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, 19 (7), 4843-54

BRCA1 Is Phosphorylated at Serine 1497 in Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

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BRCA1 Is Phosphorylated at Serine 1497 in Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

H Ruffner et al. Mol Cell Biol.

Abstract

BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

Figures

FIG. 1
FIG. 1
Endogenous and overexpressed BRCA1 reveal similar phosphorylation patterns in vivo. Shown are the two-dimensional tryptic phosphopeptide maps of endogenous BRCA1 from HBL-100 (A), 293T (B), and M059J (C) cells and of overexpressed full-length (D) and truncated BRCA1 lacking aa 772 to 1050 (E) from transiently transfected 293T cells. BRCA1 was immunoprecipitated with Ab-D (for maps B to E) or Ab-C plus Ab-D (for map A) from lysates of [32P]phosphoric acid-labeled cells. The immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis, and the labeled BRCA1 protein species were enzymatically hydrolyzed with trypsin. The resulting peptides were separated in the first and second dimensions by electrophoresis and chromatography, respectively, as indicated by the arrows. Diamonds mark sample origins; circles (labeled p1 to p7, p9, p11 to p13 [57], and p17 to p20) indicate phosphopeptides detected in the individual maps. Map D represents a combination of phosphopeptide maps of endogenous and overexpressed BRCA1 protein from 293T cells because Ab-D immunoprecipitated both proteins, which have similar mobilities on SDS-polyacrylamide gels (unlike the faster-running truncated BRCA1 lacking aa 772 to 1050 [data not shown]); however, since overexpressed BRCA1 is far more abundant than the endogenous protein (data not shown), the contribution of endogenous BRCA1 in map D is negligible.
FIG. 2
FIG. 2
BRCA1 serine residue 1497 is phosphorylated in vivo. (A) The C-terminal half of BRCA1 (aa 1051 to 1863), either wild type (lane 1) or with the S1497A substitution (lane 2), was expressed in 293T cells that were labeled with [32P]phosphoric acid. The fragments were immunoprecipitated with Ab-D and separated on SDS-polyacrylamide gels. Protein marker bands (200 and 97.4 kDa) are indicated on the left. (B) Two-dimensional tryptic phosphopeptide maps of the BRCA1 fragments shown in panel A. The asterisk (*) in the left panel denotes a fraction of phosphopeptide p7 (§) having an abnormal mobility in the chromatographic dimension, possibly due to a thin-layer chromatography plate artifact. (C) Manual Edman degradation of phosphopeptide p4. The procedure was performed as described elsewhere (72), and the reaction products were analyzed on a PhosphorImager (Molecular Dynamics). Numbers above the panel indicate cycles of degradation (0 denotes starting material). M, free [32P]phosphate applied as a marker. The ratio of free [32P]phosphate to phosphopeptide p4 was determined by using the ImageQuaNT software (Molecular Dynamics). About 50% of [32P]phosphate was liberated after the second cycle (compared to the fifth cycle), whereas most of the remaining [32P]phosphate was gradually released over cycles 3 to 5, probably due to incomplete reactions. (D) Matrix-assisted laser desorption mass spectrum of the purified Lys-C peptide 1490-1500 derived from BRCA1 aa 1314 to 1652. x axis, mass-to-charge ratio (m/z); y axis, relative intensity.
FIG. 3
FIG. 3
BRCA1 is phosphorylated by CDK2-cyclin complexes in vitro. (A) Myc-BRCA1 wt was transiently transfected into 293T cells and immunoprecipitated with antibody 9E10. The immunoprecipitate was phosphorylated in vitro in the absence (lane 1) or presence of a recombinant CDK-cyclin complex: CDK2-cyclin E (lane 2), CDK6-cyclin D1 (lane 3), CDK2-cyclin A (lane 4), or CDC2-cyclin B1 (lane 5). The reaction products were visualized by autoradiography following SDS-polyacrylamide gel electrophoresis and transfer to a membrane. The 200-kDa protein marker is indicated on the left. (B) Two-dimensional tryptic phosphopeptide maps derived from BRCA1 protein depicted in panel A. a, b, and c, maps of BRCA1 from lanes 1, 3, and 5, respectively. Phosphopeptides p4, pA, and pB are depicted in Fig. 4B.
FIG. 4
FIG. 4
An endogenous protein kinase activity as well as recombinant CDK2-cyclin complexes phosphorylate BRCA1 residues T967 and S1497 in vitro. (A) 293T cells were transiently transfected with Myc-BRCA1 that was either wild type (Wt; lanes 1 and 2) or mutated as indicated. The different BRCA1 species were immunoprecipitated with antibody 9E10 and subjected to in vitro kinase reactions in the absence (lanes 1, 3, 5, 7, and 9) or presence (lanes 2, 4, 6, 8, and 10) of the recombinant CDK2-cyclin A complex. The 200-kDa protein marker is shown on the left. (B) Two-dimensional tryptic phosphopeptide maps derived from the overexpressed BRCA1 protein species depicted in panel A. a to j, maps of BRCA1 from lanes 1, 3, 5, 2, 4, 6, 7, 9, 8, and 10, respectively. (C) Two-dimensional maps of mixtures of tryptic phosphopeptides derived from in vitro-phosphorylated and in vivo-labeled BRCA1. a, in vitro-phosphorylated BRCA1 alone; b, in vivo-labeled endogenous BRCA1 from HBL-100 cells alone; c, mixture of in vitro-phosphorylated BRCA1 and in vivo-labeled endogenous BRCA1 from HBL-100 cells; d, in vivo-labeled overexpressed BRCA1 in 293T cells alone; e, mixture of in vitro-phosphorylated BRCA1 and in vivo-labeled overexpressed BRCA1 in 293T cells. Numbered circles indicate phosphopeptides p3, p4, p9, p11, p18, p20, pA, and pB (for simplicity, only a few phosphopeptides were circled); unlabeled circles indicate the lack of the respective peptides; circles labeled by an asterisk mark phosphopeptides related to pA (see text); T (threonine) and S (serine) indicate the phosphoamino acid content of the corresponding phosphopeptide.
FIG. 4
FIG. 4
An endogenous protein kinase activity as well as recombinant CDK2-cyclin complexes phosphorylate BRCA1 residues T967 and S1497 in vitro. (A) 293T cells were transiently transfected with Myc-BRCA1 that was either wild type (Wt; lanes 1 and 2) or mutated as indicated. The different BRCA1 species were immunoprecipitated with antibody 9E10 and subjected to in vitro kinase reactions in the absence (lanes 1, 3, 5, 7, and 9) or presence (lanes 2, 4, 6, 8, and 10) of the recombinant CDK2-cyclin A complex. The 200-kDa protein marker is shown on the left. (B) Two-dimensional tryptic phosphopeptide maps derived from the overexpressed BRCA1 protein species depicted in panel A. a to j, maps of BRCA1 from lanes 1, 3, 5, 2, 4, 6, 7, 9, 8, and 10, respectively. (C) Two-dimensional maps of mixtures of tryptic phosphopeptides derived from in vitro-phosphorylated and in vivo-labeled BRCA1. a, in vitro-phosphorylated BRCA1 alone; b, in vivo-labeled endogenous BRCA1 from HBL-100 cells alone; c, mixture of in vitro-phosphorylated BRCA1 and in vivo-labeled endogenous BRCA1 from HBL-100 cells; d, in vivo-labeled overexpressed BRCA1 in 293T cells alone; e, mixture of in vitro-phosphorylated BRCA1 and in vivo-labeled overexpressed BRCA1 in 293T cells. Numbered circles indicate phosphopeptides p3, p4, p9, p11, p18, p20, pA, and pB (for simplicity, only a few phosphopeptides were circled); unlabeled circles indicate the lack of the respective peptides; circles labeled by an asterisk mark phosphopeptides related to pA (see text); T (threonine) and S (serine) indicate the phosphoamino acid content of the corresponding phosphopeptide.
FIG. 5
FIG. 5
BRCA1 coimmunoprecipitates with CDK2 and cyclin A. (A) Myc-BRCA1 wt coimmunoprecipitates with CDK2. 293T cells were transfected with expression plasmids for the following proteins: lane 1, Myc-BRCA1 wt; lane 2, Myc-BRCA1 wt, cyclin A, and CDK2; lane 3, Myc-BRCA1 wt, cyclin E, and CDK2; lane 4, cyclin A and CDK2; lane 5, cyclin E and CDK2; lane 6, Myc-BRCA1 wt and CDK2; and lane 7, CDK2. CDK2-containing complexes were immunoprecipitated (I.P.) from each sample with a rabbit CDK2 antibody (α-CDK2) and analyzed after separation on SDS-polyacrylamide gels by Western blot analysis for the presence of Myc-BRCA1 wt, using antibody 9E10 (a, bottom row), and CDK2, using a mouse anti-CDK2 monoclonal antibody (b, bottom row). The two top rows in panels a and b (Input) represent the relative amounts of Myc-BRCA1 wt and CDK2 in the cell lysates before immunoprecipitation. Panel c shows that in a duplicate experiment, Myc-BRCA1 wt from cell lysates overexpressing proteins as indicated in lanes 1, 2, 3, and 6 (in panels, a and b) was further resolved on an SDS-polyacrylamide gel (compared to panel a, top row) in order to increase resolution. (B) Untagged, full-length wild-type BRCA1 coimmunoprecipitates with CDK2 and cyclin A. Lysates from a HeLa cell line stably overexpressing BRCA1 (57a) were subjected to immunoprecipitations using the following antibodies: lane 1, an aliquot before immunoprecipitation; lane 2, anti-CDK2; lane 3, anti-CDK2, after preincubation with the blocking peptide; lane 4, anti-NF-κB p65 as a control; lane 5, anti-cyclin A; and lane 6, anti-cyclin E. Western blot analysis revealed the presence of BRCA1 and CDK2, as shown in the upper and lower panels, respectively. The 250- and 42-kDa markers are indicated on the left.
FIG. 6
FIG. 6
Phosphorylation of BRCA1 is decreased by inhibition of CDK2. (A) CDK2 dn decreases the phosphorylation state of endogenous BRCA1. 293T cells were transiently transfected with a control plasmid (lane 1) or a plasmid expressing CDK2 dn (lane 2). Endogenous BRCA1 from both sources was immunoprecipitated with Ab-D prior to treatment with (lanes 4 and 6) or without (lanes 3 and 5) BAP before analysis by immunoblotting with Ab-D. Filled arrow, BRCA1 from mock-transfected cells; open arrow, faster-migrating BRCA1 species due to expression of CDK2 dn; asterisk, BAP-treated BRCA1. (B) BRCA1 is phosphorylated in vitro by a cellular kinase activity that is sensitive to CDK2 dn. 293T cells were cotransfected with a plasmid expressing Myc-BRCA1 wt and either a control plasmid (lane a) or CDK2 dn (lane b). BRCA1 was immunoprecipitated under native conditions with antibody 9E10, and in vitro kinase reactions were performed on the immunoprecipitates. a and b, two-dimensional tryptic phosphopeptide maps of the BRCA1 protein species shown in lanes a and b, respectively, from a duplicate experiment. (C) p21 and butyrolactone I but not p16 inhibit the endogenous protein kinase activity that phosphorylates BRCA1 at T967 and S1497 in vitro. Myc-BRCA1 wt was subjected to in vitro kinase reactions as described above. Lane a, no CDI added; lanes b, c, and d, p16, p21, and butyrolactone I (Butyr), respectively, added. a to d, phosphopeptide maps of BRCA1 protein shown in lanes a to d, respectively. The top portions of panels B and C represent the amount of (Western blot analysis using antibody 9E10 [bottom rows]) and the in vitro kinase activity on (radioactive incorporation onto BRCA1 [top rows]) BRCA1. The 250- and 200-kDa markers are shown on the left.

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