Analysis of protein kinase in bladder cancer

• Main Authors: Lu, Te-Ling, 陸德齡
• Other Authors: Lai Ming-Der
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/35612725199372678565

碩士 === 國立成功大學 === 生物化學研究所 === 85 === In response to the environmental change, cells can transduce signals by using kinases to transfer g-phosphate of ATP or GTP to the hydroxyl groups(Ser, Thr or Tyr) of their protein substrates. Hundreds of kinases sequence have been determined. Comparison between amino acid sequence of these kinase revealed that the kinases share a common structure, the kinase domain. The kinase domain consists of 250-300 amino acid residues and impart catalytic activity. The kinase domain has 3 specific roles: 1) to bind and orientate ATP and GTP with divalent cation (usually Mg2+ or Mn2+). 2) to bind and orientate the substrate. 3) to transfer g-phosphate of ATP or GTP to the hydroxyl groups( Ser, Thr or Tyr) of their protein substrate. The kinase domain are further divided into 12 subdomains. Subdomains VIB,VIII and IX are particularly well conserved among the individual members of different protein kinase families.Many oncogenes and proto- oncogenes are known to be tyrosine kinases, and tyrosine kinase play important roles in cell growth of normal and tumor cells. Therefore we wish to study the tyrosine kinase in bladder cancer. RT-PCR amplified of RNA from BFTC 905 human bladder cancer cell line were performed with two degenerate primers, R1 and F3, which were designed to the correspond region of subdomain VII and IX. The 180 bp PCR products were ligated into pCRTMII vector with TA cloning kit and transformed into E. coli. DNA sequence was performed with insert-positive clones. The DNA sequence of all these clones revealed the expression pattern of tyrosine kinase and possible novel kinase genes. Among the 460 clones screened by PCR, 354 clones were insert-positive . Sequencing of 100 clones revealed that:1) The highest expression kinases are trkE and arg. The sequence of trkE gene appealed in 24 clones of the 100 clones (26%), and arg gene appealed in 16 clones (16%). 2) The DNA sequence of two clones, 240 and 246 can't be matched in BLAST research. Clone 240 is significantly homologous to a serine/threonine kinase Sok-1 in BLAST search.In 1996, Thomas Force identified a human Ste20 like serine/ threonine kinase Sok-1(Ste20/oxidant stress response kinase-1). Sok-1 was not activated by growth factors, alkylating agents, cytokines or environmental stress including heat shock and osmolar stress, but activated by oxidant stress like H2O2 or menadione.As Sok-1 plays an important role in tranducing signals in response to environmental stress, our clone Sok-1 like gene may also play an important role in a similar pathway. We then clone the full-length cDNA of the novel Sok-1 like kinase with the use of RACE method (rapid amplification of cDNA ends). The DNA sequence of the novel clone is 88 % homology with Sok-1 in kinase domain.