The N-terminal moiety of CDC25(Mm), a GDP/GTP exchange factor of Ras proteins, controls the activity of the catalytic domain. Modulation by calmodulin and calpain.
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 approximately 100 times higher IC50 than in the case of CDC25(Mm) ( approximately 10 microM versus 0.1 microM, 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.
Baouz
S
S
Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France.
Jacquet
E
E
Bernardi
A
A
Parmeggiani
A
A
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000502:physiology*; D002154:Calpain / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D020666:ras-GRF1
D002147:Calmodulin; D018797:Cell Cycle Proteins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D020666:ras-GRF1; D004533:Egtazic Acid; D010749:Phosphoprotein Phosphatases; D002154:Calpain; D002118:Calcium