Protein kinase CK2 phosphorylation of SAPS3 subunit increases PP6 phosphatase activity with Aurora A kinase

Abstract

Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.

PPP6C negatively regulates oncogenic ERK signaling through dephosphorylation of MEK

“…We found that MEK1 co-immunoprecipitated with each of the PP6 regulatory subunits (Figure 5D), with Article ll OPEN ACCESS significantly less associating with FLAG-tagged PPP6C. These results suggest that PP6 regulatory subunits serve to recruit MEK to the PP6 complex for dephosphorylation by PPP6C, consistent with a general role for non-catalytic subunits of PP6 and other PP2A family phosphatases in substrate binding (Brautigan and Shenolikar, 2018;Heo et al, 2020;Hosing et al, 2012;Stefansson and Brautigan, 2006;Zhong et al, 2011). In keeping with our observation that MEK binds to each of the regulatory subunits, we found that combined siRNA silencing of PPP6R1, PPP6R2, and PPP6R3, but not knockdown of individual subunits, significantly elevated MEK phosphorylation in 501mel cells (Figure S5D).…”

“…While we did not observe changes in levels of PPP6C upon inhibition of BRAF-MEK-ERK signaling, we cannot rule out transcriptional control of PP6 regulatory or scaffolding subunits as a mechanism of feedback regulation. Furthermore, because phosphorylation of PP6 regulatory subunits can mediate recruitment to substrates and other interaction partners (Heo et al, 2020;Kettenbach et al, 2018), targeting of MEK may be impacted by ERK-dependent phosphorylation or other modifications.…”

“…The capacity for specific complexes to restrain cell proliferation is key to the activity of recently developed PP2A small-molecule activators, which stabilize specific holoenzymes (Leonard et al, 2020;Morita et al, 2020). As with PP2A, at least some PP6 substrates are recruited through interaction with individual regulatory subunits (Heo et al, 2020;Hosing et al, 2012;Stefansson and Brautigan, 2006;Zhong et al, 2011), suggesting the potential for developing PP6 activators with therapeutic benefit.…”

PPP6C negatively regulates oncogenic ERK signaling through dephosphorylation of MEK

“…Significantly less MEK1 associated with FLAG-tagged PPP6C. These results suggest that PP6 regulatory subunits serve to recruit MEK to the PP6 complex for dephosphorylation by PPP6C, consistent with a general role for non-catalytic subunits of PP6 and other PP2A family phosphatases in substrate binding (Brautigan and Shenolikar, 2018; Heo et al, 2020; Hosing et al, 2012; Stefansson and Brautigan, 2006; Zhong et al, 2011).…”

A conserved acetylation switch enables pharmacological control of tubby-like protein stability

“…1E), a ubiquitin ligase involved in DNA double strand break repair (34), and PP6R3/SAPS3 ( Fig. 1F), a regulatory subunit of protein phosphatase 6 (35).…”