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Identification of phosphorylation sites in the multifuctional enzmye CAD
Author(s) -
Wauson Eric M,
Graves Paul R,
Higgins Matthew J.,
Graves Lee M.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1122-b
Subject(s) - phosphorylation , biology , kinase , protein phosphorylation , biochemistry , serine , protein kinase a , mutant , gene
CAD, the highly regulated multienzyme complex, catalyzes the rate‐limiting step in de novo pyrimidine synthesis. CAD expression and activity are highly correlated with cell proliferation and CAD is reported to be necessary for the early embryological development of specific tissues in zebrafish and drosophila. CAD is a phospho‐protein and a substrate for the MAP Kinase ERK and protein kinase A (PKA). ERK phosphorylated CAD on Thr456 and PKA phosphorylated Ser1406 and Ser1859. The phosphorylation by both of these kinases affected the allosteric regulation of CAD. Additionally, serum was shown to induce CAD phosphorylation in intact cells. However, CAD deficient cells (G9C) transfected with the CAD Thr456Ala mutant still display CAD phosphorylation in response to serum, suggesting that Thr456 is not the major serum‐induced phosphorylation site. In this study we re‐evaluate CAD phosphorylation in intact cells using 32 ‐P metabolic labeling, 2‐dimensional phospho‐peptide mapping, and mass spectrometry. Our data shows that there are numerous phospho‐peptides in CAD from intact cells grown in low and normal serum concentrations. We identified an amino acid phosphorylated in response to serum stimulation and have created alanine and glutamic acid mutants of residues in CAD that are phosphorylated in intact cells. Currently the effects of these mutations on enzyme activity, enzyme localization, and protein‐protein interactions are being examined. Additionally, the identity of kinases responsible for the phosphorylation of specific CAD residues are being investigated.