Biochemistry and Pharmacology - Research Publications

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    Regulation and function of protein kinases and phosphatases.
    Cheng, H-C ; Qi, RZ ; Paudel, H ; Zhu, H-J (Hindawi Limited, 2011)
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    Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)
    Gunawardana, D ; Cheng, H-C ; Gayler, KR (OXFORD UNIV PRESS, 2008-01)
    The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn(2+)- or Mg(2+)-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.
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    Structural elements and allosteric mechanisms governing regulation and catalysis of CSK-family kinases and their inhibition of Src-family kinases
    Ia, KK ; Mills, RD ; Hossain, MI ; Chan, K-C ; Jarasrassamee, B ; Jorissen, RN ; Cheng, H-C (TAYLOR & FRANCIS LTD, 2010-10)
    C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) are endogenous inhibitors constraining the activity of the oncogenic Src-family kinases (SFKs) in cells. Both kinases suppress SFKs by selectively phosphorylating their consensus C-terminal regulatory tyrosine. In addition to phosphorylation, CHK can suppress SFKs by a unique non-catalytic inhibitory mechanism that involves tight binding of CHK to SFKs to form stable complexes. In this review, we discuss how allosteric regulators, phosphorylation, and inter-domain interactions interplay to govern the activity of CSK and CHK and their ability to inhibit SFKs. In particular, based upon the published results of structural and biochemical analysis of CSK and CHK, we attempt to chart the allosteric networks in CSK and CHK that govern their catalysis and ability to inhibit SFKs. We also discuss how the published three-dimensional structure of CSK complexed with an SFK member sheds light on the structural basis of substrate recognition by protein kinases.
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    Allosteric networks governing regulation and catalysis of Src-family protein tyrosine kinases: Implications for disease-associated kinases
    Cheng, H-C ; Johnson, TM ; Mills, RD ; Chong, Y-P ; Chan, K-C ; Culvenor, JG (WILEY, 2010-01)
    1. The Src-family protein tyrosine kinases (SFKs) are multidomain oncogenic protein tyrosine kinases. Their overactivation contributes to cancer formation and progression. Thus, synthetic inhibitors of SFKs are being developed as therapeutics for cancer treatment. Understanding the regulatory and catalytic mechanisms of SFKs is necessary for the development of therapeutic SFK inhibitors. 2. Although many upstream regulators and protein substrates of SFKs have been identified, both the mechanisms of activation and catalysis of SFKs are not fully understood. In particular, it is still unclear how the inactive SFKs undergo conformational transition during activation. The mechanism governing the binding of substrates and the release of products during catalysis is another area that requires investigation. 3. Several recent publications indicate the presence of a 'hydrophobic spine' formed by four conserved interacting hydrophobic residues in the kinase domain of SFKs. In the present review, we discuss how the assembly and disassembly of the hydrophobic spine residues may govern conformational transition of SFKs during activation. In addition to regulation of kinase activity, the hydrophobic spine is implicated to be involved in catalysis. It has been postulated recently that perturbation of the hydrophobic spine residues is a key step in catalysis. 4. Further investigations to decipher the roles of the hydrophobic spine residues in regulation and catalysis of SFKs will benefit the development of therapeutic SFK inhibitors for cancer treatment.
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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 β-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)
    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.
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    Analysis of LRRK2 accessory repeat domains: prediction of repeat length, number and sites of Parkinson's disease mutations
    MILLS, R ; Mulhern, TD ; Cheng, HC ; Culvenor, JG ( 2012)
    Various investigators have identified the major domain organization of LRRK2 (leucine-rich repeat kinase 2), which includes a GTPase ROC (Ras of complex proteins) domain followed by a COR (C-terminal of ROC) domain and a protein kinase domain. In addition, there are four domains composed of structural repeat motifs likely to be involved in regulation and localization of this complex protein. In the present paper, we report our bioinformatic analyses of the human LRRK2 amino acid sequence to predict the repeat size, number and likely boundaries for the armadillo repeat, ankyrin repeat, the leucine-rich repeat and WD40 repeat regions of LRRK2. Homology modelling using known protein structures with similar domains was used to predict structures, exposed residues and location of mutations for these repeat regions. We predict that the armadillo repeats, ankyrin repeats and leucine-rich repeats together form an extended N-terminal flexible 'solenoid'-like structure composed of tandem repeat modules likely to be important in anchoring to the membrane and cytoskeletal structures as well as binding to other protein ligands. Near the C-terminus of LRRK2, the WD40 repeat region is predicted to form a closed propeller structure that is important for protein complex formation.
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    Aberrant regulation and function of Src family tyrosine kinases: Their potential contributions to glutamate-induced neurotoxicity
    Hossain, MI ; Kamaruddin, MA ; Cheng, H-C (WILEY, 2012-08)
    Excitotoxicity, a major cause of neuronal death in acute and chronic neurodegenerative diseases and conditions such as stroke and Parkinson's disease, is initiated by overstimulation of glutamate receptors, leading to calcium overload in affected neurons. The sustained high concentration of intracellular calcium constitutively activates a host of enzymes, notably the calcium-activated proteases calpains, neuronal nitric oxide synthase (nNOS) and NADPH oxidase (NOX), to antagonise the cell survival signalling pathways and induce cell death. Upon overactivation by calcium, calpains catalyse limited proteolysis of specific cellular proteins to modulate their functions; nNOS produces excessive amounts of nitric oxide (NO), which, in turn, covalently modifies specific enzymes by S-nitrosylation; and NOX produces excessive amounts of reactive oxygen species (ROS) to inflict damage to key metabolic enzymes. Presumably, key regulatory enzymes governing cell survival and cell death are aberrantly modified and regulated by calpains, NO and ROS in affected neurons; these aberrantly modified enzymes then cooperate to induce the death of affected neurons. c-Src, an Src family kinase (SFK) member, is one of the aberrantly regulated enzymes involved in excitotoxic neuronal death. Herein we review how SFKs are functionally linked to the glutamate receptors and the biochemical and structural basis of the aberrant regulation of SFKs. Results in the literature suggest that SFKs are aberrantly activated by calpain-mediated truncation and S-nitrosylation. Thus, the aberrantly activated SFKs are targets for therapeutic intervention to reduce the extent of brain damage caused by stroke.
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    Analysis of the Regulatory and Catalytic Domains of PTEN-Induced Kinase-1 (PINK1)
    Sim, CH ; Gabriel, K ; Mills, RD ; Culvenor, JG ; Cheng, H-C (WILEY, 2012-10)
    Mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene can cause early-onset familial Parkinson disease (PD). PINK1 encodes a neuroprotective protein kinase localized at the mitochondria, and its involvement in regulating mitochondrial dynamics, trafficking, structure, and function is well documented. Owing to the lack of information on structure and biochemical properties for PINK1, exactly how PINK1 exerts its neuroprotective function and how the PD-causative mutations impact on PINK1 structure and function remain unclear. As an approach to address these questions, we conducted bioinformatic analyses of the mitochondrial targeting, the transmembrane, and kinase domains of PINK1 to predict the motifs governing its regulation and function. Our report sheds light on how PINK1 is targeted to the mitochondria and how PINK1 is cleaved by mitochondrial peptidases. Moreover, it includes a potential optimal phosphorylation sequence preferred by the PINK1 kinase domain. On the basis of the results of our analyses, we predict how the PD-causative mutations affect processing of PINK1 in the mitochondria, PINK1 kinase activity, and substrate specificity. In summary, our results provide a conceptual framework for future investigation of the structural and biochemical basis of regulation and the neuroprotective mechanism of PINK1.