title: The Functional Significance of Posttranslational Modifications on Polo-Like Kinase 1 Revealed by Chemical Genetic Complementation.
abstract: Mitosis is coordinated by carefully controlled phosphorylation and ubiquitin-mediated proteolysis. Polo-like kinase 1 (Plk1) plays a central role in regulating mitosis and cytokinesis by phosphorylating target proteins. Yet, Plk1 is itself a target for posttranslational modification by phosphorylation and ubiquitination. We developed a chemical-genetic complementation assay to evaluate the functional significance of 34 posttranslational modifications (PTMs) on human Plk1. To do this, we used human cells that solely express a modified analog-sensitive Plk1 (Plk1AS) and complemented with wildtype Plk1. The wildtype Plk1 provides cells with a functional Plk1 allele in the presence of 3-MB-PP1, a bulky ATP-analog inhibitor that specifically inhibits Plk1AS. Using this approach, we evaluated the ability of 34 singly non-modifiable Plk1 mutants to complement Plk1AS in the presence of 3-MB-PP1. Mutation of the T-loop activating residue T210 and adjacent T214 are lethal, but surprisingly individual mutation of the remaining 32 posttranslational modification sites did not disrupt the essential functions of Plk1. To evaluate redundancy, we simultaneously mutated all phosphorylation sites in the kinase domain except for T210 and T214 or all sites in the C-terminal polo-box domain (PBD). We discovered that redundant phosphorylation events within the kinase domain are required for accurate chromosome segregation in anaphase but those in the PBD are dispensable. We conclude that PTMs within the T-loop of Plk1 are essential and nonredundant, additional modifications in the kinase domain provide redundant control of Plk1 function, and those in the PBD are dispensable for essential mitotic functions of Plk1. This comprehensive evaluation of Plk1 modifications demonstrates that although phosphorylation and ubiquitination are important for mitotic progression, many individual PTMs detected in human tissue may have redundant, subtle, or dispensable roles in gene function.
authors.affiliation: University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, 53705, United States of America.|Department of Medicine, Hematology/Oncology Division, University of Wisconsin-Madison, Madison, Wisconsin, 53705, United States of America.
authors.forename: Amber L
authors.initials: AL
authors.lastname: Lasek
authors.affiliation: University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, 53705, United States of America.|Department of Medicine, Hematology/Oncology Division, University of Wisconsin-Madison, Madison, Wisconsin, 53705, United States of America.
authors.forename: Brittany M
authors.initials: BM
authors.lastname: McPherson
authors.affiliation: University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, 53705, United States of America.|Department of Medicine, Hematology/Oncology Division, University of Wisconsin-Madison, Madison, Wisconsin, 53705, United States of America.
authors.forename: Natalie G
authors.initials: NG
authors.lastname: Trueman
authors.affiliation: University of Wisconsin Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, 53705, United States of America.|Department of Medicine, Hematology/Oncology Division, University of Wisconsin-Madison, Madison, Wisconsin, 53705, United States of America.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Burkard
mesh_terms: D019943:Amino Acid Substitution; D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002455:Cell Division; D020090:Chromosome Segregation; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004789:Enzyme Activation; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010766:Phosphorylation; D017354:Point Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054875:Ubiquitination; D000097869:Polo-Like Kinase 1
chemical_list: D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins; D018844:Cyclin-Dependent Kinases; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D000097869:Polo-Like Kinase 1; C528899:CDK1 protein, human