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

Polo-like kinase 1 (Plk1) is an essential protein kinase that promotes faithful mitotic progression in eukaryotes. The subcellular localization and substrate interactions of Plk1 are tightly controlled and require its binding to phosphorylated residues. To identify phosphorylation-dependent interactions within the Plk1 network in human mitotic cells, we performed quantitative proteomics on HeLa cells cultured with kinase inhibitors or expressing a Plk1 mutant that was deficient in phosphorylation-dependent substrate binding. We found that many interactions were abolished upon kinase inhibition; however, a subset was protected from phosphatase opposition or was unopposed, resulting in persistent interaction of the substrate with Plk1. This subset includes phosphoprotein phosphatase 6 (PP6), whose activity toward Aurora kinase A (Aurora A) was inhibited by Plk1. Our data suggest that this Plk1-PP6 interaction generates a feedback loop that coordinates and reinforces the activities of Plk1 and Aurora A during mitotic entry and is terminated by the degradation of Plk1 during mitotic exit. Thus, we have identified a mechanism for the previously puzzling observation of the Plk1-dependent regulation of Aurora A.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase 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.

LRRK2 Facilitates tau Phosphorylation through Strong Interaction with tau and cdk5.

Leucine-rich repeat kinase 2 (LRRK2) and tau have been identified as risk factors of Parkinson's disease (PD). As LRRK2 is a kinase and tau is hyperphosphorylated in some LRRK2 mutation carriers of PD patients, the obvious hypothesis is that tau could be a substrate of LRRK2. Previous reports that LRRK2 phosphorylates free tau or tubulin-associated tau provide direct support for this proposition. By comparing LRRK2 with cdk5, we show that wild-type LRRK2 and the G2019S mutant phosphorylate free recombinant full-length tau protein with specific activity 480- and 250-fold lower than cdk5, respectively. More strikingly tau binds to wt LRRK2 or the G2019S mutant 140- or 200-fold more strongly than cdk5. The extremely low activity of LRRK2 but strong binding affinity with tau suggests that LRRK2 may facilitate tau phosphorylation as a scaffold protein rather than as a major tau kinase. This hypothesis is further supported by the observation that (i) cdk5 or tau coimmunoprecipitates with endogenous LRRK2 in SH-SY5Y cells, in mouse brain tissue, and in human PBMCs; (ii) knocking down endogenous LRRK2 by its siRNA in SH-SY5Y cells reduces tau phosphorylation at Ser396 and Ser404; (iii) inhibiting LRRK2 kinase activity by its inhibitors has no effect on tau phosphorylation at these two sites; and (iv) overexpressing wt LRRK2, the G2019S mutant, or the D1994A kinase-dead mutant in SH-SY5Y cells has no effect on tau phosphorylation. Our results suggest that LRRK2 facilitates tau phosphorylation indirectly by recruiting tau or cdk5 rather than by directly phosphorylating tau.