BRCA1 was found to co-immunoprecipitate with ATM and ATM was found to co-immunoprecipitate with BRCA1 from HeLa cell nuclear extracts.
Gamma-irradiation of cells was found to cause endogenous BRCA1 to migrate more slowly on SDS-PAGE, suggesting phosphorylation of BRCA1. This effect was not seen in ATM-deficient cells, but was restored by addition of an ATM expression vector.
Phosphorylation of glutathione-S-transferase (GST)
-BRCA1 fusion proteins in vitro by ATM occurred mostly between amino acids 1351 and 1552. Mass spectroscopy of a fusion protein consisting of GST and BRCA1 residues 1021-1552, phosphorylated in vitroby ATM revealed two different phosphopeptides when analysed before and after phosphatase treatment. Peptide sequencing of these peptides indicated that serine 1423 and serine 1524 were phosphorylated.
Gamma-irradiation of cells expressing a BRCA1 fragment (residues 1351-1552) caused a shift in its mobility on SDS-PAGE, and this effect was accentuated by the co-expression of ATM. However, mobility of a BRCA1 fragment, identical except for the mutation of serines 1423 and 1524 to alanines (which cannot be phosphorylated) was only slightly shifted by gamma-irradiation, and this effect was not accentuated by expression of active ATM.
Introduction of wild type, full length BRCA1 into BRCA1-mutant HCC1937 cells decreased their sensitivity to gamma-irradiation by both cell viability and colony forming assays. However, introduction of a BRCA1 mutant (S1423A and S1524A) did not.