A limited screen for protein interactions reveals new roles for protein phosphatase 1 in cell cycle control and apoptosis.
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.
Flores-Delgado
Guillermo
G
Division Of Hematology/Oncology, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, 4650 Sunset Boulevard, Los Angeles, California 90027, USA.
Liu
Cathy W Y
CW
Sposto
Richard
R
Berndt
Norbert
N
D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D044842:Cullin Proteins / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000378:metabolism* / Q000502:physiology*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D054645:Protein Phosphatase 1; D040901:Proteomics / Q000379:methods; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D044786:S-Phase Kinase-Associated Proteins; D051028:bcl-2-Associated X Protein; D050760:Cyclin-Dependent Kinase Inhibitor p27; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1