The N-terminal Moiety of CDC25^Mm, a GDP/GTP Exchange Factor of Ras Proteins, Controls the Activity of the Catalytic Domain

This work describes the in vitro properties of full-length CDC25^Mm (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21. CDC25^Mm, isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p21·GDP dissociation rate; however, its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25^Mm285) and 5 times lower than the activity of the C-terminal half-molecule (631 residues). This reveals a negative regulation of the catalytic domain by other domains of the molecule. Accordingly, the GEF activity of CDC25^Mm was increased severalfold by the Ca²⁺-dependent protease calpain that cleaves around a PEST-like region (residues 798-853), producing C-terminal fragments of 43-56 kDa. In agreement with the presence of an IQ motif on CDC25^Mm (residues 202-229), calmodulin interacted functionally with the exchange factor. Depending on the calmodulin concentration an inhibition up to 50% of the CDC25^Mm-induced nucleotide exchange activity on H-ras p21 was observed, an effect requiring Ca²⁺ ions. Calmodulin also inhibited C-CDC25^Mm285 but with a ∼100 times higher IC₅₀ than in the case of CDC25^Mm (∼10 μM versus 0.1 μM, respectively). Together, these results emphasize the role of the other domains of CDC25^Mm in controlling the activity of the catalytic domain and support the involvement of calmodulin and calpain in the in vivo regulation of the CDC25^Mm activity.