Sites of phosphorylation by protein kinase A in CDC25Mm/GRF1, a guanine nucleotide exchange factor for Ras.
J Biol Chem
; 276(3): 1742-9, 2001 Jan 19.
Article
en En
| MEDLINE
| ID: mdl-11018028
Activation of the neuronal Ras GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 is known to be associated with phosphorylation of serine/threonine. To increase our knowledge of the mechanism involved, we have analyzed the ability of several serine/threonine kinases to phosphorylate CDC25Mm in vivo and in vitro. We could demonstrate the involvement of cAMP-dependent protein kinase (PKA) in the phosphorylation of CDC25Mm in fibroblasts overexpressing this RasGEF as well as in mouse brain synaptosomal membranes. In vitro, PKA was found to phosphorylate multiple sites on purified CDC25Mm, in contrast to protein kinase C, calmodulin kinase II, and casein kinase II, which were virtually inactive. Eight phosphorylated serines and one threonine were identified by mass spectrometry and Edman degradation. Most of them were clustered around the Ras exchanger motif/PEST motifs situated in the C-terminal moiety (residues 631-978) preceding the catalytic domain. Ser745 and Ser822 were the most heavily phosphorylated residues and the only ones coinciding with PKA consensus sequences. Substitutions S745D and S822D showed that the latter mutation strongly inhibited the exchange activity of CDC25Mm on Ha-Ras. The multiple PKA-dependent phosphorylation sites on CDC25Mm suggest a complex regulatory picture of this RasGEF. The results are discussed in the light of structural and/or functional similarities with other members of this RasGEF family.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Quinasas Dependientes de AMP Cíclico
/
Ras-GRF1
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
J Biol Chem
Año:
2001
Tipo del documento:
Article
País de afiliación:
Francia
Pais de publicación:
Estados Unidos