A Limited Screen for Protein Interactions Reveals New Roles for Protein Phosphatase 1 in Cell Cycle Control and Apoptosis

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.

Identification of genes associated with non-small-cell lung cancer promotion and progression

“…PHACTR3 inhibits protein phosphatase-1 (PP1) activity, which was found to dephosphorilate a number of proteins important for cell proliferation and survival including the tumor suppressor pRb. Due to its interaction with pRb, PP1 can trigger apoptosis and can inhibit oncogenic signaling [38,39]. It is not a surprise then, that in our study patients with altered E2F4 and PHACTR3 lived significantly shorter than those without it (P = 0.03).…”

PTEN: a new guardian of the genome

“…Furthermore, this cooperative interaction between PTEN and p53 was applied to an animal model in which loss of one allele of Pten dramatically accelerated tumor onset in p53 þ /À mice (Freeman et al, 2003). The physical association between PTEN and p53 was confirmed by other studies, which give further evidence of their functional cross talk (Tang and Eng, 2006;Flores-Delgado et al, 2007). Recently, PTEN was found to physically associate with p300 in the nucleus, which maintains p53 in a highly acetylated state in response to DNA damage (Bilbao et al, 2006).…”

Molecules that modulate Apaf‐1 activity

“…regulation of transcriptional responses to hypoxia 99 and some HIF-regulated proteins showed putative Apaf-1 binding properties. 100,101 Apaf-1 has also been described as target for both protein kinases and phosphatases, [102][103][104][105][106] and the phosphorylation state of Apaf-1 could have relevance on apoptosome activity regulation.…”