Biochemistry and Pharmacology - Research Publications

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Author: KAMARUDDIN, MOHD × Author: Cheng, Heung × Author: HOSSAIN, IQBAL KAZI MOHAMMAD ×

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    Defining the Substrate Specificity Determinants Recognized by the Active Site of C-Terminal Src Kinase-Homologous Kinase (CHK) and Identification of beta-Synuclein as a Potential CHK Physiological Substrate
    Ia, KK ; Jeschke, GR ; Deng, Y ; Kamaruddin, MA ; Williamson, NA ; Scanlon, DB ; Culvenor, JG ; Hossain, MI ; Purcell, AW ; Liu, S ; Zhu, H-J ; Catimel, B ; Turk, BE ; Cheng, H-C (AMER CHEMICAL SOC, 2011-08-09)
    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.
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    A facile, click chemistry-based approach to assembling fluorescent chemosensors for protein tyrosine kinases
    Kamaruddin, MA ; Ung, P ; Hossain, MI ; Jarasrassamee, B ; O'Malley, W ; Thompson, P ; Scanlon, D ; Cheng, H-C ; Graham, B (PERGAMON-ELSEVIER SCIENCE LTD, 2011-01-01)
    A group of fluorophore-labeled peptide substrates of Src kinases have been synthesized with the aid of click chemistry. Some of the generated peptides exhibit an increase in fluorescence upon phosphorylation and are capable of detecting Src kinases with high sensitivity and specificity. Their availability permits real-time activity measurement of aberrantly activated oncogenic Src kinases in the crude lysate of chronic myelogenous leukemia cells. These new chemosensor peptides are highly useful tools that can be used for high-throughput screening to search for small molecule inhibitors of Src kinases as potential therapeutics for cancer treatment.