博士 / 國立臺灣大學 / 動物學研究所 / 88 / Abstract
The identity and the specific location of the point mutations in the guanidine nucleotide-binding region of Ras protein N-termini affected the Ras GTP-locked ability, GTPase activity, and moreover the efficiency of cellular transformation. In order for the Ras protein to promote cell proliferation it has to be recruited to the cell membrane for activation. Prenylation of the Ras C terminus is a pivotal event, since it introduces a hydrophobic tail for insertion into the cell membrane. Therefore, I attempted to study the effect of point mutations in the guanidine nucleotide binding region on the C-terminal geranylgeranylation of Penaeus (Marsupenaeus) japonicus Ras.
First, I designed the degenerate primer set base on the homologous region of the vertebrate H-, K-, and N-Ras amino acid sequences from the Genetics Computer Group Data Bank. I obtained a 275 bp PCR product from the hepatopancreas cDNA of the banded prawn Penaeus (Marsupenaeus) japonicus. I used this PCR product as a probe to screen the cDNA library from the hepatopancreas of the banded prawn Penaeus (Marsupenaeus) japonicus and obtained a ras cDNA with a full length of 1,294 bp and a 564 bp open reading frame. Translation of the coding region gave a to 187-residue shrimp Ras protein(S-Ras). S-Ras shared at least 79 % identity with Artemia Ras, Drosophila Ras1 and mammalian(human, mouse, rat) KB-Ras. The C-terminus of the S-Ras contains two signal motifs: one motif (CIVF box) comprising the last four amino acids of the C-terminus was the protein geranylgeranyltransferase I recognition and reaction site. The other motif contains four lysines and six arginines that is located at the 165~187 residues of the C-terminus, and this motif was different from the poly-lysine region of the C-terminus of the mammalian(human, mouse, rat) KB-Ras protein, implying that the polybasic amino acid region of the Ras proteins interacted with species specific membrane anchorage domains or sites.
I expressed the S-ras gene in E.coli with a 4 kDa calmodulin-binding peptide tag at the N-terminus. The p25 S-Ras fusion protein was purified with the calmodulin affinity column. The optimal GTP/GDP binding condition was found to be at pH 8.5; the saturated binding rate of GTP was 75 % and that of GDP was 12.5 %. The dissociation rate constant of the S-Ras-GTP complex (K2=1.46 x 10-3S-1) was smaller than the S-Ras-GDP complex (K2=5.42 x 10-3S-1), which means that the S-Ras fusion protein prefers to bind to GTP over GDP. The GTP binding activity of the S-Ras fusion protein was strongly inhibited by Mg2+ or Mn2+, in contrast with the previous studies done with the mammalian(human, mouse, rat) Ras proteins. The optimal pH for GTPase activity was pH6.0; under this condition, adding 0.5~1 mM Mg2+enhanced the GTPase activity, and the activity was increased 2.5 folds when Mg2+ was replaced with Mn2+.
I constructed several mutants of the S-ras with single nucleotide point mutation: G12V, Q61K and N116I. The same mutations in the mammalian(human, mouse, rat) Ras proteins had the ability to transform the NIH/3T3 cells. The structure of Ras protein bound with GTP or GDP has been analyzed by x-ray crystallography(Sprang,1997), and it has been shown that G12 was located at the G1 Box (GXXXXGKS/T) and associated with α-,β-phosphate, that Q61 was nearby the G3 Box (DXXG) and associated with γ-phosphate and Mg2+, and that N116 was located at the G4 Box (NKXD) and recognized the guanine ring. The G12V and Q61K mutants have a reduced intrinsic GTPase activity, and thus became GTP-locked, active form of Ras. The GDP/GTP exchange rate of N116I was increased. Therefore, the mutants of Ras were constitutively activated and passed down the growth signal in cells. I detected the GTP-locked ability of the wildtype and mutant Ras fusion proteins with thin layer chromatography. The results showed that the GTP binding activities were Q61K > G12V > wildtype and that N116I did not have the GTP/GDP binding activity. The protein geranylgeranyltransferase I(PGGT I) partially purified from the hepatopancreas of the shrimp Penaeus (Marsupenaeus) japonicus was able to react with the wildtype and mutant S-Ras fusion proteins, the extent of geranylgeranylation positively correlates with the GTP-locked ability. It was thus proven that the point mutations in the guanidine nucleotide-binding region of the S-Ras protein N-termini affected the C-terminal geranylgeranylation efficiency.
Finally, the wildtype and mutant S-ras genes were transfected to the BALB/3T3 cells and tested for transformed foci formation. The S-ras(Q61K) had the ability to transform the BALB/3T3 cells and this result shows the S-ras had biological activity. The other two mutants: the S-ras(G12V) and the S-ras(N116I) did not form transformed foci, which differs from the mammalian(human, mouse, rat) Ras mutants, which with the same mutant sites and could transform the NIH/3T3 cells. When I analyzed the S-ras(Q61K) transformed foci cells, I found the mRNA level of the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be 50% higher than non-transformed controlled cells. This enzyme is involved in the glycolysis pathways and is related with the proliferation of cancer cells. Although data from the BALB/3T3 cells did not provide convincing evidence for my hypothesis, that increasing the GTP-locked ability of the S-Ras proteins could enhance their C-termini geranylgeranylation, and thus enhance the transforming efficiency, it should be noted here that using the mammalian cells to study shrimp Ras cellular function may be unsuitable. Since the C-termini of the S-Ras and the mammalian(human, mouse, rat) Ras are very different, they may prefer to bind specific components of their own cell types, for example, cell membrane or docking proteins. The postion and identity of the mutation I introduced may affect S-Ras''s affinity for its regulatory proteins and/or down-stream effectors in addition to GTP-locked ability. This may explain the lack of correlation between GTP-locked ability and transformation efficiency of the different mutants I studied. We would therefore like to establish a shrimp cell line for further studies.
| Identifer | oai:union.ndltd.org:TW/088NTU00312005 |
| Date | January 2000 |
| Creators | Chein-Fuang Huang, 黃千芳 |
| Contributors | Nin-Nin Chuang, 莊 寧 寧 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 142 |
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