A Limited Screen for Protein Interactions Reveals New Roles for

A Limited Screen for Protein Interactions Reveals New Roles for Protein Phosphatase 1 in Cell Cycle Control and Apoptosis Guillermo Flores-Delgado, Cathy W. Y. Liu, Richard Sposto, and Norbert Berndt* Division Of Hematology/Oncology, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, 4650 Sunset Boulevard, Los Angeles, California 90027 Received September 25, 2006

Protein phosphatase 1 (PP1) catalytic subunits typically combine with other proteins that modulate their activity, direct them to distinct substrates, or serve as substrates for PP1. More than 50 PP1interacting proteins (PIPs) have been identified so far. Given there are approximately 10 000 phosphoproteins in mammals, many PIPs remain to be discovered. We have used arrays containing 100 carefully selected antibodies to identify novel PIPs that are important in cell proliferation and cell survival in murine fetal lung epithelial cells and human A549 lung cancer cells. The antibody arrays identified 31 potential novel PIPs and 11 of 17 well-known PIPs included as controls, suggesting a sensitivity of at least 65%. A majority of the interactions between PP1 and putative PIPs were isoform- or cell typespecific. We confirmed by co-immunoprecipitation that 9 of these proteins associate with PP1: APAF1, Bax, E-cadherin, HSP-70, Id2, p19Skp1, p53, PCNA, and PTEN. We examined two of these interactions in greater detail in A549 cells. Exposure to nicotine enhanced association of PP1 with Bax (and Bad), but also induced inhibitory phosphorylation of PP1. In addition to p19Skp1, PP1R antibodies also coprecipitated cullin 1, suggesting that PP1R is associated with the SCF1 complex. This interaction was only detectable during the G1/S transition and S phase. Forced loss of PP1 function decreased the levels of p27Kip1, a well-known SCF1 substrate, suggesting that PP1 may rescue proteins from ubiquitin/ proteasome-mediated destruction. Both of these novel interactions are consistent with PP1 facilitating cell cycle arrest and/or apoptosis. Keywords: Protein phosphatase 1 • SCF complex • Bad • Bax • p27Kip1 • cell cycle • apoptosis • antibody array • siRNA • co-immunoprecipitation

Introduction Protein phosphatase 1 (PP1) is a major mammalian phosphatase encoded by three genes, giving rise to four isoforms. PP1R, PP1β (also designated PP1δ), and PP1γ1 are ubiquitously expressed, while another, PP1γ2, is a testes-specific splice variant. The known primary structures of a given isoform are identical across mammalian species, and the isoforms differ significantly only in their extreme C-terminal regions. The human genome encodes approximately 30 000 proteins, onethird of which is believed to be regulated by reversible protein phosphorylation.1 As there are approximately 25 genes encoding serine/threonine-specific protein phosphatases,2 each phosphatase may have more than 300 physiological substrate proteins, which may increase if we consider individual phosphorylation sites as substrates. Thus, it is not surprising that, over the last 10 years, PP1 has been implicated in many cellular processes, including, but not limited to, cell cycle control, apoptosis, transcription, adhesion, motility, metabolism, and memory. However, very few substrates for PP1 have been established so far. Besides proteins engaged in glycogen * Corresponding author. Current address: Moffitt Cancer Center & Research Institute, SRB-2, 12902 Magnolia Drive, Tampa, FL 33612. Phone: (813) 745-3675. E-mail: [email protected]. 10.1021/pr060504h CCC: $37.00

 2007 American Chemical Society

metabolism, muscle contraction, and protein synthesis that were recognized as PP1 substrates early on, more recently, PP1 was found to dephosphorylate a number of proteins important for cell proliferation and survival, for example, the tumor suppressor pRb (reviewed in ref 3), lamin B,4 Bad,5 Aurora,6,7 focal adhesion kinase,8 Nek2,9,10 Cdc25,11 and Chk1.12 To perform dephosphorylation reactions that are meaningful in time and space, the diverse functions of PP1 must be independently regulated (see ref 13 for a review). Post-translationally, mammalian PP1 can be regulated in several ways. First, PP1 binds to other proteins that modulate PP1 activity and/or target PP1 to distinct substrates. These heterodimers may be part of larger multimeric protein complexes (reviewed in refs 13-17). Second, many regulatory subunits of PP1 are regulated by phosphorylation. Third, PP1 catalytic subunits undergo inhibitory phosphorylation by cyclin-dependent kinases (Cdks),18-21 Nek2,9 and KPI-222 (reviewed in ref 3). A variety of approaches has identified more than 50 PP1interacting proteins (PIPs). These proteins function as modulators, targeting subunits, substrates, or a combination thereof (reviewed in refs 13 and 17). Recalling the numbers of phosphatases and phosphoprotein substrates encoded by the human genome, a large number of PIPs remains unknown. Journal of Proteome Research 2007, 6, 1165-1175

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research articles In this study, we sought to identify novel molecular partners of PP1 that could possibly provide new insights into the functions of PP1 in cell proliferation, differentiation, and survival. One goal of this study was to evaluate antibody arrays for protein-protein interaction screening. After we confirmed several putative PIPs identified with the array by co-immunoprecipitation, we proceeded to examine two selected interactions in more detail. Our results demonstrate that this technique is capable of setting the stage for new lines of inquiry providing insights into undiscovered roles of PP1 in cellular regulation.

Experimental Procedures Small Interfering RNAs (siRNA). RNA duplexes were synthesized using human PP1 cDNA sequences as templates. Since cDNAs encoding PP1 isozymes are very similar to one another, to maximize isoform specificity, we sought to identify sequences that were sufficiently mismatched to each of the other isoforms. For PP1R, we selected the sequence 445-AACCGCATCTATGGTTTCTAC (accession no. BC008010), which showed 6 and 4 mismatches to the corresponding sequence for PP1β and PP1γ1, respectively. For PP1β, we selected the sequence 543-AACTATGATGAATGCAAACGA (accession no. AF092905), which showed 6 and 5 mismatches to the corresponding sequence for PP1R and PP1γ1, respectively. The leading two nucleotides for the PP1β sequence are not derived from PP1, but were “added” in the siRNA duplex. For PP1γ1, we selected the sequence 827-AAATGACAGAGGAGTGTCCTT (accession no. BC014073), which showed 7 and 5 mismatches to the corresponding sequence for PP1R and PP1β, respectively. The efficiency and specificity of the siRNAs are depicted in Figure 1 (Supporting Information). Antibodies. Polyclonal antibodies to the three mammalian PP1 isoforms were generated following a similar strategy. We used the following PP1-derived sequences as immunogenic peptides: 299-ADKNKGKYGQFSGLNPG-315 for PP1R, 298SEKKAKYGYGGLNG-312 for PP1β, and 299-AEKKKP NATRPVTP-312 for PP1γ1. Immunizations and bleeds for the three antibodies were performed at Genemed Synthesis, South San Francisco, CA. Polyclonal antibodies to mammalian PP2A have been generated and characterized as described previously.23 Antibodies were purchased from Cell Signaling (Bad, phosphoBad-S112, Bax, and p27Kip1), Santa Cruz Biotechnology (APAF1, β-actin, cullin 1, HSP-70, p53, PCNA, and ubiquitin), and BD Bioscience (E-cadherin, Id2, p19Skp1, and PTEN). Antibody Arrays. This technique has first been described by Wang et al.24 Arrays containing 100 antibodies (0.5 µg each) immobilized on PVDF membranes were custom-ordered from HyproMatrix, Inc., Worcester, MA. Eighty-one of these antibodies were supplied by HyproMatrix, 15 antibodies were purchased from Santa Cruz Biotechnology, and 4 antibodies to PP1R, PP1β, PP1γ1, and PP2A were generated in this laboratory (see above and Table 1, Supporting Information). Cell Lines. Fetal lung epithelial cells (FLECs) were isolated from murine embryonic lungs at E16 as previously described,25,26 seeded at 1 × 106 cells in a 100-mm plate, and incubated at 37 °C in a 5% CO2 atmosphere. A549 human nonsmall cell lung cancer cells (ATCC) were seeded at 2.5 × 105 cells in a 100-mm plate and incubated at 37 °C in a 5% CO2 atmosphere. Cells were synchronized using standard protocols, as described earlier.21,27,28 Cell Lysates. Before lysis, preconfluent cells were washed four times with 50 mM Tris/HCl, pH 7.5, and 150 mM NaCl. 1166

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Whole cell extracts were prepared using two alternative conditions that either preserve or destroy native protein-protein interactions. To maintain protein complexes, ice-cold lysis buffer A (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 0.1 mM DTT, 1 mM Na3VO4, 0.1 µM okadaic acid, 1 mM PMSF, 1 µM pepstatin, 1 µM E64, 0.3 mg/ mL benzamidine chloride, and 2.5% glycerol) was added to washed cell monolayers. To isolate singular proteins, 0.2 mL of 0.6% SDS in phosphate-buffered saline was used instead. Before proceeding further, the SDS was diluted at least 5 times with lysis buffer A. After 5 min on ice, cellular debris was removed by centrifugation at 14 000 rpm for 15 min at 4 °C. Protein concentrations in the supernatants were determined according to Bradford.29 The supernatants were used immediately for incubation with antibody arrays. Detection of Proteins That Directly or Indirectly Interact with PP1. All steps described below were performed at room temperature. The arrays were incubated with a blocking solution containing 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, and 5% BSA for 2 h to block unspecific binding sites. Cellular lysates containing 3 mg of protein in 3 mL were then added for 2 h. The membranes were washed three times with 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20 and incubated with biotinylated antibodies to PP1R, PP1β, or PP1γ1 at a concentration of 1 µg/mL for 2 h. The membranes were washed again three times, and horseradish peroxidase (HRP) conjugated to streptavidin was added for 1 h. Following three additional washes, the membranes were incubated with ECL substrate, and positive signals were visualized by immediate exposure to HyperFilm ECL (Amersham) for 1, 2, and 5 min. Positive signals were taken to indicate the presence of PP1, suggesting that PP1 was complexed to the protein captured by the respective immobilized antibody. The membranes were then incubated with HRP-labeled goat anti-rabbit IgG, goat anti-mouse IgG, and rabbit anti-goat IgG with the aim to count and locate the immobilized primary antibodies on the membrane. After a second exposure to film, proteins complexed to PP1 were identified by careful alignment of the experimental film with the control film showing all 100 antibodies. Immunoprecipitations (IP) and Western Blotting. IP and Western blotting were performed using standard procedures.30 For IP, cell lysates containing 0.8 mg of protein were precleared with protein A/G Sepharose beads, and then incubated for 4 h or overnight at 4 °C with 30 µL protein A/G Sepharose beads in the presence of 2 µg of the primary antibody or nonspecific IgG of the appropriate species (as a negative control). The immunoprecipitates were washed six times in the presence of freshly added protease inhibitors (see above), and half of them (0.4 mg of protein) were subjected to Western blots, which were developed using the ECL system (Amersham). PP1 Overlay Assay. To detect proteins that directly interact with PP1, we performed PP1 overlay assays according to Van Eynde and Bollen.31

Results Selection of Proteins Used To Screen for PIPs. To probe for novel molecular interactions of PP1, we selected antibodies to 100 proteins with known functions in cell cycle control, apoptosis, tumor suppression and oncogenic transformation, cell adhesion or motility, differentiation, transcription, and translation. Tables 1 and 2 summarize essential features of the antibodies, their arrangement on the array, and the functions of their cognate antigens (Supporting Information). To assess

New Roles for PP1 in Cell Cycle Control and Apoptosis

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Table 1. Properties of the Known PP1-Interacting Proteinsa

a The table was assembled after review of the primary literature. Except for AKAP149, GADD34, and p19ARF, all of these proteins are regulated by phosphorylation in Ser and/or Thr. Proteins highlighted in green have been reported to form stable interactions with PP1. Abbreviations: a targeting subunit, b regulator, c kinase phosphorylating PP1, d substrate, e rabbit reticulocyte lysate.

the reliability of this approach, we included 17 antibodies to proteins known to interact with PP1. Fourteen of these have been shown to form stable complexes with PP1, mostly by coimmunoprecipitation, GST pulldown, and/or yeast two-hybrid screens (Table 1). Characterization of Isoform-Specific Antibodies to PP1. Since the PP1 antibodies used to probe for PP1-protein interactions were most critical in executing and interpreting the antibody array assays, it was important to assess the quality and specificity of these antibodies. PP1R, PP1β, and PP1γ1 were immunoprecipitated from cell extracts under stringent conditions, using the antibodies described in Experimental Procedures. The immunoprecipitates were probed for the presence of each isoform in Western blot analyses. The results suggest that each PP1 antibody (a) only reacted with its cognate isoform and (b) showed very little if any cross-reactivity with other proteins (Figure 1). Specificity and Sensitivity of the Antibody Array. The specificity and sensitivity of the antibody array are conversely related to the false-positive rate R and false-negative rate β, important parameters to interpret the results of this assay. We performed several control experiments to determine whether technical aspects of this approach systemically contribute to R and β (Figure 2, Supporting Information). 1. False-Negative Rate. After completion of an experiment, the membrane was exposed to secondary antibodies (i) to ascertain that the immobilized antibodies are present and (ii) to facilitate identification of discreet spots. A typical result, shown in Figure 2A (Supporting Information) indicates that all 100 antibodies were present on the membrane. 2. False-Positive Rate. To assess the potential for falsepositive signals, we performed several controls. As shown in Figure 2B (Supporting Information), incubation of the membrane with HRP-streptavidin alone did not produce any signals, suggesting that the secondary reagent HRP-streptavidin does

not bind to any of the immobilized antibodies. Another membrane was incubated with purified PP1R and then probed with biotinylated PP1R antibody. Figure 2C (Supporting Information, left panel) shows only one major spot in the correct position (i5), indicating that the other 99 immobilized antibodies did not react with PP1R. This experiment was repeated for PP1β and PP1γ1, with essentially the same outcome (Figure 2C, Supporting Information, center and right panels). Next, we also prepared a cell lysate in the presence of 0.6% SDS to isolate singular proteins. Following dilution of the lysate to 0.05% SDS, it was incubated with three individual membranes, which were then probed with biotinylated antibodies to the distinct PP1 isoforms and HRP-streptavidin. This experiment showed a single spot for each of the three isoforms (positions i5-i7), suggesting that the PP1 antibodies did not react with captured antigens (Figure 2D, Supporting Information). This conclusion is also supported by the Western blots shown in Figure 1, which suggested a high degree of specificity for the PP1 antibodies. This also suggests that the likelihood of PP1 antibodies reacting with non-PP1 proteins that might be associated with the immobilized antigens is negligible. Tentative Identification of Molecular Interactions of PP1 with an Antibody Array. Cell extracts were prepared under mild conditions designed to preserve protein complexes as much as possible with the goal to identify proteins that interact with PP1 either directly or indirectly. Extracts from murine FLECs or human A549 lung cancer cells were separated into three aliquots and individually incubated with arrays, which were then probed with biotinylated antibodies to the three different isoforms of PP1 described in Figure 1. From the 17 positive controls, antibodies to 3 proteins (AKAP149, BRCA1, and CREB) only react with the human, but not the murine protein. In murine FLECs, 10 of the 14 remaining PIPs were recognized, including ATM and eIF-2R, two proteins that so far have not been demonstrated to interact stably with PP1. In addition, this Journal of Proteome Research • Vol. 6, No. 3, 2007 1167

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Table 2. PP1 Interactors Recognized by the Antibody Arraysa

a This table was compiled from the data presented in Figure 2. Proteins are separated into four groups: the first 11 proteins are well-known PP1 interactors (cf. Table 1). Although Bad was not recognized in any of the arrays, we were able to show that PP1R binds to Bad in A549 cells exposed to nicotine (cf. Figure 4A). The next 31 proteins are putative novel PP1 interactors identified by this assay. Proteins phosphorylated in S or T are also potential PP1 substrates. This list was generated by a literature survey and referral to www.phosphosite.org. The presence of one or two known canonical PP1 binding sites, RVxF and FxxKxK, is also indicated. The (+) signs indicate the strength of the signal obtained from exposure of the arrays to film: +, weak signal; ++, moderate signal; +++, strong signal. a Proteins in blue: Stable interactions suggested by the present study. b Proteins in green: Stable interactions reported in the literature. c Proteins in red: Interaction confirmed by co-IP (see Figures 3 and 4B). d Akt and p53 were reported to interact with PP1 while this work was in progress.65,66

assay identified 27 novel potential PIPs. Remarkably, individual PP1 isoforms were apparently interacting with overlapping but distinct subsets of proteins (Figure 2A). Four other proteins (Aurora, Bad, Cdc25, and GADD34) must be considered as putative false-negatives. In A549 cells, different PP1 isoforms apparently interacted with overlapping but distinct subsets of 1168


proteins (Figure 2B). However, PP1R interactions were markedly different in A549 lung cancer cells. First, the PP1R antibody reacted only with 6 known PIPs including Aurora, while 22 novel PIPs were recognized. It is noteworthy that, in A549 cells, some interactions (protein kinase B or Akt, E-cadherin, PTEN, and survivin) were uniquely present, while others (e.g., ATR,


Figure 1. Specificity of PP1 isoform-specific antibodies. PP1 isoforms were immunoprecipitated from murine FLEC extracts with isoform-specific antibodies (conjugated to Sepharose beads) indicated at the top (lanes 1-3). Stringent conditions were used to minimize protein-protein interactions. Cell extracts (lane 4) and recombinant PP1R, purified as described earlier45 (P, lane 5 in the left panel), were included as controls. Samples were separated by 12% SDS-PAGE and subjected to Western blotting with the antibody indicated at the bottom.

Bax, cyclin E, EGFR, GSK-3R, hamartin, histone deacetylase 1, Id2, MAP τ, Nek2, PP2B, and TSG101) were absent. Altogether, 5 of the tentatively identified novel PIPs bound to only one PP1 isozyme, whereas 13 other novel PIPs appeared to be specific for one isozyme and one of the two cell types (Table 2).

research articles In summary, considering the combined results for murine and human cell extracts as well as for the three isoforms, the antibody array approach identified 11 out of 17 known PIPs, and 31 novel PIPs (Table 2). We note that PP1 isoforms appeared to interact with each other, which is not surprising, as several proteins were interacting with more than one isoform. If we include the 3 PP1 isoforms in this consideration, then the array identified 14 out of 20 known PIPs. Fifty-five out of 100 proteins were not recognized under any conditions (Table 3, Supporting Information). Most importantly, 18 of the 31 novel PP1-protein interactions appeared to be dependent on the PP1 isoform and/or the cell type. Co-Immunoprecipitation Experiments Confirm Novel PP1Interacting Proteins. The above experiments revealed a catalog of potentially stable and novel PP1-interacting proteins. In the next step, we sought to confirm the most interesting PIPs by an independent method. We have reported earlier that PP1R is intricately involved in the regulation of pRb.21,27,28 When antibody arrays are used, the only interaction detected between pRb and PP1 in murine FLECs involves the γ1 isoform (cf. Figure 2). We have therefore precipitated pRb from FLECs synchronized in G1 or M phase and probed for PP1 in Western blots that were performed subsequently. Although all three PP1 isoforms were physically associated with pRb during the G1 phase of the cell cycle, the most abundant one was PP1γ1, followed by PP1β, and then PP1R (Figure 3A). Seven previously unknown PIPs, that is, APAF-1, E-cadherin, HSP-70, Id2, p19Skp1, PCNA, and PTEN, could be precipitated with the PP1R antibody, whereas one more, p53, could be precipitated with the PP1γ1 antibody (Figure 3B). Bearing in mind that the interactions detected by the array started with the putative PP1

Figure 2. Identification of PP1-binding proteins by antibody arrays. Membranes containing 100 immobilized antibodies were incubated with 5% BSA for 2 h, 3 mL of cytosolic extracts containing 3 mg of protein for 2 h, and then probed with specific biotinylated antibodies specific for PP1 isoforms and HRP-streptavidin. After addition of ECL substrate, the membranes were exposed to film. PP1 isoforms interact with overlapping but distinct subsets of proteins from (A) murine fetal lung epithelial cells or (B) human A549 lung cancer cells. The black arrows indicate the position of PP1R. The colored circles indicate proteins that were later confirmed by coimmunoprecipitation (see Figure 3). Journal of Proteome Research • Vol. 6, No. 3, 2007 1169

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Figure 3. Confirmation of PP1-binding proteins by co-immunoprecipitation. (A) Association of PP1 with pRb immunoprecipitated from murine FLECs. The dilutions of the PP1 antibodies used for Western blotting were adjusted to yield identical titers. All three PP1 isoforms coprecipitate with pRb antibodies during G1. Cell lysates containing 5 µg of protein were also directly analyzed by Western blotting to estimate how much of the PP1 isoform binds to pRb (lane 3). PP1γ1 is the most abundant in the complex, whereas on the array, only PP1γ1 yields a positive signal in murine FLECs (see Figure 2A). While consistent, these two experiments indicate that the sensitivity of the antibody array is limited. (B) Proteins that associate with immunoprecipitated PP1R. Immune complexes (the cell type is indicated on the left) were subjected to Western blotting with the antibody indicated at the bottom (lane 1). Proteins were also subjected to immunoprecipitation with IgG to account for unspecific binding (lane 2). Cell lysates containing 7.5 µg of protein were also directly analyzed by Western blotting to estimate how much of the protein in question binds to PP1 (lane 3). Eight proteins identified by the array as potential PIPs coprecipitate with PP1R or PP1γ1.

interactor, these experiments are equivalent to demonstrating an association from the second molecular partner, which is considered to be a strong argument for in vivo binding.30,46 1170



The Interaction of PP1 with Bad and Bax Depends on Prior Exposure to Nicotine. Nicotine has been known to suppress apoptosis.47 In human lung cancer cells, this effect relies upon induction of inhibitory phosphorylation of Bad48 and Bax.49 Since Bad is a substrate for PP1,5 the upregulation of Bad phosphorylation could conceivably rest upon compromising PP1 binding to, or PP1 activity toward, Bad as well. Therefore, we asked the question whether nicotine affects complex formation between PP1 and Bad or Bax. Bax was included in this experiment, since it is related to the established PP1 interactor Bad, and the antibody array identified it as a potential binding partner for PP1. Contrary to our expectations, exposure of A549 cells to nicotine appeared to induce binding between PP1R and Bad (Figure 4A) as well as Bax, a protein previously unknown to bind PP1 (Figure 4B). At the same time, nicotine induced phosphorylation of Ser112 in Bad (Figure 4A), as previously noted,48 but also Thr320 in PP1R (Figure 4C). To summarize, nicotine induced complex formation between PP1R and Bad/Bax, but inhibited PP1R activity, which is consistent with, or may even be necessary for, enhanced phosphorylation of Bad/Bax. The Role(s) of PP1 in Cell Cycle-Dependent Proteolysis. The novel PP1 interaction with p19Skp1 is of particular interest to us, as this protein is part of the Skp1-Cullin-Fbox protein (SCF) complex, thus, providing a link to cell cycle-dependent proteolysis.50,51 In accordance with the array data, PP1 was also able to interact with the ubiquitin-conjugating enzyme Cdc34p, which transiently binds to SCF.52 Seeking further evidence for PP1R’s involvement in the SCF complex, we examined whether PP1R coprecipitates other partners of SCF. Figure 5A shows that PP1R also associated with cullin 1, implicating a role for PP1R in the SCF1 complex, which plays a prominent role in recruiting proteins to the proteasome-mediated degradation during G1/ S.50,51 The interaction between PP1R and p19Skp1 was cell cycledependent. The association between p19Skp1 and PP1R was readily detectable at the G1/S transition and during S phase, but barely in G2/M and not at all during G1 (Figure 5B). Both PP1R and p19Skp1 protein levels were constant throughout the cell cycle. Although p19Skp1 bears a putative PP1-binding motif (KVIQW), PP1 overlay experiments indicated that purified PP1R was unable to bind directly to p19Skp1, suggesting that there is another protein in the SCF complex that directly binds to PP1R (Figure 5C). These findings suggest that PP1R may be a substrate for, or participate in regulating, the ubiquitin-proteasome pathway. To address these alternative scenarios, we immunoprecipitated polyubiquitinated proteins from A549 cells in the absence and presence of the proteasome inhibitor MG132 and analyzed the blots with antibodies to ubiquitin and PP1R. The results provide no evidence for PP1R itself being degraded (Figure 6). To gain further insight into the possible role(s) of PP1R in this pathway, we determined whether PP1 silencing affects the levels of p27Kip1, a Cdk inhibitor that is recruited by SCF1 before the G1/S transition to be subsequently destroyed.53-55 Treatment of A549 cells with siPP1R and siPP1γ1 led to a significant decrease in p27Kip1 levels within 24 h (Figure 7A). Next, we examined cell cycle progression of A549 cells after releasing them from a mimosine-induced mid-to-late G1 arrest. First, lack of PP1γ1 appeared to prevent an efficient synchronization in G1 by mimosine, since the cell cycle profile of siPP1γ1treated cells was very similar to that of asynchronous cells (Figure 7B). Analysis of cell cycle progression for up to 36 h

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Figure 4. Bad and Bax interact with PP1 upon stimulation with nicotine. A549 cells were exposed to 1 µM nicotine for the times indicated at the top. Cell lysates were then subjected to immunoprecipitation and/or Western blot analysis. (A) Nicotine induces coimmunoprecipitation of PP1R with Bad and, as previously reported by Jin et al.,48 phosphorylation of Ser112. (B) Nicotine induces co-immunoprecipitation of PP1R with Bax. (C) Nicotine induces inhibitory phosphorylation of PP1R.

after removal of mimosine confirmed this result. While cells treated with control siRNA were exiting G1 in nearly perfect fashion, cells treated with siPP1γ1 behaved very much like asynchronous cells. Furthermore, control cells treated with mimosine transiently accumulated in sub-G1, whereas siPP1γ1 showed very low levels of cells in sub-G1 (Figure 7C). Very similar results were obtained with siPP1R. We reported before that inhibition of PP1 did not prevent araC-induced apoptosis in HL-60 cells,28 and similarly, we did not see this protective effect in HeLa cells here (not shown). When taking into account that (i) mimosine is thought to arrest cells in G1 by accumulating p27Kip1,56 (ii) mimosine induces apoptosis in other experimental models,57,58 and (iii) overexpression of p27Kip1 triggers apoptosis,59,60 our data suggest that, at least in some lung cancer cells, PP1 may be necessary for cell cycle arrest in G1 or perhaps G0 and apoptosis, possibly by rescuing p27Kip1 from degradation.

Discussion During the past few years, interest in deciphering signal transduction networks has grown enormously.61-64 As a major Ser/Thr-specific phosphatase, PP1 has been implicated in a number of important pathways relying on reversible phosphorylation, although, given the ratio of protein kinases to phosphatases in mammalian genomes, many more protein interactions involving PP1 remain undiscovered. Here, we report the use of antibody arrays to identify 31 potential novel molecular partners of PP1. Qualitative and Statistical Evaluation of the Antibody Arrays. As the antibody array experiments were the basis for further experimentation, it is important to evaluate them critically. Several control experiments suggested that, in our hands, the specificity of our custom array was satisfactory and its sensitivity was not compromised due to intrinsic problems, for example, missing immobilized antibodies (see Figure 2, Supporting Information). However, the antibody array experiments revealed a few false-negatives, for example, AKAP149, Bad, BRCA1, and GADD34. These are proteins that have been established as PIPs, but failed to be recognized in the two murine and human cell types investigated here. Several possibilities have to be considered for the failure of detecting PP1 in these complexes. PP1 could fail to interact with a captured antigen because the immobilized cognizant antibody competes with the PP1 binding sites, or because the cell lysis conditions were too harsh to preserve the interaction. These two condi-

tions would lead to false-negative signals. Also, a protein interactor of PP1 may simply be absent from the cell type investigated; for instance, Id2 expression was not detected in murine adult lungs or human lung cancer cells (data not shown). Furthermore, the interaction could be cell type-specific (see Table 2) or it could depend on external signals. These conditions would be true negatives. To explore the latter possibility, we examined the expression levels of a number of proteins in cell extracts that produced a negative signal on the arrays: Akt, E-cadherin, and PTEN bound to PP1 only in A549 cells, but were present in similar amounts in FLECs as well (data not shown). In statistical terms, we must consider the false-negative rate, β, and more importantly, the false-positive rate, R. The probability that a positive signal on the array corresponds to a true interacting protein is the positive predictive value, PPV: PPV )

(1 - β) × θ (1 - β) × θ + R × (1 - θ)

where θ equals the prevalence of PP1 interactors on a given array, or in other words, the chance that a protein selected for study on the array is a PP1 interactor. This formula also highlights that the PPV is much more dependent on variations in specificity than variations in sensitivity. While θ cannot be known, if the PPV is near 1 for a wide range of θ values, then one can be confident a high percentage of the proteins identified by this assay are true PP1-interacting proteins. The results described above provide information about the sensitivity and specificity of the assay, as well as the prevalence of PIPs, and therefore allow us to make statements about the PPV. The following calculations are based on published knowledge at the time these experiments were completed, and take into account murine proteins only. Including PIPs published since then (see next paragraph) and/or human proteins does not significantly change these numbers. The array detected 10 of 14 known PIPs, or 13 of 17, if we include the PP1 isoforms. This provides an estimate of 1 - β ) 0.76 ( 0.10 (estimate ( SEM). The false-positive rate R is obviously more difficult to determine. As stated above, 3 antibodies that should react with well-recognized PIPs (AKAP149, BRCA1, and perhaps CREB), did not produce a signal in the experiment depicted in Figure 2A. As the antibodies in question are specific for human proteins, these 3 proteins represent a control confirming true negatives and, hence, imply a low false-positive rate. In addition, the control experiments depicted in Figure 2 (SupJournal of Proteome Research • Vol. 6, No. 3, 2007 1171

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Figure 5. Characterization of the PP1R-SCF1 interaction in A549 cells. (A) Immunoprecipitated PP1R is associated with cullin 1. (B) PP1R was immunoprecipitated from A549 cells synchronized in various phases of the cell cycle and processed as in Figure 3. Blots with antibodies to p19Skp1 (top) suggested that the interaction is cell cycle-dependent, while both PP1R (center) and p19Skp1 expression levels (bottom) were unaltered during the cell cycle. A loading control for β-actin has also been included. (C) p19Skp1 was immunoprecipitated, divided into three aliquots, separated by SDS-PAGE, and transferred to PVDF membranes. The first lane, which was probed with a p19Skp1 antibody, determined the position of the p19Skp1 protein. The next two lanes were probed with the PP1R antibody without (lane 2) or with (lane 3) prior PP1R overlay. Since this was a negative result, we proceeded to subject a cell lysate to a PP1R overlay analysis (lanes 4 and 5), demonstrating that PP1R is capable of binding to numerous (unknown) proteins.

porting Information) and the Western blots with our PP1 antibodies (see Figure 1) rule out systemic errors to a large extent, again suggesting a small value for R. Co-immunopre1172



cipitation experiments have shown that at least 9 among the remaining 80 candidate proteins were in fact PIPs, indicating that a reasonable lower bound for the prevalence θ is 9/80 ) 11.25%. Using this value for θ and assuming the false-positive rate is near 1%, then we can see from the graph in Figure 3 (Supporting Information) that the PPV for this assay is exceeding 90%. Comments on Other PP1-Protein Interactions: The identification of PIPs and the functional characterization of novel PP1-protein complexes is an area of research that is rapidly moving forward, which is illustrated by the fact that, while this work was in progress, at least four more proteins have been demonstrated to form physiologically relevant complexes with PP1: Akt,65 p53,66 the oncogenic protein tyrosine kinase Fer,67 and caspase-9.68 Of these, only Akt and p53 were included in, and recognized as PIPs by, the array (Figure 2). According to Chen et al., histone deacetylase (HDAC) inhibitors promote PP1-mediated Akt dephosphorylation by dissociating PP1 from HDAC-PP1 complexes.65 Although the PP1 isoform complexing with p53 was not identified by Li et al.,66 we also found that p53 associates with immunoprecipitated PP1γ1 (Figure 3B). According to these investigators, PP1 dephosphorylates specific serines in p53, thus, inhibiting its apoptotic activity and suggesting a pro-survival function for PP1.66 Oncogenic Fer inhibits the pRb phosphatase activity of PP1R by upregulating PP1R phosphorylation in Thr-320.67 Most interestingly, PP1R activates caspase-9 by dephosphorylation.68 Although the antibody array failed to provide a signal for caspase-9, it recognized APAF-1 and HSP-70 as potential PIPs. Both of these proteins were confirmed by co-immunoprecipitation (Figure 3B). Given that HSP-70 negatively regulates APAF-1,69,70 and APAF-1 positively regulates caspase-9,71-73 it would appear important to further dissect the functional roles of PP1 in this complex. Although other interactions identified by the array would deserve a comment, we will restrict these to the Cdks, the cyclins, and Id2. Like the vast majority of all proteins listed in Table 2, these are phosphoproteins and, therefore, potential PP1 substrates. That PP1 is apparently associated with Cdk/ cyclin complexes is hardly surprising if we recall the multiple roles for PP1 in cell cycle control. As Id2 is activated by Cdk2, inhibitory phosphorylation of this protein by PP1 would be consistent with the requirement of PP1R for differentiation we reported earlier.74 Potential Roles for PP1 in Concert with SCF and Bad/Bax. PP1 is required for entry into mitosis,75 and the dephosphorylation of Ser287 in Cdc25, which allows Cdc25 to activate Cdk1, is a critical function of PP1, at least in Xenopus11 In addition, PP1 is required for exit from M phase.76-79 This is likely due to PP1 being necessary for the rebuilding of the nuclear envelope, acting through the dephosphorylation of nuclear lamin B.4,32,33 On the other hand, due to its interaction with pRb, PP1 activity also maintains cells in the G1 phase of the cell cycle,27 can trigger apoptosis,28 and can inhibit oncogenic signalling.80 Another pro-apoptotic function of PP1 rests upon dephosphorylation of Bad.5,35,81 The present study adds two more interesting functions to the PP1 repertoire. First, PP1 appears to be a transient member of the SCF1 complex and protect certain proteins, for example, p27Kip1, from cell cycledependent proteolysis. A recent report by Gallego et al. has shown that PP1 also prevents the ubiquitin/proteasomedependent degradation of a circadian clock protein.82 Interestingly, unlike HeLa cells, A549 lung cancer cells seemed to

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Figure 6. No evidence for PP1R proteolysis. A549 cells, with or without prior exposure to 0.5 µM of the proteasome inhibitor MG132, were subjected to immunoprecipitation with an antibody to ubiquitin (first lane of each panel), followed by Western blot analysis of ubiquitin or PP1R, respectively. As in Figure 2, we included IPs with unspecific IgG (lane 2) or total cell lysates (third lane of each panel) as controls. Left panel: MG132 increases the intensity of high Mr proteins, both in lysates and ubiquitin immune complexes, indicating the presence of polyubiquitinated proteins. Right panel: MG132 does not induce the appearance of high Mr forms of PP1R.

Figure 7. Effects of PP1 knockdown on A549 cell cycle progression and survival in G1. Following transfection of cells with 100 nM PP1 siRNAs, we determined several parameters. (A) Twenty-four hours after transfection with PP1 siRNAs, p27Kip1 levels are significantly decreased. (B) Cells were transfected with siPP1γ1, and 24 h later, they were exposed to mimosine for another 24 h to induce G1 arrest. Cell cycle distribution was then determined by FACS analysis. Downregulation of PP1γ1 via RNAi apparently prevents efficient synchronization. (C) Cell cycle progression was monitored for 36 h after removal of mimosine by FACS analysis. Most strikingly, siPP1γ1 prevented cells from DNA fragmentation and apparently protected cells from apoptosis.

require the presence of at least one PP1 isozyme for efficient synchronization in G1. If this dependence on PP1 in some cancer cells can be confirmed, maintaining this function of PP1 may be a promising therapeutic target. Second, our data suggest that the pro-apoptotic protein Bax is a substrate for PP1. In addition to upregulating the appropriate protein kinases, the ability of nicotine to suppress apoptosis47-49 may depend on inhibiting PP1 activity that is directed toward Bad and/or Bax. The induction of PP1-Bad/Bax complex formation and inhibition of PP1 activity toward these two proteins (see Figure 4A,B) would suggest that PP1 may have a role as a guardian that, upon reactivation, can quickly turn a cell already committed to survival to switch to the apoptotic program. Although further confirmation is needed for these anticipated roles for PP1, they are consistent with previously reported functions of PP1 in cell cycle control and cell death.

Conclusions In this manuscript, we describe and evaluate an antibody array to tentatively identify several PIPs, an approach that might be applicable to other proteins of interest. It is worth recalling that this experimental approach has its limitations. For example, suitable antibodies for a number of proteins of interest may not currently be available. Second, this assay alone cannot distinguish between direct and indirect interactions. As shown in Table 2, only 12 out of 31 novel PIPs bear a canonical PP1binding site. This raises the question whether these PIPs bind PP1 directly, possibly via novel interfaces. Alternatively, a third protein could mediate binding between PP1 and the PIPs tentatively identified here. Finally, interactions with a low stoichiometry may be missed. When applying this technique to PP1, or other proteins, we recommend to include a reasonable number of known interacting proteins to provide a value Journal of Proteome Research • Vol. 6, No. 3, 2007 1173

research articles for the prevalence θ > 0.15. More work, using independent methodology, is definitely needed to establish most of the proteins identified by the array as genuine PP1 interactors and to determine the functional significance of these interactions. However, this approach was able to set the stage for new lines of inquiry, which, as our study demonstrates, led to new insights into the functions of PP1 in cell cycle control and apoptosis, processes that are relevant for carcinogenesis and cancer progression.

Acknowledgment. This work was supported in part by grants from the NIH (R01-CA54167) and the TJ Martell Foundation (to N.B.). Supporting Information Available: Figure 1, silencing of human PP1 catalytic subunits via siRNA; Figure 2, control incubations to evaluate the antibody arrays (Pdf files); Table 1, properties of all antibodies and proteins; Table 2, arrangement of the antibodies on the array; Table 3, list of proteins that did not interact with PP1; Table 4, statistical evaluation (data used to generate Figure 3); Figure 3, statistical evaluation of the antibody array data (Microsoft Excel file). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Johnson, S. A.; Hunter, T. Kinomics: methods for deciphering the kinome. Nat. Methods 2005, 2, 17-25. (2) Cohen, P. T. W. Overview of protein serine/threonine phosphatases. In Protein PhosphatasessTopics in Current Genetics; Arin ˜ o, J., Alexander, D., Eds.; Springer-Verlag: Berlin, Germany, 2004; Vol. 5, pp 1-20. (3) Berndt, N. Roles and regulation of serine/threonine-specific protein phosphatases in the cell cycle. In Progress in Cell Cycle Research; Meijer, L., Je´ze´quel, A., Roberge, M., Eds.; Editions “Life in Progress”: Roscoff, France, 2003; Vol. 5, pp 497-510. (4) Thompson, L. J.; Bollen, M.; Fields, A. P. Identification of protein phosphatase 1 as a mitotic lamin phosphatase. J. Biol. Chem. 1997, 272, 29693-29697. (5) Aylloń, V.; Martıńez-A, C.; Garcıá, A.; Cayla, X.; Rebollo, A. Protein phosphatase 1R is a ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis. EMBO J. 2000, 19, 2237-2246. (6) Hsu, J. Y.; Sun, Z. W.; Li, X. M.; Reuben, M.; Tatchell, K.; Bishop, D. K.; Grushcow, J. M.; Brame, C. J.; Caldwell, J. A.; Hunt, D. F.; Lin, R. L.; Smith, M. M.; Allis, C. D. Mitotic phosphorylation of histone H3 is governed by IpI1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 2000, 102, 279-291. (7) Katayama, H.; Zhou, H.; Li, Q.; Tatsuka, M.; Sen, S. Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle. J. Biol. Chem. 2001, 276, 46219-46224. (8) Fresu, M.; Bianchi, M.; Parsons, J. T.; Villa-Moruzzi, E. Cell-cycledependent association of protein phosphatase 1 and focal adhesion kinase. Biochem. J. 2001, 358, 407-414. (9) Helps, N. R.; Luo, X.; Barker, H. M.; Cohen, P. T. W. NIMA-related kinase 2 (Nek2), a cell cycle-regulated protein kinase localized to centrosomes, is complexed to protein phosphatase 1. Biochem. J. 2000, 349, 509-518. (10) Meraldi, P.; Nigg, E. A. Centrosome cohesion is regulated by a balance of kinase and phosphatase activities. J. Cell Sci. 2001, 114, 3749-3757. (11) Margolis, S. S.; Walsh, S.; Weiser, D. C.; Yoshida, M.; Shenolikar, S.; Kornbluth, S. PP1 control of M phase entry exerted through 14-3.-3-regulated Cdc25 dephosphorylation. EMBO J. 2003, 22, 5734-5745. (12) Den Elzen, N. R.; O’Connell, M. J. Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1. EMBO J. 2004, 23, 908-918. (13) Cohen, P. T. W. Protein phosphatase 1stargeted in many directions. J. Cell Sci. 2002, 115, 241-256. (14) Hubbard, M. J.; Cohen, P. On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem. Sci. 1993, 18, 172-177.

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A Limited Screen for Protein Interactions Reveals New Roles for

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