Overexpression and functional analysis of a mitogen-inducible nuclear GTPase activating protein, Spa-1.
The two Ras-related GTPases called Rap1 and Rsr1, which share 50% sequence identity with Ras GTPases are known to be activated by two distinct mammalian GAPs, i.e. cytosolic GAP3c of 55 kDa and membrane-bound GAP3m of 85 kDa. Recently we have cloned a gene encoding a 68 kDa (p68) protein product, which is associated with chromosomes during interphase. The N-terminal 190 amino acids share 43% sequence identity with the second half of the GTPase activating domain (residues 210-397) of GAP3m. The N-terminal fragment of 209 amino acids of Spa-1 (called Span-N) was overproduced in E. coli as a glutathione S-transferase (GST) fusion protein and affinity purified. Rap1 and Rsr1 GTPase stimulatory activity of Spa-1 was tested and compared with GAP3m. Spa-1 preferentially stimulates Rsr1 GTPase rather than Rap1 GTPase, while GAP3m has a preference for Rap1 GTPase. This suggests that although Spa-1 and GAP3m stimulate GTPase of Rap1 family members, they differ in affinity for them. By mutational analysis it was also found that amino acid residues 10-183 are enough for Rap GAP activity of Spa-1.
Nur-e-Kamal
M S
MS
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
D000818:Animals; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D066298:In Vitro Techniques; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D015394:Molecular Structure; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D020691:rab GTP-Binding Proteins; D020668:rap GTP-Binding Proteins
D020690:GTPase-Activating Proteins; D008934:Mitogens; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C091108:Sipa1 protein, mouse; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C084013:RSR1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins; D020668:rap GTP-Binding Proteins