A Study of the Interactions among GNMT, DEPDC6 and P-Rex2

• Main Authors: Chung-Hsien Li, 李忠憲
• Other Authors: Yi-Ming Arthur Chen
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/84000966323155174750

碩士 === 國立陽明大學 === 公共衛生研究所 === 98 === Glycine N-methyltransferase (GNMT) is abundant in liver. It affects cellular methylation potential and DNA stability by regulating the ratio of S-adenosylmethionine to S-adenosylhomocysteine and serving as the major folate binding protein in the liver. The expression of GNMT is down-regulated in human hepatocellular carcinoma (HCC). In addition, GNMT knockout mice developed HCC spontaneously. Therefore, GNMT is a tumor suppressor gene for liver cancer. However, it has been reported that sarcosine, a metabolic product of GNMT enzymatic activity, can serve as a biomarker of prostate cancer progression and metastasis. In order to study the role of GNMT in carcinogenesis, we used yeast two hybrid screen to identify a GNMT interacting protein, DEP domain containing 6 (DEPDC6). It has been reported that DEPDC6 is an mTOR binding protein that normally functions to inhibit the mTOR activity. Results from a phylogenetic analysis indicate that the first DEP domain of DEPDC6 clustered with P-Rex2, a regulator of the small guanosine triphosphatase Rac, linking mTOR signaling to Rac activation and cell migration. P-REX2 has been identified as a direct regulator of PTEN activity and as a potential oncoprotein. In this study, we study the interactions among GNMT, DEPDC6 and P-Rex2. Results from reciprocal co-immunoprecipitation assays demonstrated that P-Rex2, GNMT, DEPDC6 and mTOR could interact with each other. In addition, the DH-PH, PDZ and InsPx4-Phosphatase domains of P-Rex2 were involved in the interactions with GNMT and DEPDC6. Moreover, the PDZ domain of DEPDC6 mediated the interaction with P-Rex2. Results from gel filtration assay demonstrated cofractionation of P-Rex2, GNMT, DEPDC6 and mTOR in 293T cell extracts. Together, these indicated that P-Rex2, GNMT, DEPDC6 and mTOR could present in the same complex. In regard to the functional roles of these interactions, we demonstrated that GNMT could disrupt the interaction between mTOR and DEPDC6. In addition, overexpression of P-Rex2 could positively regulate the expression of DEPDC6. Moreover, overexpression of GNMT and DEPDC6 had no effects on the cell cycle progression of 293T cells. Finally, results from Rac-GEF assay indicated that DEPDC6 could interact with activated Rac and activate the Rac slightly.