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.
Kurotsuchi
Ai
A
Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Murakumo
Yoshiki
Y
Jijiwa
Mayumi
M
Kurokawa
Kei
K
Itoh
Yasutomo
Y
Kodama
Yoshinori
Y
Kato
Takuya
T
Enomoto
Atsushi
A
Asai
Naoya
N
Terasaki
Hiroko
H
Takahashi
Masahide
M
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000502:physiology*; D000818:Animals; D045744:Cell Line, Tumor; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009447:Neuroblastoma / Q000473:pathology*; D017434:Protein Structure, Tertiary; D051096:Proto-Oncogene Proteins c-ret / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C492987:DOK6 protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human