Sites of phosphorylation by protein kinase a in CDC25Mm/GRF1, a guanine nucleotide exchange factor for Ras

Activation of the neuronal Ras GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 i s known to be associated with phosphorylation of serine/threonine. To incre ase our knowledge of the mechanism involved, we have analyzed the ability o f several serine/threonine kinases to phosphorylate CDC25Mm in vivo and in vitro. We could demonstrate the involvement of cAMP-dependent protein kinas e (PKA) in the phosphorylation of CDC25Mm in fibroblasts overexpressing thi s RasGEF as well as in mouse brain synaptosomal membranes. In vitro, PKA wa s found to phosphorylate multiple sites on purified CDC25Mm, in contrast to protein kinase C, calmodulin kinase II, and casein kinase II, which were v irtually inactive. Eight phosphorylated serines and one threonine were iden tified by mass spectrometry and Edman degradation. Most of them were cluste red around the Ras exchanger motif/PEST motifs situated in the C-terminal m oiety (residues 631-978) preceding the catalytic domain. Ser(745) and Ser(8 22) were the most heavily phosphorylated residues and the only ones coincid ing 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 sugges t 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.