Ken-ichi Iida

The Role of Amino Acids Surrounding Tyrosine 1062 in Ret in Specific Binding of the Shc Phosphotyrosine- Binding Domain*

We investigated the role of the I-E-N-K-L (amino acids 1057‐1061) sequence amino-terminal to Tyr 1062 in Ret for binding of the Shc phosphotyrosine-binding (PTB) domain. Substitution of Ser for Ile 1057 (I1057S), Ala for Asn 1059 (N1059A), or Pro for Leu 1061 (L1061P) in this sequence significantly decreased the transforming activity of Ret with the multiple endocrine neoplasm type 2A (MEN2A) mutation as well as the binding affinity of Shc to it in vivo and in vitro, indicating that these three amino acids play a role in Shc binding. In addition, as the RET protooncogene is translated as three isoforms of 1114 amino acids (Ret 51), 1106 amino acids (Ret 43), and 1072 amino acids (Ret 9) that differ from one another in the sequence carboxyl-terminal to Tyr 1062 , we examined whether these sequence differences influence the binding affinity of Shc to Ret. As a result, we found that the transforming activity of Ret 43 isoform with the MEN2A mutation and the binding affinity of Shc to it were very low, although the consensus sequence for the binding of the Shc PTB domain is conserved in the Ret 43 isoform. This finding suggested that the sequence carboxyl-terminal to Tyr 1062 in Ret could also influence the binding affinity to Shc. (Endocrinology 140: 3992‐3998, 1999)

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.

Elevated expression of PDI family proteins during differentiation of mouse F9 teratocarcinoma cells

We investigated the expression of protein disulfide isomerase family proteins (PDI, ERp61, and ERp72) in mouse F9 teratocarcinoma cells during differentiation induced by treatment with retinoic acid and dibutyryl cAMP. Each member of this family was expressed at a constitutive level in undifferentiated F9 cells. During differentiation of F9 cells to parietal or visceral endodermal cells the protein level of all these enzymes increased, although the extent of this increase in both protein and mRNA levels varied among the enzymes. Certain proteins were found to be co‐immunoprecipitated with PDI, ERp61, and ERp72 in the presence of a chemical crosslinker. Type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Thus, the induction of PDI family proteins during the differentiation of F9 cells and their association with different proteins may implicate specific functions of each member of this family despite the common redox activity capable of catalyzing the disulfide bond formation. J. Cell. Biochem. 68:436–445, 1998.