title: Design of a protein kinase-inducible domain.
abstract: Protein phosphorylation is a critical regulatory strategy. New tools are necessary which may be used to interrogate and are responsive to the activities of protein kinases and phosphatases. We have used protein design to develop a protein motif, termed a protein kinase-inducible domain, whose structure is dependent on its phosphorylation state. Based on an EF hand calcium-binding loop, the key design element is the replacement of a structurally critical Glu residue, which binds metal in a bidentate manner, with a serine residue, which is expected to bind metal tightly when phosphorylated but poorly when not phosphorylated. The design comprises an EF hand consensus sequence, a tryptophan at residue 7 to sensitize lanthanide luminescence, and the recognition sequence of a serine/threonine kinase. Designed peptides, which contain minimal substrate recognition motifs of the protein kinases PKA, PKC, or the MAP kinase Erk, form complexes with Tb3+ when phosphorylated, showing strong Tb3+ luminescence emission at 544 nm, but show weak luminescence when not phosphorylated. The change in fluorescence on phosphorylation is comparable to or greater than that observed in described kinase sensors. Site-specific lanthanide binding was confirmed by NMR with diamagnetic and paramagnetic metals. The kinase-inducible domain peptides comprise an expressible sequence, potentially enabling their use as genetically encoded tags of protein kinase activity. The motif is general and potentially applicable to the majority of serine/threonine kinases.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
authors.forename: Shalini
authors.initials: S
authors.lastname: Balakrishnan
authors.forename: Neal J
authors.initials: NJ
authors.lastname: Zondlo
mesh_terms: D000595:Amino Acid Sequence; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D011494:Protein Kinases