A Genetic Toggle for Chemical Control of Individual Plk1 Substrates

James M Johnson; Alexander S Hebert; Quentin H Drane; Robert F Lera; Jun Wan; Beth A Weaver; Joshua J Coon; Mark E Burkard

doi:10.1016/j.chembiol.2020.01.007

A Genetic Toggle for Chemical Control of Individual Plk1 Substrates

Cell Chem Biol. 2020 Mar 19;27(3):350-362.e8. doi: 10.1016/j.chembiol.2020.01.007. Epub 2020 Feb 3.

Authors

James M Johnson¹, Alexander S Hebert², Quentin H Drane¹, Robert F Lera¹, Jun Wan³, Beth A Weaver⁴, Joshua J Coon⁵, Mark E Burkard⁶

Affiliations

¹ Department of Medicine, Division of Hematology/Oncology, University of Wisconsin, 1111 Highland Avenue, WIMR 6059, Madison, WI 53705, USA; University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, WI 53705, USA.
² Genome Center, University of Wisconsin, Madison, WI 53705, USA; Morgridge Institute for Research, Madison, WI 53705, USA.
³ Physiology Training Program, University of Wisconsin, Madison, WI 53705, USA.
⁴ University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, WI 53705, USA; Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI 53705, USA; Department of Oncology/McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53705, USA.
⁵ Genome Center, University of Wisconsin, Madison, WI 53705, USA; Department of Chemistry, University of Wisconsin, Madison, WI 53705, USA; Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI 53705, USA; Morgridge Institute for Research, Madison, WI 53705, USA.
⁶ Department of Medicine, Division of Hematology/Oncology, University of Wisconsin, 1111 Highland Avenue, WIMR 6059, Madison, WI 53705, USA; University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, WI 53705, USA; Department of Oncology/McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53705, USA. Electronic address: mburkard@wisc.edu.

Abstract

Polo-like kinase 1 has hundreds of substrates and multiple functions that operate within the ∼60 min of mitosis. Herein, we describe a chemical-genetic system that allows particular substrates to be "toggled" into or out of chemical control using engineered phosphoacceptor selectivity. Biochemical assays and phosphoproteomic analysis of mitotic cell extracts showed that Plk1^S (L197F) and Plk1^T (L197S/L211A) selectively phosphorylate Ser and Thr, respectively. Plk1^S but not Plk1^T sustains mitotic progression to anaphase, affording the opportunity to toggle substrate residues between Ser and Thr to place them under chemical control. Using this system, we evaluated Kif2b, a known substrate of Plk1 that regulates chromosome alignment. Toggling Ser to Thr on Kif2b places these phosphorylation sites under reversible chemical control, as indicated by a sharp increase in the frequency of misaligned chromosomes and prometaphase arrest. Thus, we demonstrate the ability to chemically control a single substrate by a genetic Ser/Thr toggle.

Keywords: Kif2b; chemical biology; chemical-genetics; genetic toggle; kinase; mitosis; phosphoacceptor selectivity; phosphorylation.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Cell Cycle Proteins / genetics
Cell Cycle Proteins / metabolism*
Humans
Mitosis
Phosphorylation
Polo-Like Kinase 1
Protein Engineering
Protein Serine-Threonine Kinases / genetics
Protein Serine-Threonine Kinases / metabolism*
Proto-Oncogene Proteins / genetics
Proto-Oncogene Proteins / metabolism*
Serine / genetics*
Serine / metabolism
Substrate Specificity
Threonine / genetics*
Threonine / metabolism

Substances

Cell Cycle Proteins
Proto-Oncogene Proteins
Threonine
Serine
Protein Serine-Threonine Kinases

Abstract

Publication types

MeSH terms

Substances

Grants and funding