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Updated: Jul 17, 2026
A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
Published on: April 29, 2022
A limited screen for protein interactions reveals new roles for protein phosphatase 1 in cell cycle control and
Guillermo Flores-Delgado1, Cathy W Y Liu, Richard Sposto
1Division Of Hematology/Oncology, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, 4650 Sunset Boulevard, Los Angeles, California 90027, USA.
Abstract:
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 PP1-interacting 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 type-specific. We confirmed by co-immunoprecipitation that 9 of these proteins associate with PP1: APAF-1, 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, PP1alpha antibodies also coprecipitated cullin 1, suggesting that PP1alpha 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.
Insights
Researchers identified novel protein phosphatase 1 (PP1)-interacting proteins (PIPs) crucial for cell proliferation and survival. These interactions are often specific to cell type and PP1 isoform, impacting cell cycle regulation and apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Protein phosphatase 1 (PP1) catalytic subunits interact with numerous proteins to regulate their function, substrate specificity, and localization.
- Over 50 PP1-interacting proteins (PIPs) are known, but many more likely exist given the vast number of phosphoproteins in mammals.
- Identifying novel PIPs is crucial for understanding PP1's diverse roles in cellular processes.
Purpose of the Study:
- To identify novel PIPs involved in cell proliferation and survival using antibody arrays.
- To investigate the cell type and isoform specificity of PP1 interactions.
- To elucidate the functional significance of novel PP1 interactions in cancer cells.
Main Methods:
- Utilized antibody arrays with 100 selected antibodies to screen for novel PIPs in murine fetal lung epithelial and human A549 lung cancer cells.
- Confirmed novel interactions using co-immunoprecipitation assays.
- Investigated specific PP1 interactions with Bax and the PP1alpha-SCF1 complex in A549 cells.
Main Results:
- Identified 31 potential novel PIPs and 11 known PIPs, indicating an array sensitivity of at least 65%.
- Confirmed interactions of 9 proteins with PP1, including APAF-1, Bax, E-cadherin, HSP-70, Id2, p19Skp1, p53, PCNA, and PTEN.
- Demonstrated cell type- and isoform-specific interactions, with nicotine exposure affecting PP1-Bax association and PP1alpha associating with the SCF1 complex during specific cell cycle phases.
Conclusions:
- Novel PIPs were identified, expanding the known interactome of PP1.
- PP1 interactions are highly specific, contributing to its diverse regulatory functions.
- Novel PP1 interactions with Bax and the SCF1 complex suggest roles in cell cycle arrest and apoptosis, potentially offering therapeutic targets.
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