Biochemistry and Pharmacology - Research Publications
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Itemp32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology, cell metabolism and survival(PORTLAND PRESS LTD, 2013-08-01)p32 [also known as HABP1 (hyaluronan-binding protein 1), gC1qR (receptor for globular head domains complement 1q) or C1qbp (complement 1q-binding protein)] has been shown previously to have both mitochondrial and non-mitochondrial localization and functions. In the present study, we show for the first time that endogenous p32 protein is a mitochondrial protein in HeLa cells under control and stress conditions. In defining the impact of altering p32 levels in these cells, we demonstrate that the overexpression of p32 increased mitochondrial fibrils. Conversely, siRNA-mediated p32 knockdown enhanced mitochondrial fragmentation accompanied by a loss of detectable levels of the mitochondrial fusion mediator proteins Mfn (mitofusin) 1 and Mfn2. More detailed ultrastructure analysis by transmission electron microscopy revealed aberrant mitochondrial structures with less and/or fragmented cristae and reduced mitochondrial matrix density as well as more punctate ER (endoplasmic reticulum) with noticeable dissociation of their ribosomes. The analysis of mitochondrial bioenergetics showed significantly reduced capacities in basal respiration and oxidative ATP turnover following p32 depletion. Furthermore, siRNA-mediated p32 knockdown resulted in differential stress-dependent effects on cell death, with enhanced cell death observed in the presence of hyperosmotic stress or cisplatin treatment, but decreased cell death in the presence of arsenite. Taken together, our studies highlight the critical contributions of the p32 protein to the morphology of mitochondria and ER under normal cellular conditions, as well as important roles of the p32 protein in cellular metabolism and various stress responses.
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ItemTracking protein aggregation and mislocalization in cells with flow cytometry(NATURE PUBLISHING GROUP, 2012-05)We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.
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ItemDual role of Src kinase in governing neuronal survival(ELSEVIER, 2015-01-12)BACKGROUND: Src-family kinases (SFKs) are involved in neuronal survival and their aberrant regulation contributes to neuronal death. However, how they control neuronal survival and death remains unclear. OBJECTIVE: To define the effect of inhibition of Src activity and expression on neuronal survival. RESULTS: In agreement with our previous findings, we demonstrated that Src was cleaved by calpain to form a 52-kDa truncated fragment in neurons undergoing excitotoxic cell death, and expression of the recombinant truncated Src fragment induced neuronal death. The data confirm that the neurotoxic signaling pathways are intact in the neurons we used for our study. To define the functional role of neuronal SFKs, we treated these neurons with SFK inhibitors and discovered that the treatment induced cell death, suggesting that the catalytic activity of one or more of the neuronal SFKs is critical to neuronal survival. Using small hairpin RNAs that suppress Src expression, we demonstrated that Src is indispensable to neuronal survival. Additionally, we found that neuronal death induced by expression of the neurotoxic truncated Src mutant, treatment of SFK inhibitors or knock-down of Src expression caused inhibition of the neuroprotective protein kinases Erk1/2, or Akt. CONCLUSIONS: Src is critical to both neuronal survival and death. Intact Src sustains neuronal survival. However, in the excitotoxic condition, calpain cleavage of Src generates a neurotoxic truncated Src fragment. Both intact Src and the neurotoxic truncated Src fragment exert their biological actions by controlling the activities of neuroprotective protein kinases.
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ItemSelective STAT3-α or -β expression reveals spliceform-specific phosphorylation kinetics, nuclear retention and distinct gene expression outcomes(PORTLAND PRESS LTD, 2012-10-01)Phosphorylation of STAT3 (signal transducer and activator of transcription 3) is critical for its nuclear import and transcriptional activity. Although a shorter STAT3β spliceform was initially described as a negative regulator of STAT3α, gene knockout studies have revealed that both forms play critical roles. We have expressed STAT3α and STAT3β at comparable levels to facilitate a direct comparison of their functional effects, and have shown their different cytokine-stimulated kinetics of phosphorylation and nuclear translocation. Notably, the sustained nuclear translocation and phosphorylation of STAT3β following cytokine exposure contrasted with a transient nuclear translocation and phosphorylation of STAT3α. Importantly, co-expression of the spliceforms revealed that STAT3β enhanced and prolonged the phosphorylation and nuclear retention of STAT3α, but a STAT3β R609L mutant, with a disrupted SH2 (Src homology 2) domain, was not tyrosine phosphorylated following cytokine stimulation and could not cross-regulate STAT3α. The physiological importance of prolonged phosphorylation and nuclear retention was indicated by transcriptome profiling of STAT3(-/-) cells expressing either STAT3α or STAT3β, revealing the complexity of genes that are up- and down-regulated by the STAT3 spliceforms, including a distinct set of STAT3β-specific genes regulated under basal conditions and after cytokine stimulation. These results highlight STAT3β as a significant transcriptional regulator in its own right, with additional actions to cross-regulate STAT3α phosphorylation and nuclear retention after cytokine stimulation.
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ItemOxidative stress impairs multiple regulatory events to drive persistent cytokine-stimulated STAT3 phosphorylation(ELSEVIER SCIENCE BV, 2014-03)Although cytokine-driven STAT3 phosphorylation and activation are often transient, persistent activation of STAT3 is a hallmark of a range of pathologies and underpins altered transcriptional responses. As triggers in disease frequently include combined increases in inflammatory cytokine and reactive oxygen species levels, we report here how oxidative stress impacts on cytokine-driven STAT3 signal transduction events. In the model system of murine embryonic fibroblasts (MEFs), combined treatment with the interleukin-6 family cytokine Leukemia Inhibitory Factor (LIF) and hydrogen peroxide (H2O2) drove persistent STAT3 phosphorylation whereas STAT3 phosphorylation increased only transiently in response to LIF alone and was not increased by H2O2 alone. Surprisingly, increases in transcript levels of the direct STAT3 gene target SOCS3 were delayed during the combined LIF + H2O2 treatment, leading us to probe the impact of oxidative stress on STAT3 regulatory events. Indeed, LIF + H2O2 prolonged JAK activation, delayed STAT3 nuclear localisation, and caused relocalisation of nuclear STAT3 phosphatase TC-PTP (TC45) to the cytoplasm. In exploring the nuclear import/ export pathways, we observed disruption of nuclear/cytoplasmic distributions of Ran and importin-alpha3 in cells exposed to H2O2 and the resultant reduced nuclear trafficking of Classical importin-alpha/3-dependent protein cargoes. CRM1-mediated nuclear export persisted despite the oxidative stress insult, with sustained STAT3 Y705 phosphorylation enhancing STAT3 nuclear residency. Our studies thus reveal for the first time the striking impact of oxidative stress to sustain STAT3 phosphorylation and nuclear retention following disruption of multiple regulatory events, with significant implications for STAT3 function.
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ItemIdentification and characterization of bi-thiazole-2,2′-diamines as kinase inhibitory scaffolds(ELSEVIER SCIENCE BV, 2013-06)Based on bioinformatics interrogation of the genome, >500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4'-methyl-N(2)-3-pyridinyl-4,5'-bi-1,3-thiazole-2,2'-diamine (JNK Docking (JD) compound 123, but not the related compound (4'-methyl-N~2~-(6-methyl-2-pyridinyl)-4,5'-bi-1,3-thiazole-2,2'-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10μM JD123, identified inhibition of kinases including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.
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ItemDifferences in c-Jun N-terminal kinase recognition and phosphorylation of closely related stathmin-family members(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2014-03-28)The stathmin (STMN) family of tubulin-binding phosphoproteins are critical regulators of interphase microtubule dynamics and organization in a broad range of cellular processes. c-Jun N-terminal kinase (JNK) signalling to STMN family proteins has been implicated specifically in neuronal maturation, degeneration and cell stress responses more broadly. Previously, we characterized mechanisms underlying JNK phosphorylation of STMN at proline-flanked serine residues (Ser25 and Ser38) that are conserved across STMN-like proteins. In this study, we demonstrated using in vitro kinase assays and alanine replacement of serine residues that JNK phosphorylated the STMN-like domain (SLD) of SCG10 on Ser73, consistent with our previous finding that STMN Ser38 was the primary JNK target site. In addition, we confirmed that a JNK binding motif ((41)KKKDLSL(47)) that facilitates JNK targeting of STMN is conserved in SCG10. In contrast, SCLIP was phosphorylated by JNK primarily on Ser60 which corresponds to Ser25 on STMN. Moreover, although the JNK-binding motif identified in STMN and SCG10 was not conserved in SCLIP, JNK phosphorylation of SCLIP was inhibited by a substrate competitive peptide (TI-JIP) highlighting kinase-substrate interaction as required for JNK targeting. Thus, STMN and SCG10 are similarly targeted by JNK but there are clear differences in JNK recognition and phosphorylation of the closely related family member, SCLIP.
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ItemNo Preview AvailablecAMP-dependent Protein Kinase and c-Jun N-terminal Kinase Mediate Stathmin Phosphorylation for the Maintenance of Interphase Microtubules during Osmotic Stress(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2014-01-24)Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.
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ItemNo Preview AvailableIntracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress(ELSEVIER SCIENCE BV, 2014-02)The c-Jun N-terminal kinases (JNKs) are a group of stress-activated protein kinases that regulate gene expression changes through specific phosphorylation of nuclear transcription factor substrates. To address the mechanisms underlying JNK nuclear entry, we employed a semi-intact cell system to demonstrate for the first time that JNK1 nuclear entry is dependent on the importin α2/β1 heterodimer and independent of importins α3, α4, β2, β3, 7 and 13. However, quantitative image analysis of JNK1 localization following exposure of cells to either arsenite or hyperosmotic stress did not indicate its nuclear accumulation. Extending our analyses to define the dynamics of nuclear trafficking of JNK1, we combined live cell imaging analyses with fluorescence recovery after photobleaching (FRAP) protocols. Subnuclear and subcytoplasmic bleaching protocols revealed the slowed movement of JNK1 in both regions in response to hyperosmotic stress. Strikingly, while movement into the nucleus of green fluorescent protein (GFP) or transport of a GFP-T-antigen fusion protein as estimated by initial rates and time to reach half-maximal recovery (t1/2) measures remained unaltered, hyperosmotic stress slowed the nuclear entry of GFP-JNK1. In contrast, arsenite exposure which did not alter the initial rates of nuclear accumulation of GFP, GFP-T-antigen or GFP-JNK1, decreased the t1/2 for nuclear accumulation of both GFP and GFP-JNK1. Thus, our results challenge the paradigm of increased nuclear localization of JNK broadly in response to all forms of stress-activation and are consistent with enhanced interactions of stress-activated JNK1 with scaffold and substrate proteins throughout the nucleus and the cytosol under conditions of hyperosmotic stress.
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ItemNo Preview AvailableCardioprotective 3′,4′-dihydroxyflavonol attenuation of JNK and p38MAPK signalling involves CaMKII inhibition(PORTLAND PRESS LTD, 2013-12-01)DiOHF (3',4'-dihydroxyflavonol) is cardioprotective against I/R (ischaemia/reperfusion) injury. The biological activities of flavonols are associated with kinase modulation to alter cell signalling. We thus investigated the effects of DiOHF on the activation of MAPKs (mitogen-activated protein kinases) that regulate the cardiac stress response. In an ovine model of I/R, JNK (c-Jun N-terminal kinase), p38(MAPK), ERK (extracellular-signal-regulated kinase) and Akt were activated, and NP202, a pro-drug of DiOHF, reduced infarct size and inhibited JNK and p38(MAPK) activation, whereas ERK and Akt phosphorylation were unaltered. Similarly, in cultured myoblasts, DiOHF pre-treatment preserved viability and inhibited activation of JNK and p38(MAPK), but not ERK in response to acute oxidative and chemotoxic stress. Furthermore, DiOHF prevented stress-activation of the direct upstream regulators MKK4/7 (MAPK kinase 4/7) and MKK3/6 respectively. We utilized small-molecule affinity purification and identified CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) as a kinase targeted by DiOHF and demonstrated potent CaMKII inhibition by DiOHF in vitro. Moreover, the specific inhibition of CaMKII with KN-93, but not KN-92, prevented oxidative stress-induced activation of JNK and p38(MAPK). The present study indicates DiOHF inhibition of CaMKII and attenuation of MKK3/6→p38(MAPK) and MKK4/7→JNK signalling as a requirement for the protective effects of DiOHF against stress stimuli and myocardial I/R injury.