The three Type 2A protein phosphatases, PP2Ac, PP4c and PP6c, are differentially regulated by Alpha4
Section snippets
Background
Alpha4 is a highly conserved protein with similarity to Tap42 from Saccharomyces cervisiae [1], [2]. In yeast, Tap42 plays an integral role in the Target of Rapamycin (TOR) pathway that regulates cellular growth and metabolism in response to growth factors and nutrients [2], [3]. Consistent with this role in growth and metabolism, Alpha4 is upregulated in a number of cancers and transformed cell lines correlating with increased rates of cellular migration and proliferation [4], [5].
Plasmids
We used a second generation lentiviral transfection system consisting of three plasmids: a packaging plasmid (psPAX2; gift from Didier Trono, Addgene #12260), an envelope plasmid (pMD2.G; gift from Didier Trono, Addgene #12259), and a transfer plasmid (pLKO.1-TRC; gift from David Root, AddGene #10878) [23]. The scrambled shRNA in pLKO.1 was a gift from David Sabatini (Addgene #1864) [24]. The shRNAs directed to the 3′UTR (NM_001551.x-1110s1c1) and coding regions of Alpha4 (NM_001551.2–752s21c1)
Association of Alpha4 with Type 2A phosphatases
To determine the fraction of each phosphatase catalytic subunit (PP2Ac, PP4c, and PP6c) bound to Alpha4 in HEK293T cell lysates, we conducted successive rounds of immunodepletion using an Alpha4-specific antibody. The resulting supernatants were probed for PP2Ac, PP4c, and PP6c (Fig. 1). Levels of PP6c were significantly reduced in the immunodepleted supernatants as compared to controls, while neither PP2Ac nor PP4c showed any significant reductions in levels following immunodepletion (Fig. 1).
Discussion
Knockout of Alpha4 causes dramatic reductions in expression levels of all three Type 2A phosphatases, leading to the hypothesis that Alpha4 plays a role in stabilizing nascent catalytic subunit and allowing for proper folding [14]. This hypothesis is consistent with observations from recently determined crystal structures showing Alpha4 bound to partially unfolded PP2Ac [17]. If Alpha4 is a protein specific foldase, it would explain the dramatic effects of Alpha4 knockout on the expression of
Acknowledgements
This work was supported by National Institutes of Health Public Health Service grants R01 AI108778 to BWS and R01 DK070787 and R01 GM051366 to BEW. MLN was supported by a predoctoral fellowship F31 AG039947 from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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