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.