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

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Science Signaling  13 Oct 2015:
Vol. 8, Issue 398, pp. rs12
DOI: 10.1126/scisignal.aab3138

Exploring mitotic phosphatase function

To study the function of the serine-threonine protein phosphatase PP6, Rusin et al. analyzed the effect of depleting HeLa cells of the catalytic subunit of PP6 on the phosphoproteome when the cells were arrested in mitosis. Motif analysis of the sequences of the sites that changed in the PP6c-depleted cells suggested kinases that PP6 activity may oppose. In addition to the known role of PP6 in reducing the activity of the Aurora kinase A, a kinase that is important for chromosome segregation and spindle formation, network analysis of the proteins that exhibited differences in phosphorylation identified roles for PP6 in the regulation of RNA splicing, rRNA processing, and translation. Biochemical analysis showed that a subunit of the complex necessary for chromosome condensation was phosphorylated by casein kinase 2 and dephosphorylated by PP6. Thus, this study identified a mitotically regulated phosphorylation event in this critical complex and provided many other potential direct substrates of PP6 and pathways regulated by PP6 in mitotic cells.


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.

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