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Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of enzymes with important functions in physiology and disease. HAD phosphatases can target diverse metabolites, lipids, DNA, and serine/threonine or tyrosine phosphorylated proteins with often high specificity (Seifried et al., 2013). These enzymes thus markedly enlarge the repertoire and substrate spectrum of mammalian phosphatases. However, the basis of HAD phosphatase substrate specificity is still elusive and a number of mammalian HAD phosphatases remain uncharacterized to date. This study characterizes the biochemical and structural properties of AUM (aspartate-based, ubiquitous, Mg2+-dependent phosphatase), a previously unexplored mammalian HAD phosphatase.
In vitro phosphatase assays of purified, recombinant AUM showed phosphatase activity towards para-nitrophenyl phosphate and adenine and guanine nucleotide di- and triphosphates. Inhibitor studies indicated that similar to other HAD superfamily members, the AUM-catalyzed dephosphorylation reaction proceeds via a pentacovalent phosphoaspartate intermediate. In line with an aspartate-based catalytic mechanism, AUM was insensitive to inhibitors of serine/threonine phosphatases. The characterization of the purified recombinant murine enzyme also revealed that AUM exists in equilibrium between dimers and tetramers.
AUM was identified as the closest, yet functionally distinct relative of chronophin, a pyridoxal 5’-phosphate and serine/threonine-directed phosphatase. Phylogenetic analyses showed that AUM and chronophin evolved via duplication of an ancestral gene at the origin of the vertebrates. In contrast to chronophin, AUM acts as a tyrosine-specific HAD-type phosphatase in vitro and in cells. To elucidate how AUM and chronophin achieve these distinct substrate preferences, comparative evolutionary analyses, biochemical approaches and structural analyses were combined. Swapping experiments of less homologous regions between AUM and chronophin were performed. The mutational analysis revealed residues important for AUM catalysis and specificity. A single differently conserved residue in the cap domain of AUM or chronophin is crucial for phosphatase specificity (AUML204, chronophinH182). The X-ray crystal structure of the AUM cap fused to the catalytic core of chronophin (CAC, PDB: 4BKM) was solved to 2.65 Å resolution. It presents the first crystal structure of the murine AUM capping domain. The detailed view of the catalytic clefts of AUM and chronophin reveals the structural basis of the divergent substrate specificities. These presented findings provide insights into the design principles of capped HAD phosphatases and show that their substrate specificity can be encoded by a small number of predictable residues. In addition, the catalytic properties of AUM were investigated, identifying a mechanism of reversible oxidation regulating the activity of AUM in vitro. AUM phosphatase activity is inhibited by oxidation and can be recovered by reduction. The underlying molecular mechanism was revealed by mutational analyses. The cysteines C35, C104 and C243, located in the AUM core domain, are responsible for the inhibition of AUM by oxidation. C293 mediates the redox-dependent tetramerization of AUM in vitro. Based on the chronophin and CAC structure, a direct impact of the oxidation of C35 on the nucleophile D34 is proposed. In addition, a redox-dependent disulfide bridge (C104, C243), connecting the core and cap domain of AUM may be important for an open/close-mechanism. This hypothesis is supported by CD spectroscopy experiments that demonstrate a structural change in AUM upon reduction. These data present the first evidence for the regulation of AUM catalysis by reversible oxidation. This finding is so far unique in the field of HAD phosphatases.
In this context, the first cell-based AUM activity assay was developed. For this, the artificial substrate pNPP was combined with the reducing agent DTT to create a specific AUM activity readout. This fractionation-based assay is the first tool to differentiate between cell lines or tissues with different AUM concentrations or activities.
Taken together, the presented biochemical characterization reveals the specificity determinants and catalytic properties of AUM. General insights into structural determinants of mammalian HAD phosphatase substrate recognition are provided and reversible oxidation as possible regulatory mechanism for AUM is proposed. These findings constitute a framework for further functional analyses to elucidate the biomedical importance of AUM.
In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work.
Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases.
Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity.
Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1.
Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.
The haloacid dehalogenase (HAD) family of phosphatases is an ancient, ubiquitous group of enzymes, and their emerging role in human health and disease make them attractive targets for detailed analyses.
This thesis comprises the biochemical and structural characterization of chronophin, an HAD-type
phosphatase, which has been shown to act on Ser3-phosphorylated cofiln-1, a key regulator of actin dynamics, and on the Ser/Thr-phosphorylated steroid receptor co-activator 3 (SRC-3). Besides being a specific phosphoprotein phosphatase, chronophin also acts on the small molecule pyridoxal 5'-phosphate (PLP, vitamin B6), implying that chronophin serves as a regulator of a variety important physiological pathways. The analysis of chronophin was performed on different levels, ranging from intrinsic regulatory mechanisms, such as the allosteric regulation via dimerization or the characterization of specificity determinants, to modes of extrinsic modulation, including the association with putative interacting proteins or the generation of chronophin-specific inhibitors.
The association of the previously identified putative chronophin interactors calcium- and integrinbinding protein 1 (CIB1) and calmodulin was investigated using recombinantly expressed and purified proteins. These studies revealed that the interaction of chronophin with CIB1 or calmodulin is mutually exclusive and regulated by calcium. Neither CIB1 nor calmodulin had an effect on the in vitro chronophin phosphatase activity towards PLP or phospho-cofilin-1, but might regulate other functions of this important phosphatase.
The role of chronophin dimerization was studied by generating a constitutively monomeric variant,
which showed reduced PLP hydrolyzing activity. X-ray crystallographic studies revealed that dimerization is essential for the positioning of the substrate specificity loop in chronophin, unraveling a previously unknown mechanism of allosteric regulation through a homophilic interaction. This mechanism potentially applies to other enzymes of the C2a subfamily of HAD-type phosphatases, as all structurally characterized members show a conserved mode of dimerization.
The general determinants of substrate specificity in the C2a subfamily of HAD phosphatases were
investigated by performing domain swapping experiments with chronophin and its paralog AUM and
subsequent biochemical analyses of the hybrid proteins. The X-ray crystallographic structure
determination of the chronophin catalytic domain equipped with the AUM capping domain revealed the first partial structure of AUM. This structural information was then used in subsequent studies that analyzed the divergent substrate specificities of AUM and chronophin in an evolutionary context.
Finally, a set of four chronophin inhibitors were generated based on the structure of PLP and
characterized biochemically, showing moderate inhibitory effects with IC50-values in the micromolar range. These compounds nevertheless constitute valuable tools for future in vitro experiments, such as studies concerning the structure-function relationship of chronophin as a PLP phosphatase. In addition, the crystal structure of one inhibitor bound to chronophin could be solved. These results provide the basis for the further development of competitive chronophin inhibitors with increased specificity and potency.
Abstract
Glioblastomas, primary brain tumors, represent a tumor entity with a dismal prognosis and a median survival of only about one year. Invasion into the healthy brain parenchyma contributes substantially to the malignancy of this type of brain tumor. Therefore, a better understanding of the mechanisms promoting the invasive behavior of these brain tumors is needed to identify new therapeutic targets.
Cofilin, an actin regulatory protein, has been shown to be an important regulator of the invasive behavior of tumor cells in other types of cancer and the actin cytoskeleton is involved in the formation of a variety of cellular structures important for cell migration and invasion. Cofilin is regulated by phosphorylation on a single residue, serine 3. The aim of this thesis was to examine the role of the cofilin regulatory phosphatase chronophin for glioma cell migration and invasion.
First, it was established that chronophin depletion in the cell line GBM6840 leads to an increase in the ratio of phosphorylated cofilin to total cofilin. Higher chronophin levels were correlated with a decrease in F-actin in the cell lines GBM6840 and U87 as measured in an actin spin down assay and in a flow cytometry based assay.
Furthermore, it was shown that knockdown of chronophin in two different cell lines, GBM6840 and DBTRG-05-MG, strongly increased their invasiveness in vitro. Expression of human chronophin in the cell line U87 decreased its invasiveness substantially. There was no difference in cell proliferation between GBM6840 and DBTRG-05-MG cells expressing a chronophin targeting shRNA or a control shRNA and U87 cells transfected with an empty vector or a human chronophin encoding plasmid. The increase in invasiveness after chronophin depletion could be correlated with an increase in directionality in cell migration under 2D culture conditions in the cell lines U87 and GBM6840. Moreover, treatment with the ROCK inhibitor Y-27632 decreased directionality in GBM6840 cells under 2D culture conditions and reduced the invasiveness of GBM6840 chronophin shRNA cells back to control levels.
Expression of a non-phosphorylatable cofilin mutant, the S3A mutant, was able to reduce invasiveness and to reduce directionality under 2D culture conditions back to control levels in GBM6840 chronophin shRNA cells.
This provides important evidence for the involvement of cofilin phosphoregulation in the phenotypes described above.
In vivo, when injected into NOD-SCID mice, chronophin depleted cells showed a dramatic growth reduction as compared to control and rescue cells.
Transciptomic characterization of GBM6840 cells by microarray analysis and subsequent comparison of the data with microarray profiles of normal brain tissues and different glioma entities identified two specifically chronophin regulated transcripts potentially involved in tumor progression and invasion, MXI1 and EDIL3. Moreover, c-myc was identified as a significantly altered transcription factor after chronophin deregulation based on the number of c-myc target molecules in the microarray dataset.
MXI1 is a potential negative regulator of c-myc dependent transcription, and was strongly downregulated after chronophin knockdown in GBM6840. In line with this, the activity of a c-myc reporter plasmid was increased after chronophin depletion in GBM6840 and reduced after chronophin expression in U87 cells.
However, the protein level of the c-myc protein was reduced after chronophin depletion in GBM6840.
Finally, anaylsis of the expression of proteases known to be important for glioblastoma pathogenesis revealed no major changes in protease expression between chronophin depleted and control cells.
Therefore, a comprehensive analysis of chronophin in the context of glioma pathogenesis has been performed in this thesis. It has been shown that chronophin depletion strongly enhanced invasiveness of glioma cells and that it induced transcriptomic changes potentially involved in tumor progression. The proteins regulating cofilin phosphorylation are therefore valuable therapeutic targets for anti-invasive therapy in glioblastomas. Inhibitors for kinases upstream of cofilin, e.g. LIMKs and ROCKs, are available, and might be promising agents for anti-invasive therapy.