The N-terminal Moiety of CDC25Mm, a GDP/GTP Exchange Factor of Ras Proteins, Controls the Activity of the Catalytic Domain: MODULATION BY CALMODULIN AND CALPAIN

Soria Baouz,Eric Jacquet,Alberto Bernardi,Andrea Parmeggiani

doi:10.1074/jbc.272.10.6671

Abstract

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.

Highlights

This work describes the in vitro properties of fulllength CDC25Mm (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21
Farnsworth et al [24] reported that in vivo the activity of the homologous p140rasGRF 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 CDC25Mm of an IQ domain, a sequence frequently found in proteins interacting with calmodulin [25, 26]

Summary

Introduction

This work describes the in vitro properties of fulllength CDC25Mm (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21. CDC25Mm, isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p211⁄7GDP dissociation rate; its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25Mm285) and 5 times lower than the activity of the C-terminal half-molecule (631 residues). The GEF activity of CDC25Mm 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 CDC25Mm (residues 202–229), calmodulin interacted functionally with the exchange factor. The purified GEF shows a specific activity much lower than its isolated catalytic domain or the C-terminal half-molecule; it is inhibited by calmodulin and is cleaved by calpain

Methods

Results

Conclusion