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[Cancer Research 62, 6357-6361, November 15, 2002]
© 2002 American Association for Cancer Research

Advances in Brief

Regulation of BRCA1 Phosphorylation by Interaction with Protein Phosphatase 1{alpha}1

Ying Liu, David M. Virshup, Raymond L. White and Lih-Ching Hsu2

Department of Oncological Sciences, University of Utah, Salt Lake City, Utah 84112


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Numerous reports have revealed that the tumor suppressor BRCA1 may play an important role in DNA damage repair. BRCA1 is expressed and phosphorylated during cell cycle progression and after DNA damage. BRCA1 is hypophosphorylated in G0-G1 and probably during mitosis as well. Kinases known to phosphorylate BRCA1 include cyclin-dependent kinase 2, as well as ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related kinase (ATR), which function in G2 checkpoint control. However, protein phosphatases responsible for dephosphorylation of BRCA1 had yet to be identified. hCds1, which acts downstream of ATM, also phosphorylates a BRCA1 fragment containing amino acids 759-1064 [BRCA1 fragment 4 (BF4)]. We have used a GST-BF4 protein phosphorylated by hCds1 [glutathione S-transferase (GST)-BF4-P] as a substrate to identify potential phosphatases responsible for BRCA1 dephosphorylation. Data presented here show that both recombinant protein phosphatase 1 {alpha} (PP1{alpha}) catalytic subunit and endogenous PP1{alpha} dephosphorylate GST-BF4-P. Inhibitor 2 abolishes this activity. Overexpression of PP1{alpha} partially inhibits hyperphosphorylation of BRCA1 after ionizing radiation, indicating that PP1{alpha} dephosphorylates BRCA1 in vivo. BRCA1 and PP1{alpha} reciprocally coimmunoprecipitate, and a glutathione S-transferase pull-down assay shows that PP1{alpha} catalytic subunit associates directly with the BF4 region of BRCA1. In addition, BRCA1 inhibits PP1{alpha} activity. Therefore, BRCA1 is both a substrate and a regulator of PP1{alpha}. The interaction between BRCA1 and PP1{alpha} thus may play a role in DNA damage repair and cell cycle progression.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Inherited BRCA1 mutations are responsible for the majority of familial breast and ovarian cancer cases (reviewed in Ref. 1 ). BRCA1 is a tumor suppressor protein consisting of 1863 amino acids with an apparent molecular weight of Mr ~220,000. It is mainly localized to the nucleus where it displays a nuclear dot pattern (2) . BRCA1 is believed to play an important role in DNA repair and/or cell cycle regulation. Evidence indicates that BRCA1 associates with DNA repair proteins such as Rad51 and Rad50 (3 , 4) , which participate in the repair of double-strand breaks.

Phosphorylation of BRCA1 occurs mainly at multiple Ser3 sites and is tightly regulated by cell cycle progression and DNA damage. In early G1, BRCA1 is hypophosphorylated. It has been shown that BRCA1 becomes phosphorylated during the G1-S transition and remains phosphorylated during S and M phases (5, 6, 7) . BRCA1 interacts with cyclin-dependent kinase 2 and cyclins A, suggesting a role for cyclin-dependent kinase 2-cyclin complexes in the increase of BRCA1 phosphorylation at G1-S (6 , 7) . In addition, multiple DNA damage checkpoint kinases are involved in the regulation of BRCA1 phosphorylation. Both ATM and ATR participate in the phosphorylation of BRCA1 after {gamma}-irradiation (8, 9, 10) .

However, one report has demonstrated that BRCA1 protein is hypophosphorylated in MCF7 cells delayed in G2-M by colchicine (11) . We have confirmed this finding using COS-7 cells arrested in G2-M by sequential treatments with thymidine and nocodazole.4 In addition, we have shown that hypophosphorylated BRCA1 associates with {gamma}-tubulin and the centrosome during mitosis (12) . Therefore, it is likely that dephosphorylation occurs during G2-M progression. Identification of the PPs involved in dephosphorylation of BRCA1 will provide additional insight into BRCA1 functions during cell cycle progression and in response to DNA damage.

Protein phosphorylation and dephosphorylation are major mechanisms controlling cell cycle progression (reviewed in Refs. 13 , 14 ). Ser/threonine PPs regulate cell cycle control and help ensure correct chromosome segregation during mitosis. The four known types of Ser/threonine phosphatases, PP1, PP2A, PP2B, and PP2C, are classified based on their relative activity toward the {alpha}- and ß-subunits of phosphorylase kinase and sensitivity to inhibitor 1 and I-2. It has been reported that nuclear and chromatin-associated PP1 activity peaks during G0-G1 and mitosis (15) and is involved in mitotic progression (13) . Three major isoforms of PP1 have distinct subcellular localizations (16) . During mitosis, PP1{alpha} is localized to the centrosome, whereas PP1{gamma}1 and PP1{delta} associate with the mitotic spindle and chromosomes, respectively. BRCA1 protein is also associated with the centrosome during mitosis, as detected by immunofluorescence staining of whole cells and biochemical analysis of isolated centrosomes (12) .

BRCA1 is hyperphosphorylated after DNA damage (11 , 17) . The protein kinase hCds1 (hChk2), which is the mammalian homologue of Rad53 in Saccharomyces cerevisiae and Cds1 in Schizosaccharomyces pombe, acts downstream of ATM and is involved in G2-M checkpoint control after DNA damage (18, 19, 20) . Lee et al. (21) have identified Ser 988 in BRCA1 as the major hCds1 phosphorylation site. We have confirmed this finding, demonstrating that hCds1 phosphorylates BRCA1, primarily within BF4, which spans amino acids 759-1064. Extending these results, we have used hCds1-phosphorylated BF4 to demonstrate that PP1{alpha} interacts with and dephosphorylates BRCA1. Interestingly, BRCA1 inhibits PP1{alpha} activity and may serve as a regulator of PP1{alpha}.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Construction of Vectors.
The full-length hCds1 cDNA sequence was obtained from GenBank (GenBank Accession No. 4206720). The hCds1 cDNA was PCR amplified from a human colon phage cDNA library using primer pairs 5'-GGGGAATTCATGTCTCGGGAGTCGGATGTT-3' and 5'-GGCTCGAGTCACAACACAGCAGCACACACA-3'. The resulting EcoRI-XhoI fragment containing the hCds1 cDNA was cloned into pBluescript, sequence verified, and then subcloned into pGEX-5X-1 (Amersham Pharmacia Biotech) to generate an expression construct encoding a GST-hCds1 fusion protein. To generate a PP1{alpha}GFP expression vector, PP1{alpha} cDNA was PCR amplified using primer pairs 5'-gggctcgaggccaccatgtccgacagcgagaagctc-3' and 5'-gggaagctttttcttggctttggcagagtt-3' and a rabbit PP1{alpha} cDNA as a template and subcloned into the XhoI and HindIII sites of pEGFP-N1-KS(-) (22) .

Cell Culture and Transfection.
COS-7 and 293T cells were grown in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and fed twice a week. 293T cells were transfected overnight with PP1{alpha}GFP or the pEGFP-N1-KS(-) control vector using the calcium phosphate method. Twenty-four h after transfection, cells were either untreated or irradiated with 5 Gy of {gamma}-radiation and harvested for Western analysis 1–3 h after ionizing irradiation.

GST-Fusion Proteins.
GST-fusion proteins were expressed in Escherichia coli and purified using glutathione Sepharose beads (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Constructs of GST-BFs 1–6 were obtained from Drs. Junjie Chen and David Livingston at Dana-Farber Cancer Institute (3) .

hCds1 Kinase Assay.
The hCds1 kinase reaction was performed in a 25 µl-reaction volume containing 50 mM Tris (pH 7.5), 10 mM MgCl2, 2 mM DTT, 50 µM cold ATP, 5 µCi [{gamma}-32P]ATP (3000 Ci/mmol; 1 Ci = 37 MBq), 2 µg of GST-hCds1, 2 µg of GST-BF4, and glutathione beads. The reaction was incubated for 30 min at 30°C to generate 32P-labled GST-BF4-P.

Phosphatase Assay Using Phosphorylated GST-BF4 as a Substrate.
After the kinase reaction, 32P-labled GST-BF4-P on glutathione beads were washed three times with phosphatase reaction buffer and used as a substrate for the phosphatase assay. Phosphatases used were 0.67 ng (i.e., 0.01 units) of recombinant PP1{alpha} catalytic subunit of rabbit skeletal muscle (2.5 units/µl, specific activity: ~15 units/µg; New England BioLabs) and 20 or 180 ng of purified PP2A catalytic subunit in 25-µl reactions. One unit of PP1{alpha} is defined as the amount of enzyme that hydrolyzes 1 nmol of p-nitrophenyl phosphate (50 mM) in 1 min at 30°C in a total volume of 50 µl. PP1{alpha} reaction buffer contained 50 mM Tris (pH 7.0), 0.1 mM Na2EDTA, 5 mM DTT, 0.01% Brij 35, and 1 mM MnCl2. PP2A reaction buffer contained 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% BSA, and 0.1% 2-mercaptoethanol. COS-7 cell extract containing 0.1 µg of total protein and a PP1{alpha} immunoprecipitate from COS-7 cell lysate were also used as sources of PP1{alpha} in the phosphatase assay. I-2 (New England BioLabs) was added at a concentration of 400 ng/ml. The phosphatase assay was carried out in 25–50-µl reaction volume for 30 min at 30°C and stopped by adding an equal volume of 2x SDS sample buffer and boiled for 5 min. Proteins were separated by 4–20% SDS-PAGE (Invitrogen) and stained with Coomassie Blue. Gels were dried and autoradiographed. 32P-labled GST-BF4-P was quantified using ImageQuant software (Molecular Dynamics).

PP Assay Using Phosphorylase a as a Substrate.
The PP assay was performed according to the manufacturer’s instructions (Life Technologies, Inc.). The assay was carried out for 10 min at 30°C in a final volume of 30 µl with phosphatase and 30 µg of 32P-labeled phosphorylase a in a PP assay buffer containing 0.1 mM EDTA, 1 mg/ml BSA, 20 mM imidizole-HCl (pH 7.63), and 0.1% ß-mercaptoethanol. The cpm released was detected by a scintillation counter. Under the assay conditions, there was a linear correlation between the amount of enzyme and phosphatase activity.

BRCA1 and PP1{alpha} Immunoprecipitation.
COS-7 cells were lysed in NETN buffer containing 150 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8.0), 0.5% NP40, or L buffer (22) supplemented with protease inhibitors and phosphatase inhibitors (1 mM sodium orthovanadate and 50 mM sodium fluoride). COS-7 cell extract was incubated for 2 h at 4°C with BRCA1 rabbit polyclonal antibody C-20 (Santa Cruz Biotechnology), control rabbit IgG, or goat polyclonal PP1{alpha} antibody (raised against the COOH terminus of the catalytic subunit of human PP1{alpha}; Santa Cruz Biotechnology) with or without PP1{alpha} blocking peptide, and protein A or protein G beads. Immunoprecipitation was performed as described previously (22) , and Western blot analysis was performed using mouse monoclonal BRCA1 antibody MS110 (Calbiochem) or PP1{alpha} antibody (Santa Cruz Biotechnology).

GST Pull-Down Using GST-BRCA1.
GST-BRCA1 fusion proteins were expressed and purified as described above. COS-7 cells were lysed in NETN buffer with protease inhibitors. GST-BRCA1 fusion protein was incubated with 500 µg of COS-7 cell lysate and glutathione Sepharose beads in GST binding buffer for 2 h at 4°C, washed with L buffer, and subjected to SDS-PAGE. Western blot analysis was performed using PP1{alpha} antibody (Santa Cruz Biotechnology). To determine whether BF4 and PP1{alpha} catalytic subunit interact directly, 1 µg of GST-BF4, GST-BF4-P, GST-BF5, or GST-BF6 was incubated with 1 µl of recombinant PP1{alpha} catalytic subunit (2.5 units/µl) and glutathione beads for 2 h at 4°C. Beads were washed with L buffer and resuspended in SDS sample buffer. Precipitates were subjected to SDS-PAGE and followed by Western analysis using PP1{alpha} antibody.

BRCA1 S988-P-specific Antibody.
The antibody was generated as previously described by Lee et al. (21) . Briefly, Ser 988 (CRIPPLFPIKSFVKTK) and S988-P (CRIPPLFPIKSpFVKTK) peptides were custom synthesized by Bob Schackmann at the University of Utah. S988-P peptide was coupled to keyhole limpet hemocyanin and used for raising rabbit antisera. S988-P specific antibody was affinity-purified using Ser 988 and S988-P affinity columns. Production and purification of the antibody was a custom service provided by Covance Research Products, Inc.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Recombinant PP1{alpha} Catalytic Subunit Dephosphorylates BF4 of BRCA1.
BRCA1 is hyperphosphorylated in response to DNA damage (11 , 17) and is involved in S-phase and G2-phase checkpoints (23) . It is likely that BRCA1 is mainly hypophosphorylated in M phase (11) . We have also reported that a hypophosphorylated isoform of BRCA1 is associated with the centrosome during mitosis (12) . Hypophosphorylated BRCA1 may be generated by de novo synthesis or dephosphorylation of BRCA1. A working hypothesis is that dephosphorylation of BRCA1 may serve as a signal for completion of DNA repair and play a role in cell cycle progression into mitosis. We therefore are interested in identifying phosphatases involved in dephosphorylation of BRCA1. It has been reported that hCds1 phosphorylates BRCA1 at Ser 988 of BF4 (amino acids 759-1064) after DNA damage. Phosphorylation at Ser 988 is required for the release of BRCA1 from hCds1 and is important for BRCA1 to restore survival after DNA damage in BRCA1-mutated breast cancer cell line HCC1937 (21) . We first confirmed that hCds1 phosphorylated BRCA1 mainly on BF4 and then used GST-BF4 phosphorylated by GST-hCds1 (GST-BF4-P) as a substrate to search for PPs that could dephosphorylate BRCA1.

We first tested whether a recombinant PP1{alpha} catalytic subunit or a purified PP2A catalytic subunit could dephosphorylate GST-BF4-P. As shown in Fig. 1ACitation , 0.67 ng (0.01 units) of recombinant PP1{alpha} dephosphorylated GST-BF4-P efficiently (10% of the signal remained compared with the buffer control). In contrast, 20 ng of PP2A had no effect on GST-BF4-P phosphorylation level. Although 180 ng of PP2A did dephosphorylate GST-BF4-P (~30% signal remained), it was not as effective as 0.67 ng of PP1{alpha}. To exclude the possibility that the purified PP2A might have lost its phosphatase activity, we conducted a PP assay comparing the activity of 0.67 ng of recombinant PP1{alpha} to 20 ng of purified PP2A using 32P-labeled phosphorylase a as a substrate. Twenty ng of PP2A exhibited >5-fold phosphorylase phosphatase activity than 0.67 ng of PP1{alpha} (Fig. 1B)Citation . Taken together, these results suggest that GST-BF4-P may be a specific substrate of PP1{alpha}.



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  		Fig. 1. Recombinant PP1{alpha} catalytic subunit but not purified PP2A catalytic subunit dephosphorylates BF4. A, PP1{alpha} dephosphorylates BF4 much more efficiently than PP2A. The recombinant catalytic subunit of PP1{alpha} (0.67 ng or 0.01 units) and purified catalytic subunit of PP2A (20 and 180 ng) were incubated with GST-BF4-P. GST-BF4-P in reaction buffer was used as a negative control. The top panel shows phosphorylation of GST-BF4, and the bottom panel shows relative intensity of GST-BF4-P (% of the negative control). B, protein phosphatase assay of PP1{alpha} and PP2A. Activities of recombinant PP1{alpha} and purified PP2A catalytic subunits were determined by the release of 32P from 32P-labeled phosphorylase a. The assay was performed three times in duplicate. The phosphorylase phosphatase activity of 20 ng of PP2A is 5.6-fold of that of 0.67 ng of PP1{alpha}.

 
Endogenous PP1{alpha} Dephosphorylates BF4 of BRCA1.
The action of PP catalytic subunit is modulated by formation of heteromeric complexes with regulatory subunits that target the catalytic subunit to specific subcellular compartments and localize the catalytic subunit close to its substrates. Therefore, regulatory subunits of PPs can determine their substrate specificities. Results shown in Fig. 1Citation were obtained using recombinant or purified catalytic subunits of PP1{alpha} and PP2A, which lack regulatory subunits. To establish that PP1{alpha} holoenzymes were also active on GST-BF4-P, COS-7 cell extract and PP1{alpha} immunoprecipitates were used as sources for endogenous PP1{alpha} complex. A COS-7 cell extract removed the phosphate groups on GST-BF4-P to the same extent as recombinant PP1{alpha} catalytic subunit (Fig. 2A)Citation . Four hundred ng/ml (~17.5 nM) I-2, which specifically inhibits PP1 (13 , 14) , reduced the phosphatase activity of the COS-7 cell extract toward GST-BF4-P (~70% inhibition), indicating that the major phosphatase activity in COS-7 cell extract was because of PP1 (Fig. 2A)Citation . Immunoprecipitation of PP1{alpha} with an antibody specific for PP1{alpha} catalytic subunit might bring down the PP1{alpha} holoenzyme, which contains regulatory subunit(s) and possesses substrate specificity. As shown in Fig. 2BCitation , a PP1{alpha} immunoprecipitate also exhibited phosphatase activity toward GST-BF4-P, whereas protein G beads alone did not have this activity.



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  		Fig. 2. Endogenous PP1{alpha} specifically dephosphorylates BF4. A, crude extract from COS-7 cells (0.1 µg protein) was incubated with GST-BF4-P in the absence or presence of 400 ng/ml I-2. GST-BF4-P in reaction buffer was used as a negative control. B, immunoprecipitated PP1{alpha} was incubated with GST-BF4-P. Precipitate with protein G beads alone was used as a negative control. The top and middle panels show phosphorylation of GST-BF4, and the bottom panel show relative intensity of GST-BF4-P (percentage of the negative control).

 
Taken together, these data indicate that endogenous PP1{alpha} specifically dephosphorylates BRCA1, which is phosphorylated by hCds1.

PP1{alpha} Partially Inhibits Hyperphosphorylation of BRCA1 Induced by IR.
To provide evidence that PP1{alpha} could dephosphorylate BRCA1 in vivo, 293T cells were transfected with pEFGP-N1-KS(-) or PP1{alpha}GFP, followed by ionizing irradiation. As shown in Fig. 3Citation , PP1{alpha}GFP (Lanes 1 and 2) but not pEFGP-N1-KS(-)-transfected cells (Lanes 3 and 4) expressed PP1{alpha}GFP, which was detected by PP1{alpha} antibody. BRCA1 was hyperphosphorylated in pEFGP-N1-KS(-)-transfected cells after exposure to IR, as illustrated by slower migrating bands (Lane 4) compared with that in the untreated control (Lane 3). The mobility shift of BRCA1 in PP1{alpha}GFP-transfected cells (Lane 2) was less profound than that in pEFGP-N1-KS(-)-transfected cells (Lane 4). In addition, a BRCA1 band in Lane 2 comigrating with that in the control (Lane 1) was obvious and equally intense as the slower migrating band induced by IR (Lane 2). These results suggest that hyperphosphorylation of BRCA1 after IR is partially dephosphorylated by overexpression of PP1{alpha}GFP and PP1{alpha} is capable of dephosphorylating BRCA1 in vivo.



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  		Fig. 3. PP1{alpha} dephosphorylates BRCA1 in vivo. 293T cells transfected with PP1{alpha}GFP (Lanes 1 and 2) or the pEGFP-N1KS(-) control vector (Lanes 3 and 4) were untreated (Lanes 1 and 3) or irradiated with 5 Gy of {gamma}-radiation (Lanes 2 and 4). Cells were harvested 1–3 h after IR, lysed in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors, and subjected to SDS-PAGE and Western analysis. BRCA1 was detected by MS110 antibody and PP1{alpha}GFP was detected by PP1{alpha} antibody.

 
Direct Interaction between BRCA1 and PP1{alpha} Catalytic Subunit.
We next investigated whether there was a physical interaction between endogenous PP1{alpha} and BRCA1. Fig. 4ACitation demonstrates that PP1{alpha} antibody specific for PP1{alpha} catalytic subunit coimmunoprecipitated PP1{alpha} catalytic subunit (Mr ~37,500) and BRCA1 from a COS-7 cell lysate. Coimmunoprecipitation of PP1{alpha} and BRCA1 was blocked by addition of the immunogenic PP1{alpha} peptide. Conversely, BRCA1 immunoprecipitation using C-20 antibody also precipitated PP1{alpha} catalytic subunit but rabbit immunoglobulin did not (Fig. 4A)Citation . The reciprocal coimmunoprecipitation of BRCA1 and PP1{alpha} supports an association between the two proteins. To identify the domain in BRCA1 responsible for the interaction with PP1{alpha}, we performed a GST pull-down assay using GST-BFs 1–6 and COS-7 whole cell lysate. As shown in Fig. 4BCitation , only the GST-BF4 brought down endogenous PP1{alpha} catalytic subunit from COS-7 cell lysate. Coomassie Blue staining of GST-BFs on the blot indicates that approximately equal amounts of GST-fusion proteins were used (Fig. 4BCitation , bottom panel).



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  		Fig. 4. Interaction between PP1{alpha} and BRCA1. A, PP1{alpha} coimmunoprecipitates reciprocally with BRCA1. B, PP1{alpha} interacts with BF4, as determined by the GST pull-down assay using COS-7 whole cell lysate (WCL). Coomassie Blue staining of GST-BFs 1–6 is shown in the bottom panel. C, BF4 binds directly to the catalytic subunit of PP1{alpha}. The GST pull-down assay was performed using recombinant PP1{alpha} catalytic subunit. Western analysis was carried out using PP1{alpha} antibody. Coomassie Blue staining of the blot is shown in the bottom panel. D, PP1{alpha} coimmunoprecipitates with BRCA1 regardless of DNA damage. Control (C) and IR-treated COS-7 cells were harvested for immunoprecipitation (IP) 3 h after treatment with 5 Gy of {gamma}-radiation. E, I-2 inhibits PP1{alpha} activity and allows GST-BF4 phosphorylated by GST-hCds1. The hCds1 kinase assay was performed using GST-BF4 and GST-hCds1 (1 µg of each in each 25 µl-reaction). 0.25 µl of recombinant PP1{alpha} (2.5 units/µl) and 0.5 µl of I-2 (1 mg/ml) were added as indicated. GST-BF4-P was detected by Western analysis using an S988-P antibody.

 
GST-BF4 may interact with PP1{alpha} catalytic subunit directly, or it may interact with another subunit in the PP1{alpha} holoenzyme and bring down PP1{alpha} catalytic subunit indirectly. To answer this question, GST-BF4 was incubated with recombinant PP1{alpha} catalytic subunit. As shown in Fig. 4CCitation , GST-BF4 but not GST-BFs 5 or 6 interacted directly with recombinant PP1{alpha} catalytic subunit. Because unphosphorylated GST-BF4 is capable of interacting with PP1{alpha} in vitro, we next tested whether hyperphosphorylated BRCA1 also associated with PP1{alpha} by coimmunoprecipitation. BRCA1 is hyperphosphorylated after ionizing irradiation indicated by slower gel mobility. PP1{alpha} antibody consistently immunoprecipitated BRCA1 in both control and irradiated COS-7 cell lysates, although BRCA1 levels varied slightly in different experiments (Fig. 4D)Citation . These results indicate that PP1{alpha} may associate with BRCA1 regardless of its phosphorylation state.

Data shown above suggest that PP1{alpha} may associate with BRCA1 all of the time, raising one question regarding how BRCA1 becomes phosphorylated. PP1{alpha} may be regulated by an inhibitor in the BRCA1-PP1{alpha} complex and activated only when BRCA1 needs to be dephosphorylated. To test this hypothesis, recombinant PP1{alpha} catalytic subunit and I-2 were included in a kinase assay with GST-BF4 and GST-hCds1. Phosphorylation of GST-BF4 by GST-hCds1 was detected by an S988-P specific antibody. As illustrated in Fig. 4ECitation , S988-P antibody recognized GST-BF4 phosphorylated by GST-hCds1 (Lane 2) much better than unphosphorylated GST-BF4 (Lane 1). In the presence of recombinant PP1{alpha}, the phosphorylation of GST-BF4 by GST-hCds1 was removed (Lane 3). However, in the presence of recombinant PP1{alpha} and I-2 (Lane 4), PP1{alpha} was unable to dephosphorylate GST-BF4-P. These in vitro data support that PP1{alpha} activity can be inhibited by an inhibitor while associated with BRCA1, allowing BRCA1 to be phosphorylated, for example, after DNA damage.

BRCA1 Regulates PP1{alpha} Activity through the BF4 Region.
It has been reported that the tumor suppressor pRB interacts with PP1{alpha} catalytic subunit. pRB is a substrate as well as a regulator of PP1{alpha}. By binding to PP1{alpha}, pRB inhibits PP1{alpha} catalytic activity (24, 25, 26) . To determine whether BRCA1 regulated PP1{alpha} activity, BRCA1 was generated by in vitro transcription/translation and incubated with recombinant PP1{alpha}. PP activity was then measured using phosphorylase a as a substrate. Phosphatase activity of PP1{alpha} was inhibited by 30% in the presence of full-length BRCA1 or BRCA1CT, which lacks the COOH-terminal 11 amino acid residues, but still contains PP1{alpha} binding region, compared with reticulocyte lysate control (Fig. 5A)Citation . Many proteins are present in reticulocyte lysate that may interfere with the PP assay and attenuate the inhibitory effect of BRCA1. We then used purified GST-BF4 to repeat the experiment. As shown in Fig. 5BCitation , GST-BF4 clearly inhibited PP1{alpha} activity in a dose-dependent manner (up to 80% inhibition at 500 nM of GST-BF4), whereas GST-BF5, which does not interact with PP1{alpha}, did not have this inhibitory effect. GST-BF4 inhibited phosphorylase phosphatase activity of PP1{alpha} with IC50 of ~150 nM (3 nM recombinant PP1{alpha}). Therefore, BRCA1 is a regulator as well as a substrate of PP1{alpha}.



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  		Fig. 5. BF4 of BRCA1 inhibits PP1{alpha} activity. PP assay was performed using 0.05 units of recombinant PP1{alpha} and 30 µg of 32P-labeled phosphorylase a in the presence of 3 µl of in vitro transcription/translation reaction (A) or 0–500 nM of GST proteins (B) in the assay buffer supplemented with 1 mM MnCl2. RL is a rabbit reticulocyte lysate control, BRCA1 is a full-length protein, and BRCA1CT is a truncated BRCA1 lacking the COOH-terminal 11 amino acid residues. The assays were performed in triplicate.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
We have demonstrated that hCds1 phosphorylates BRCA1 mainly on BF4 (amino acids 759-1064), confirming the report by Lee et al. (21) . In addition, we have used hCds1-phosphorylated BF4 (GST-BF4-P) to show that PP1{alpha} is involved in dephosphorylation of GST-BF4-P. We used different sources of PP1{alpha}, including recombinant catalytic subunit COS-7 cell lysate, and a PP1{alpha} immunoprecipitate to demonstrate that PP1{alpha} specifically dephosphorylates GST-BF4-P. This activity was reduced by I-2, a specific inhibitor of PP1. Furthermore, PP2A did not exhibit the same activity toward GST-BF4-P. Overexpression of PP1{alpha}GFP partially inhibits hyperphosphorylation of BRCA1 induced by IR, suggesting that indeed BRCA1 is a substrate of PP1{alpha} in vivo. It is not yet known whether other subtypes of PP1 can dephosphorylate GST-BF4-P or whether PP1{alpha} is involved in dephosphorylation of BRCA1 phosphorylated by kinases other than hCds1.

One intriguing question is whether the association between BRCA1 and PP1{alpha} is initiated after DNA damage. A GST pull-down assay demonstrated that unphosphorylated GST-BF4 interacted with PP1{alpha}. Coimmunoprecipitation experiments also indicated that endogenous BRCA1 associated with PP1{alpha} either with or without DNA damage induced by ionizing radiation. These results suggest that BRCA1 and PP1{alpha} may associate stably, independent of DNA damage. It is possible that PP1{alpha} is constantly active and BRCA1 phosphorylation is primarily controlled by kinase activity. Alternatively, because the catalytic subunit of PP1 is controlled by numerous regulatory subunits, it is more likely that PP1{alpha} activity is inhibited in this complex by a separate inhibitor. Activation of PP1{alpha} to dephosphorylate BRCA1 may only occur after DNA repair is completed. Our in vitro data support that indeed I-2 can inhibit PP1{alpha} and allowing BRCA1 to be phosphorylated by hCds1. Further study is needed to identify signal transduction pathways regulating PP1{alpha} activity on dephosphorylation of BRCA1 and to delineate its correlation with cell cycle progression.

Interestingly, the tumor suppressor pRB also interacts with PP1{alpha} catalytic subunit. This interaction is detected in G2-M-enriched cells. Hypophosphorylated pRB has been shown to coimmunoprecipitate with PP1{alpha} catalytic subunit, and pRB is also a substrate for dephosphorylation by PP1{alpha} (24 , 25) . By binding to PP1{alpha}, pRB also serves as a PP1{alpha} regulator and inhibits PP1{alpha} catalytic activity (26) . A similar scenario may also apply to BRCA1. Indeed, the BF4 region of BRCA1 showed profound inhibition of PP1{alpha} activity.

Many kinases have been shown to phosphorylate BRCA1. We have demonstrated for the first time that the PP, PP1{alpha} associates with and dephosphorylates BRCA1. Furthermore, by binding to PP1{alpha}, BRCA1 inhibits PP1{alpha} activity, probably serves as a feedback regulation of BRCA1 phosphorylation state or modulates PP1{alpha} activity toward other substrates. Our work lays the foundation for additional investigation regarding how the phosphorylation state of BRCA1 is involved in DNA damage repair and cell cycle progression.


   ACKNOWLEDGMENTS
 
We thank Drs. Junjie Chen and David Livingston for providing the GST-BRCA1 constructs, Dr. Roberta Melis for the phage cDNA library, Dr. Hsien-Bin Huang for the PP1{alpha} cDNA, and Dr. Ruey-Min Lee for helpful comments and suggestions. We also thank Dr. Jennifer Logan for editing this manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the United States Army Breast Cancer Research Program Grant DAMD17-99-1-9416 (to L. C. H.) and funding from the Huntsman Cancer Institute (to D. M. V., R. L. W.). Back

2 To whom requests for reprints should be addressed, at Magee-Women’s Research Institute, 204 Craft Avenue, Room 320, Pittsburgh, PA 15213. Phone: (412) 641-6200; Fax: (412) 641-5373; E-mail: lhsu{at}genetics.utah.edu Back

3 The abbreviations used are: Ser, serine; ATM, ataxia telangiectasia-mutated; ATR, ATM and Rad3-related; PP, protein phosphatase; I-2, inhibitor 2; BF, BRCA1 fragment; GST, glutathione S-transferase; S988-P, phosphorylated Ser 988; IR, ionizing radiation. Back

4 L-C. Hsu and R. L. White, unpublished data. Back

Received 5/21/02. Accepted 10/ 3/02.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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