title: Defining the substrate specificity determinants recognized by the active site of C-terminal Src kinase-homologous kinase (CHK) and identification of β-synuclein as a potential CHK physiological substrate.
abstract: C-Terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK-related functions by phosphorylating other cellular protein substrates. To define these non-SFK-related functions of CHK, we used the "kinase substrate tracking and elucidation" method to search for its potential physiological substrates in rat brain cytosol. Our search revealed β-synuclein as a potential CHK substrate, and Y127 in β-synuclein as the preferential phosphorylation site. Using peptides derived from β-synuclein and positional scanning combinatorial peptide library screening, we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ and x represent hydrophobic residues and any residue, respectively. Besides β-synuclein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail sequence of SFKs, indicating that interactions between the CHK active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK. Our results imply that recognition of SFKs by CHK is mainly governed by interactions between motifs located distally from the active site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings shed new light on how CHK recognizes SFKs and other protein substrates.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Kim K
authors.initials: KK
authors.lastname: Ia
authors.forename: Grace R
authors.initials: GR
authors.lastname: Jeschke
authors.forename: Yang
authors.initials: Y
authors.lastname: Deng
authors.forename: Mohd Aizuddin
authors.initials: MA
authors.lastname: Kamaruddin
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Williamson
authors.forename: Denis B
authors.initials: DB
authors.lastname: Scanlon
authors.forename: Janetta G
authors.initials: JG
authors.lastname: Culvenor
authors.forename: Mohammed Iqbal
authors.initials: MI
authors.lastname: Hossain
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Purcell
authors.forename: Sheng
authors.initials: S
authors.lastname: Liu
authors.forename: Hong-Jian
authors.initials: HJ
authors.lastname: Zhu
authors.forename: Bruno
authors.initials: B
authors.lastname: Catimel
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Turk
authors.forename: Heung-Chin
authors.initials: HC
authors.lastname: Cheng
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D020134:Catalytic Domain; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D051846:beta-Synuclein / Q000737:chemistry* / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases
chemical_list: D019151:Peptide Library; D051846:beta-Synuclein; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human