Article
- The EMBO Journal (2007) 26, 2262 - 2273
- doi:10.1038/sj.emboj.7601683
Published online: 19 April 2007
Subject Category:
Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate
Drew M Lowery1,a, Karl R Clauser2,a, Majbrit Hjerrild1,3,a, Dan Lim1, Jes Alexander1, Kazuhiro Kishi1, Shao-En Ong2, Steen Gammeltoft3, Steven A Carr2 and Michael B Yaffe1,2
- Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Department of Clinical Biochemistry, Glostrup Hospital, Glostrup, Denmark
Correspondence to:
Steven A Carr, Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge MA 02142, USA. E-mail: scarr@broad.mit.edu
Michael B Yaffe, MB Yaffe, Departments of Biology and Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Building E18-580, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Tel.: +1 617 452 2103; Fax: +1 617 452 4978; E-mail: myaffe@mit.edu
aThese authors contributed equally to this work
Received 27 August 2006; Accepted 14 March 2007
Abstract
Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine–threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled–coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.
Keywords:
- phosphopeptide-binding domain,
- Polo-box domain,
- Polo-like kinase,
- proteomics,
- signal transduction
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