title: Systematic investigation of hierarchical phosphorylation by protein kinase CK2.
abstract: Although multiple phosphorylation sites are often clustered in substrates, the mechanism of phosphorylation within clusters has not been systematically investigated. Intriguingly, in addition to acidic residues, protein kinase CK2 can use phosphoserine residues as consensus determinants suggesting that CK2 may act in concert with other kinases. We used a peptide array approach to outline optimal consensus sequences for hierarchical phosphorylation by CK2, both in the context of processive, multisite phosphorylation, and in concert with a priming proline-directed kinase. Results suggest that hierarchical phosphorylation involving CK2 requires precise positioning of either multiple phosphodeterminant residues or specific combinations of canonical determinants and phosphodeterminants, and can be as enzymatically favorable as canonical CK2 phosphorylation. Over 1600 human proteins contain at least one CK2 hierarchical consensus motif, and ~20% of these motifs contain at least one reported in vivo phosphorylation site. These motifs occur non-randomly in the human proteome, with significant enrichment in proteins controlling specific cellular processes. Taken together, our results provide strong in vitro evidence that hierarchical phosphorylation may contribute to the regulation of crucial biological processes. In addition, the results suggest a mechanism by which CK2, a constitutively active kinase, can be a regulatory participant in cellular processes.
  
  
  CONCLUSIONS
  Phosphorylation is a crucial regulatory mechanism governing cellular signal transduction pathways, and despite the large number of identified sites to date, most mechanistic studies remain focused on individual phosphorylation sites. This study is the first to systematically determine specific consensus sequences for hierarchical phosphorylation events. The results indicate that individual phosphorylation sites should not be studied in isolation, and that larger, multisite phosphorylation motifs may have profound impact on cellular signaling. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, ON, Canada.
authors.forename: Nicole
authors.initials: N
authors.lastname: St-Denis
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
authors.forename: Michelle
authors.initials: M
authors.lastname: Gabriel
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
authors.forename: Jacob P
authors.initials: JP
authors.lastname: Turowec
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Gloor
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
authors.forename: Shawn S-C
authors.initials: SS
authors.lastname: Li
authors.affiliation: Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; Department of Oncology, Schulich School of Medicine & Dentistry, Western University, ON, Canada. Electronic address: litchfi@uwo.ca.
authors.forename: David W
authors.initials: DW
authors.lastname: Litchfield
mesh_terms: D020816:Amino Acid Motifs; D047390:Casein Kinase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D020543:Proteome / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D020543:Proteome; D047390:Casein Kinase II
keywords: Hierarchical phosphorylation; Multisite phosphorylation; Protein kinase CK2