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RESEARCH PROFILES
Although much attention has been lavished on the study of kinases that place phosphate groups onto proteins, less has been paid to the phosphatases that remove these groups, according to Norbert Berndt, who is currently at the Moffitt Cancer Center. While he and his co-workers were at the University of Southern California, they used antibody arrays to discover novel potential substrates of protein phosphatase 1 (PP1), which is involved in many processes, including cell cycle control and apoptosis. In this issue of JPR (pp 1165–1175), they report several new PP1 interactions, some of which reveal striking insights into how lung cancer may develop. Antibodies to 100 proteins involved in cell growth and proliferation were immobilized in an array on a membrane. Three isoforms of PP1 (PP1, PP1, and PP11) are ubiquitously expressed in mammals, so three identical arrays were incubated with each lysate tested. Lysates were prepared under mild conditions to preserve native complexes. To detect interactions, each array was probed with biotinylated antibodies specific for one of the PP1 isoforms, then with horseradish peroxidase conjugated to streptavidin. The researchers tested lysates from two types of cells. “Given that our previous work has shown that PP1 is necessary for proper lung development, it was prudent to search for novel PP1 interactors in the context of development,” says Berndt. So, the researchers analyzed lysates from mouse fetal lung epithelial cells (FLECs) and from human lung cancer cells. Berndt explains the second choice by saying, “In an oversimplified way, you could say that cancer is basically development reversed, because cancer cells dedifferentiate.” With the antibody arrays, 27 novel interactions were detected from FLECs and 22 were detected from lung cancer cells. Although most proteins inter-
acted with PP1 from both cell types, some interactors were unique to one type. Berndt emphasizes that this study is only the beginning, however. The results rely heavily on the quality of each immobilized antibody, so validation experiments are necessary. By performing co-immunoprecipitation experiments, Berndt and co-workers confirmed eight of the interactions. Next, to see whether some of the interactions are biologically relevant, the researchers focused on two signaling pathways: one involved in the NORBERT BERNDT
Antibody arrays shed light on PP1’s possible role in lung cancer
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The ties that bind. Schematic of an antibody array designed to detect proteins that bind to PP1. In the diagram, an antibody to a protein called C binds to the C protein (green circle), which also binds to PP1 (yellow diamond). An antibody to PP1 (red object) is used to detect the interaction.
G1/S cell-cycle transition and the other involved in nicotine exposure. On the arrays, PP1 interacted with Skp1, which is part of the Skp1–cullin–F-box (SCF) complex that targets proteins for cell-cycle-dependent proteolysis. PP1 also interacted with Cdc34, which transiently binds the SCF complex. In co-immunoprecipitation experiments, Berndt and co-workers also discovered that PP1 binds to a second component of the SCF complex, cullin, which was not included in the array experiments. In addition, the researchers conducted RNA silencing (siRNA) experiments to turn down the levels of the PP11 isoform. In siRNA-treated lung cancer cells, the levels of the SCF substrate p27Kip1 decreased significantly. Consistent with this result, lung cancer cells treated with the siRNA continued through the cell cycle when mimosine, which normally arrests cells in G1, was
932 Journal of Proteome Research • Vol. 6, No. 3, 2007
added. “We suggest that PP1 most likely seems to work as a stabilizer to protect proteins, such as p27Kip1, from being destroyed,” explains Berndt. Finally, the researchers also followed up on array results that implicated PP1 in a nicotine exposure pathway. Studies have shown that nicotine causes Bcl-2, Bad, and Bax to become phosphorylated in lung cancer cells, thereby inhibiting cell death. In addition, other scientists have demonstrated that PP1 binds to Bcl-2 and Bad. In the current work, Berndt and co-workers observed that Bax interacts with PP1 from FLECs but not PP1 from lung cancer cells. When they treated lung cancer cells with nicotine, however, Bad and Bax bound to PP1. Although PP1 latched onto these proteins, it did not dephosphorylate them, possibly because nicotine also induced the inhibitory phosphorylation of PP1, says Berndt. “Epidemiological studies suggest that only one in 10 heavy cigarette smokers get lung cancer, though that needs to be verified,” Berndt points out. (For a review, see Cancer Cell 2002, 1, 49–52.) “There must be a reason why the other nine don’t get lung cancer,” he says. PP1 may be a modifying protein, so high PP1 activity against particular substrates may protect against certain types of cancers by placing cancer cells in a holding pattern in one phase of the cell cycle or by causing the cells to undergo apoptosis. In the future, the researchers will continue their work on apoptosis, the cell cycle, and carcinogenesis, including PP1’s role in these processes. Because Berndt doesn’t have time to follow up on all the results, he invites scientists in the field to investigate the other interactions. In addition, he predicts a bright future for antibody arrays as diagnostic tools because “many diseases, including cancer, are characterized by defects in signal transduction pathways, which rely on intricately regulated protein–protein interactions.” —Katie Cottingham
© 2007 American Chemical Society
