Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

Abstract

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity.

Global assessment of its network dynamics reveals that the kinase Plk1 inhibits the phosphatase PP6 to promote Aurora A activity

“…HeLa cells were infected with either control or PP6c shRNA baculoviruses and synchronized in mitosis using Taxol. As previously shown (55, 56), Aurora A Thr 288 phosphorylation significantly increased upon reduction of PP6c protein abundance (Figure 6A). Conversely, Aurora A Thr 288 phosphorylation significantly decreased upon treatment with Plk1 inhibitor BI2536 (Figure 6A, column 3) and was abolished in the absence of PP6c, which suggests that Plk1’s effect on Aurora A Thr 288 phosphorylation is mediated by PP6c.…”

“…Although early reports demonstrated protein phosphatase 1 (PP1c) as capable of dephosphorylating the T-loop of Aurora A in vitro (53, 54), more recent studies support a PP6 holoenzyme as the primary T-loop phosphatase for Aurora A in cells (55, 56). As introduced above, Aurora A is the activating kinase for Plk1 (13, 14).…”

“…Next, we tested our hypothesis in mitotically arrested HeLa cells transduced with pooled short hairpin RNAs (shRNAs)-containing baculoviruses previously shown to efficiently reduce PP6c protein abundance and increase Aurora A Thr 288 phosphorylation site occupancy (56). HeLa cells were infected with either control or PP6c shRNA baculoviruses and synchronized in mitosis using Taxol.…”

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C

“…The constructs allowing expression of human PP1C ( PPP1CA ), PP5C ( PPP5C ) and PP6C ( PPP6C ) were created using standard cloning methods as previously described [22,24,26,27]. Expression and purification of PP5C were performed essentially as described previously [22].…”

Condensin in Chromatid Cohesion and Segregation

“…In contrast, a positive regulatory mode of condensin I is induced by mitosis-specific Cdc2-mediated phosphorylation [Kimura et al, 1998;Takemoto et al, 2004]. Studies on the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics revealed that dephosphorylation of CAP-G by protein phosphatase 6 is opposed to CK2-dependent phosphorylation and required for the proper chromosome condensation and segregation in anaphase [Rusin et al, 2015]. Recently, based on chromatin immunoprecipitation sequencing experiments, Sutani et al [2015] reported that fission yeast condensin is localized to centromeres, ribosomal DNA (rDNA), and unwound DNA on the chromosome arms in a transcription-dependent manner in mitosis.…”