The three Type 2A protein phosphatases, PP2Ac, PP4c and PP6c, are differentially regulated by Alpha4

https://doi.org/10.1016/j.bbrc.2016.05.036Get rights and content

Abstract

Alpha4 is a non-canonical regulatory subunit of Type 2A protein phosphatases that interacts directly with the phosphatase catalytic subunits (PP2Ac, PP4c, and PP6c) and is upregulated in a variety of cancers. Alpha4 modulates phosphatase expression levels and activity, but the molecular mechanism of this regulation is unclear, and the extent to which the various Type 2A catalytic subunits associate with Alpha4 is also unknown. To determine the relative fractions of the Type 2A catalytic subunits associated with Alpha4, we conducted Alpha4 immunodepletion experiments in HEK293T cells and found that a significant fraction of total PP6c is associated with Alpha4, whereas a minimal fraction of total PP2Ac is associated with Alpha4. To facilitate studies of phosphatases in the presence of mutant or null Alpha4 alleles, we developed a facile and rapid method to simultaneously knockdown and rescue Alpha4 in tissue culture cells. This approach has the advantage that levels of endogenous Alpha4 are dramatically reduced by shRNA expression thereby simplifying interpretation of mutant phenotypes. We used this system to show that knockdown of Alpha4 preferentially impacts the expression of PP4c and PP6c compared to expression levels of PP2Ac.

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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