| Rapl belongs to Ras superfamily of small GTP-binding proteins, which is considered to control cell growth, differentiation and survival. Rapl was regarded as an antagonist of Ras because it can revert the phenotype of oncogene Ras transformed cells. Recent researches show that Rapl has its Ras-independent functions in a variety of cellular systems besides its effect as an antagonist of Ras.Rapl, like Ras, cycles between a GDP bound inactive form and a GTP-bound active form. This switching is regulated by specific guanine nucleotide exchange factors (GEFj) responsible for " on " switch and GTP^ activating proteins (GAPS) for "off" switch.In contrast to Rap GEFs, The knowledge about the RaplGAP is much less available. Rap-specific GAPs have no sequence homology with RasGAP and other GAPs such as RanGAP and RhoGAP. Rap is the only one which does not have the invariant Gin (Gin 61 in Ras) in switch II which introduce the arginine finger on GAP into active site of Ras to complement catalytic machinery. It suggests that RaplGAP has a different mechanism for both the intrinsic and stimulated GTP hydrolysis mechanism of Rap.Recent researches implicate that RaplGAP may interact with different type of alpha subunit in heterotrimeric G protein and suggest a cellular cross-talk between small G proteins and heterotrimeric G protein.To insight the function of RaplGAP and the mechanism of RaplGAP hydrolyzing GTP to GDP, Yeast two hybrid system (Y2H) was employed to look for the potential proteins that interact with RaplGAP protein. Because the sequence and functions of every chromosome of C. elegans have been studied completely. So, first we employed the RapGAP and cDNA library from Celegans to start the screen. In Y2H, RapGAP from C. elegans was used as bait to screen C. elegans cDNA library. After -LTH screen, 63 colonies were checked. By Lac Z screen, 19 positive colonies were found. After PCRchecking and electro-transformation plasmid from C. elegans into DH5a, isolating plasmid from DHSct, digesting them with restriction enzymes and DNA sequencing, six cDNA fragments, which protein products can interact with RapGAP, from cDNA library were got. Two of them were RapGAP according to DNA sequences. To confirm this finding, we re-transformed these two DNA fragments into Y190 with RapGAP individually again. After -LTH and Lac Z check, the conclusion is that two molecules of RapGAP protein from C.elegans really interacted with each other in Y2H system.To check if human Rap 1 GAP can form dimer in mammalian cells, pcDNA-myc-RaplGAP (human) and pMT2-HA-RaplGAP (human) were constructed and co-transfected into A14 cells. With co-IP method (both with IP anti-MYC, WB anti-HA and IP anti-HA, WB anti-MYC), we found that two molecules of human Rap 1 GAP protein dimerized in A14 cells.To answer the questions, which part of human Rap 1 GAP is involved in the interaction and whether the dimerization affects its catalytic activity, Goloco domain .(AG), N-terminal 75 amino acids (AN), C-terminal 247 amino acids (AC), both C-terminal and N- terminal (ANC) in human RaplGAP deleted plasmids were constructed, and Co-transfected with pPC311-MYC-RaplGAP into A14 cells. A mutant of PKA phosphorylated sites (MP) was also checked. The result showed that each of them could bind to full length of RaplGAP by co-IP.Because the 75-101 amino acids of RaplGAP is important for its catalytic activity. Deletion of this part leads to loss of the GAP activity completely. But this part is beyond the catalytic domain. Since the dimerization of RapGAP is conserved from C. elegans to human, our research focused on the conserved amino acids in this part to check which conserved amino acid was concerned with the dimerization and how was the relationship between the dimerization and catalytic activity. Four conserved amino acids in ANC of RaplGAP were chosen and site-mutants of them were made one by one. By co-transfection of the mutants with full-length RaplGAP, we found the mutant at 82 position could still bind to full length RaplGAP, while th...
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