The N-terminal Moiety of CDC25Mm, a GDP/GTP Exchange Factor of Ras Proteins, Controls the Activity of the Catalytic Domain

Abstract

This work describes the in vitro properties of fulllength 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 p21⅐GDP 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 Ca 2؉ -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 Ca 2؉ ions. Calmodulin also inhibited C-CDC25 Mm285 but with a ϳ100 times higher IC 50 than in the case of CDC25 Mm (ϳ10 M versus 0.1 M, 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.The mouse CDC25 Mm 1 protein is a guanine nucleotide exchange factor (GEF) regenerating the active form of H-ras p21, the complex with GTP (1-3). Homologous products were found in rat (p140-rasGRF) (4) and human (H-GRF) (5, 6). These rasGEFs have been described to be specific for the central nervous system (4 -9). Some evidence has also been reported for the existence of full-length and truncated forms of these exchange factors in other tissues (10,11). Experiments in vivo suggest that the upstream connection of this GEF involves G-protein-coupled receptors (9, 12, 13) and not hormone-receptor-bound tyrosine kinases via the adaptor protein GRB2, as has been found for SOS, a ubiquitous rasGEF (14 -16). CDC25 Mm contains in the N-terminal moiety two domains of pleckstrin homology (PH1 and PH2), one of DBL homology (DH) and a coiled-coil region that follows the PH1 domain (cf. (21) and the DH is a domain sharing similarity with a GDP/GTP exchange factor of members of the Rho family (2,4,22,23). Farnsworth et al. (24) reported that in vivo the activity of the homologous p140-rasGRF from rat brain is enhanced by raising the calcium concentration, an effect associated with the binding of calmodulin, and that p140-rasGRF and calmodulin form a stable complex. A direct action of calmodulin was supported by the presence in the N-terminal region of CDC25 Mm of an IQ domain, a sequence frequently found in proteins interacting with calmodulin (25, 26). Very recent experiments in vivo have indicated that PH1, ...

A Ras-induced conformational switch in the Ras activator Son of sevenless

“…Previous studies have suggested that the Rem domain of RasGRF1 is regulatory in function, containing phosphorylation sites and PEST motifs (25)(26)(27)(28). The level of activity we observe for the isolated Cdc25 domain of RasGRF1, although significantly greater than the intrinsic rate of nucleotide release from Ras, is much lower than the maximum rate observed for allosterically activated Sos cat .…”

Sites of Phosphorylation by Protein Kinase A in CDC25Mm/GRF1, a Guanine Nucleotide Exchange Factor for Ras

“…Results in vitro with CDC25Mm (13) and in vivo with SOS (14) indicated that the N-terminal portion of GEF down-regulates the activity of the catalytic domain. Several reports have shown that activation of CDC25Mm is coupled with Ca 2ϩ influx, as well as with formation of a complex with calmodulin and phosphorylation (8,15).…”

“…The cell culture, chilled to 4°C, was centrifugated at 5,000 ϫ g for 10 min and the pellet resuspended in 10 ml of 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10% glycerol, 7 mM ME, and 0.5 mM Pefabloc-SC. After sonication (five times for 15 s each), the suspension was centrifuged at 25,000 ϫ g for 20 min at 4°C and the supernatant was mixed batchwise with 5 ml of glutathione-Sepharose 4B (Amersham Pharmacia Biotech) or 5 ml of amylose resin (New England Biolabs), and gently shaken for 30 min at 4°C, as reported previously (13,22). The resin was washed four times with 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 7 mM ME and the protein eluted with glutathione (3 mg/ml) or maltose (10 mM) solutions in the same buffer.…”

Novel aspects of Ras proteins biology: regulation and implications

“…The interaction of GRP with Ca 2+ -CaM leads to GRF activation in intact cells, however, this effect does not occur in vitro. 10,11 It has therefore been proposed that the effect of Ca 2+ -CaM in vivo may be indirect, modulating the interaction of RasGRF with its targets. 10 Calcium signals could also contribute to GRF activation by promoting the proteolytic removal of the N-terminal domain, as it has been shown in vitro.…”

“…10 Calcium signals could also contribute to GRF activation by promoting the proteolytic removal of the N-terminal domain, as it has been shown in vitro. 11 A novel member of the family of Ras-GEFs has recently been cloned from brain 12 and lymphoid tissues. 4 This factor, named RasGRP, possesses calcium and diacyglycerol-binding domains and may directly couple changes in diacylglycerol and possibly calcium concentrations to Ras activation.…”