOK‐1, ‐4, ‐5, and ‐6 have been reported to play roles in the GDNF–RET signaling pathway. Although DOK‐6 has been shown to bind to RET and promote GDNF‐induced neurite outgrowth in mouse Neuro2A cells, DOK‐6 function in human cells remains unclear. In the present study, we investigated the role of DOK‐6 in GDNF–RET signaling in human cells including neuroblastoma cells. DOK‐6 was constitutively localized to the plasma membrane via its pleckstrin homology (PH) domain, and was phosphorylated following RET activation via a MEN2A mutation or GDNF stimulation. However, DOK‐6 could not significantly affect downstream signaling and neurite outgrowth in human neuroblastoma cells. The binding affinity of the DOK‐6 phosphotyrosine‐binding (PTB) domain to RET was much lower than that of the DOK‐1, DOK‐4, and SHC PTB domains to RET. These findings indicate that DOK‐6 is involved in RET signaling with less influence when compared with DOK‐1, DOK‐4, and SHC.

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 protooncogene 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 β‐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.