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

Soria Baouz,Eric Jacquet,Katia Accorsi,Codjo Hountondji,Monica Balestrini,Renata Zippel,Emmapaola Sturani,Andrea Parmeggiani

doi:10.1074/jbc.m005770200

Abstract

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.

Highlights

Ras proteins are molecular switches cycling between the active GTP-bound and the inactive GDP-bound forms [1]
The nonreceptor tyrosine kinase Src can induce on CDC25Mm a Rac1-guanine nucleotide exchange factors (GEFs) activity [18], showing that this protein is an important element for cross-regulation of Ras and Rac-dependent pathways [19], as has been reported for SOS [20] and GRF2 [21]
All of the CDC25Mm preparations used were purified in native conditions and showed a well characterized RasGEF activity

Summary

EXPERIMENTAL PROCEDURES

Materials—[␥-32P]ATP (111 Tbq/mmol) was from PerkinElmer Life Sciences; the PKA catalytic subunit, PKC, and casein kinase II were from Roche Molecular Biochemicals; and calmodulin kinase II was from Calbiochem. Samples analyzed by MALDI mass spectrometry were prepared by mixing 1.5 ␮l of matrix (saturated solution of ␣-cyano-4-hydroxycinnamic acid (Sigma) in 40% acetonitrile, 0.1% trifluoroacetic acid) with 1 ␮l of peptide (5–10 pmol) This mixture was loaded on a stainless steel sample holder and dried at room temperature. The labeled Ha-Ras1⁄7[3H]GDP complex was prepared by incubation of 2 ␮M Ha-Ras and 6 ␮M [3H]GDP (350 Bq1⁄7pmolϪ1, PerkinElmer Life Sciences) for 5 min at 30 °C in 100 ␮l of buffer B (50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 100 mM NH4Cl, and 0.5 mg1⁄7mlϪ1 bovine serum albumin) containing 3 mM EDTA. The samples (5–10 ␮l) were filtered through nitrocellulose discs (Sartorius 11306, 0.45 ␮m), washed twice with 3 ml of ice-cold buffer C (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NH4Cl), and the retained Ha-Ras-bound radioactivity counted. Molecular size markers for SDS-PAGE were from Amersham Pharmacia Biotech

RESULTS

KPMSAIPAR KLSLNIPHTGGK TPEEIDVPATIPEK KHSSDVLK

DISCUSSION