Browsing by Subject "Phosphorylierung"

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Aktivierung eines neuartigen Apoptose-Signalweges durch den Proteinkinaseinhibitor Staurosporin
(2009) Daubrawa, Merle; Graeve, Lutz
The protein kinase inhibitor staurosporine induces apoptosis via the activation of the intrinsic pathway. First staurosporine was described as a specific PKC inhibitor. Today it is known as a broad range kinase inhibitor and is used as a potent apoptosis inductor. However, the mechanism of the apoptotic effect remains elusive. Furthermore, staurosporine obviously exhibit the potential to eliminate chemotherapy resistant tumors by the induction of a novel intrinsic apoptotic signaling pathway. Different derivatives of staurosporine, e.g. UCN-01, PKC-412 or Enzastaurin are already tested in clinical trials phase I-II for cancer therapy. In the present work it could be shown that overexpression of Bcl-2 does not impede the caspase-dependent induction of apoptosis in J16- and JE6.1-Jurkat T-lymphocytes or in DT40 B-lymphocytes following staurosporine treatment . After generation of apaf-1 -/- DT40 cells it was demonstrated that staurosporine induces apoptosis despite the absence of Apaf-1 and therefore independently of the apoptosome. Together with the generated caspase-9 -/- DT40 cells, caspase-9 was identified as the central effector protein of both staurosporine-induced apoptotic pathways. The involvement of published and putative caspase-9 kinases could not be confirmed by the usage of specific inhibitors. Using phospho-mimicking and phospho-deficient caspase-9 variants, S183 could be identified as an essential phosphorylation site during staurosporine-induced apoptosis. In addition, after treatment with anticancer drugs apoptosome formation was blocked by an N-terminal tag of caspase-9. However, this tag could not prevent staurosporine-induced apoptosis. In further studies the potential role of cathepsines for this novel apoptosis signaling pathway could be analysed by their specific inhibition. In order to investigate the involvement of multiple kinases in this novel apoptotic signaling pathway, combination experiments with specific inhibitors of the respective kinases should be accomplished. Further investigations should clarify whether the influence of S183 on staurosporine-induced apoptosis is based on conformational alteration or on phosphorylation of caspase-9. The generation of additional caspase-9 variants including deltaCARD-caspase-9 or non-cleavable caspase-9 could lead to a deeper understanding of the role of caspase-9 for staurosporine-induced apoptosis. For this purpose caspase-9 -/- DT40 cells and cells reconstituted with different caspase-9 variants could be employed. The phosphorylation pattern of caspase-9 could be determined by mass spectrometric analysis. Xenograft or chorio allantois membrane models were used to investigate if the staurosporine derivative UCN-01 is also able to induce this novel apoptosis signaling pathway in vivo. The identification of both the mechanisms and the effector proteins of this staurosporine-induced apoptotic signaling pathway should provide the opportunity to develop novel agents for the elimination of chemotherapy-resistant tumors.
Effects of N-terminal mutations of human androgen receptor on polyglutamine toxicity
(2008) Funderburk, Sarah F.; Cato, Andrew C. B.
Nine neurodegenerative diseases are caused by polyglutamine (polyQ) tract amplification in different proteins. The cytotoxicity of each of these proteins is associated with a misfolding of the mutant protein, resulting in the subsequent alteration of cellular processes and interactions as well as the interrelated formation of insoluble aggregates and other conformationally toxic species. However, the diseases differ in their pathology and tissue specificity of action, which may be due to protein context/regions neighboring the polyQ stretch. For the purpose of the studies presented in ths work, the polyQ containing human androgen receptor (AR) that causes the disorder spinal and bulbar muscular atrophy (SBMA) was used to model polyQ toxicity. In previous investigations, two putative phosphorylation sites of the AR were identified, and it was demonstrated that mutation of these sites appeared to cause conformational change in the protein. Therefore, these N-terminal serine residues were exchanged to alanine in the wild type AR (ARQ22/ARQ22dm) or a receptor with an amplified polyQ stretch (ARQ77/ARQ77dm). These mutants were then used to characterize variance in types of aggregates and the associated toxic profiles due to the different protein conformations that arose from the serine mutations. Evaluating changes in aggregation and toxicity in cultured cells and in a Drosophila model of SBMA, it was found that the effects of the conformational changes differed depending on the length of the polyQ stretch. Mutations in the ARQ22 resulted in a marked increase in aggregation as well as decreased survival rates and altered locomotion behavior in Drosophila. These results were similar but not as severe as the ARQ77/SBMA model. In quite the opposite manner, mutations in the ARQ77 caused a decrease in aggregation and a lessened toxic effect in Drosophila. Moreover, it was found that inhibitor compounds used to ameliorate polyQ toxicity were not as efficient in inhibiting the varied toxicities exhibited by both the ARQ22dm and ARQ77dm. Therefore, two distinct amino acid sites that profoundly modulate polyQ toxicity in the AR have been identified. These results can be further utilized to understand the conformational changes in the AR that lead to aggregation as well as the types of aggregates that lead to toxicity.
External nutrition stimuli induced proteome and phosphoproteome responses of maize root hairs and arabidopsis root microsomal fraction
(2021) Li, Zhi; Schulze, Waltraud
This work studied how the proteome from young maize root hair cells responds to different nutrition deprivation, and gives perspectives to the possible involvement of NRT1.1 and NRT2.1 in regulating root membrane phosphoproteome responses. This work also proposes a phospho-switch model that may explain how the NRT2.1 activity was regulated.
Functional characterization of the COOH-terminal kinase activity of the TBP-associated factor TAF1
(2006) Maile, Tobias; Sauer, Frank
Activation of eukaryotic transcription involves an orchestrated interplay between transcription factors and the general RNA polymerase II (Pol II) transcription machinery (GTM), which consists of Pol II and general transcription factors (GTFs). The GTF TFIID consists of the TATA-box binding protein (TBP) and several TBP-associated factors (TAFs). The binding of TFIID to promoters can nucleate transcription. TAF1 is the largest subunit of TFIID and plays a central role within the nucleating function of TFIID in transcription. TAF1 mediates the binding of TFIID to promoters and interacts with enhancer-bound transcription factors and several GTFs. Additionally, TAF1 contains four enzymatic activities that are essential for viability of eukaryotes and mediate posttranslational modification of GTFs and histones. TAF1 is a bipartite protein kinase and contains an NH2-terminal kinase domain (NTK) and a COOH-terminal kinase domain (CTK). A previous study demonstrated that the CTK phosphorylates serine-residue 33 in histone H2B (H2BS33). However, the role of TAF1-mediated phosphorylation in transcription regulation remained unknown. In this study, the functional importance of H2BS33 phosphorylation (H2BS33P) by TAF1 was investigated by using a combination of biochemical and in vivo assays. In vitro kinase assays uncovered the two essential kinase motifs in TAF1CTK, the ATP-binding motif and the serine/threonine-specific catalytic motif, and indicate that the TAF1 CTK has intrinsic kinase-activity. Western blot analysis using an antibody to H2BS33P revealed that H2BS33 is phosphorylated in Drosophila. RNA-interference (RNAi) assays, designed to attenuate TAF1 expression (TAF1RNAi), revealed that TAF1 is a major kinase for H2BS33 in Drosophila Schneider cells. Flow-cytometry analysis of TAF1RNAi cells indicated that loss of TAF1 expression results in cell cycle arrest in G2/M-phase. Screening the transcription of cell cycle genes in TAF1RNAi cells by using reverse-transcriptase-PCR demonstrated that the transcription of the cell cycle gene string (stg) is reduced in the absence of TAF1. Chromatin immunoprecipitation assays (XChIP) indicate that H2BS33P is detectable at the transcriptionally active stg promoter but not at the silent stg promoter in TAF1RNAi cells. These results demonstrate that phosphorylation of H2BS33 is involved in stg transcription. XChIP-assays using chromatin prepared from Drosophila embryos, which express a mutant TAF1 lacking the CTK, revealed that CTK-mediated phosphorylation of H2BS33 plays an essential role in the activation of transcription of the Drosophila segmentation gene giant. In vitro kinase assays demonstrate that Bdf1 and Bdf2, the yeast homologues of the TAF1CTK, phosphorylate histones suggesting that the kinase activity of the TAF1CTK is phylogenetically conserved. The results of this work demonstrate that TAF1CTK is a major histone kinase of H2BS33 and that TAF1-mediated phosphorylation of H2BS33 plays an essential role in the transcription events during cell cycle progression and development.
Identification and functional studies of two novel serine phosphorylation sites of insulin receptor substrate (IRS)-2: Ser 675 and Ser 907
(2010) Fritsche, Louise; Schleicher, Erwin D.
Insulin receptor substrate (IRS) proteins are major transducers of the insulin and IGF-1 signal into the PI-3 kinase/PKB and the MAP kinase pathway. In addition to tyrosine phosphoryla-tion, a large number of serine/threonine phosphorylation sites enable the IRS proteins to inte-grate different extra- and intracellular stimuli resulting in positive and negative modulation of the insulin and IGF-1 signal. Chronic hyperphosphorylation of serine/threonine sites of IRS-1 is involved in the development of insulin resistance. IRS-2 is of great importance for β-cell survival and for the regulation of hepatic metabolism. The study of serine/threonine phos-phorylations is required to understand the physiological and pathophysiological regulation of this important mediator of insulin signaling. In this thesis two novel IRS-2 serine phosphoryla-tion sites have been identified and characterized (mouse amino acid numbering): Ser 675, which is located in the kinase regulatory loop binding (KRLB) domain unique to IRS-2 and Ser 907, which is adjacent to the Grb2 binding site Tyr 911. Using phospho-site specific antibod-ies both sites were demonstrated to be phosphorylated upon insulin, phorbol ester and ani-somycin treatment in Fao rat hepatoma cells. The phosphorylation was also detected in pri-mary human hepatocytes and in liver tissue of insulin treated or refed mice. The insulin-induced phosphorylation of Ser 907 was mediated by the MAP kinase ERK1/2. Simulation of a permanent phosphorylation of this site in BHK cells expressing IRS-2 Glu 907 led to a slight decrease of IRS-2 tyrosine phosphorylation with no apparent effect on insulin downstream signaling. The insulin-induced association of IRS-2 with Grb2 in HEK293 cells was abrogated by mutation of the adjacent Tyr 911 to Phe, but not influenced by mutation of Ser 907 to Ala. Of note, the activation of MAP kinase signaling was not impaired in HEK293 cells expressing IRS-2 Phe 911 and not regulated by the expression level of IRS-2 wildtype, but completely dependent on IR expression, indicating the importance of an alternative, IRS-2-Grb2-independent pathway for the activation of MAP kinase signaling in these cells. The insulin-induced phosphorylation of Ser 675 was dependent on mTOR, but not on the downstream kinase p70 S6K1. Prevention of this phosphorylation in BHK cells or HEK293 cells expressing IRS-2 Ala 675 had no effect on proximal or distal insulin signal transduction. But compared with IRS-2 wildtype, the mutated IRS-2 protein Ala 675 showed increased half life in cycloheximide-treated HEK293 cells. Thus, phosphorylation of Ser 675 could have a similar function as its homologous site Ser 632 in IRS-1 and could be involved in the regula-tion of mTOR-dependent IRS-2 proteasom-mediated protein degradation.
Novel serine phosphorylation sites of IRS2 mediate 14-3-3 binding and regulate insulin signal transduction
(2012) Neukamm, Sabine Sarah; Schleicher, Erwin D.
Insulin and insulin like growth factor (IGF)-1 mediate their metabolic and mitogenic effects on target tissues through activation of the insulin and IGF-1 receptor. Insulin receptor substrate (IRS) proteins function as intermediate docking platforms to transduce the insulin/IGF-1 signal to intracellular effector molecules that regulate glucose homoeostasis, lipid metabolism, cell proliferation and ß-cell survival. The activated receptors function as tyrosine kinases that phosphorylate IRS proteins on tyrosine residues, thereby generating interaction motifs for Src homology (SH) 2 domain containing proteins. Signal transduction via IRS proteins is furthermore regulated by their serine/threonine phosphorylation by several kinases and this can result in inhibitory or stimulatory consequences for downstream signalling. Hyperphosphorylation of IRS1 has been shown to be involved in the desensitization process that can result in insulin resistance. Both IRS1 and IRS2 undergo proteasomal degradation while particularly IRS2 levels are additionally regulated by cAMP-dependent gene activation. 14-3-3 proteins are versatile regulators of a variety of intracellular processes like control of cell cycle, cell growth, gene transcription and apoptosis. Serine/threonine phosphorylation within distinct motifs on the interaction partner is a prerequisite for 14-3-3 binding. IRS1 and IRS2 have been described as 14-3-3 interaction proteins and interaction of IRS2 with 14-3-3 proteins was specifically characterized in this thesis. Insulin/IGF-1-dependent PI 3-kinase stimulation as well as elevated cAMP levels were identified to modulate 14-3-3 binding to IRS2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS2 in transfected HEK293 cells and this bind- ing was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus or refeeding. Application of different IRS2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300-600. Mass spectrometric analysis produced a total of 24 phosphorylated serine/threonine residues on IRS2 after IGF-1 stimulation with 12 sites unique for IRS2 while the other residues are conserved in IRS1 and IRS2. The 24 identified phosphorylated residues on IRS2 included several 14-3-3 binding candidates in the region 300-600 and single alanine mutants of these candidates led to the identification of Ser573 as 14-3-3 binding site by overlay assays. A phosphosite specific antibody was generated to further characterize Ser573. IGF-1-dependent phosphorylation of Ser573 was reduced by inhibition of PI 3-kinase and Akt/PKB. The alanine mutant of Ser573 showed enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. In summary, the data presented in this thesis indicate a negative impact of Ser573 phosphorylation on downstream signalling. Binding of 14-3-3 to IRS2 upon stimulation with forskolin and the cAMP analogue CPT-cAMP (8-(4-chlorophenylthio) adenosine 3?,5?-cyclic monophosphate) was demonstrated in HEK293 cells, that was prevented with the PKA inhibitor H89. The amino acid region behind position 952 on IRS2 was identified as cAMP/PKA-dependent 14-3-3 binding region by GST-14-3-3 pulldown as- says. Mass spectrometric analyses revealed Ser1137/Ser1138 as cAMP-dependent, potential PKA phosphorylation sites. Inhibition of Akt/PKB or ERK did not prevent the cAMP-dependent phosphorylation of IRS2 on PKA consensus motifs. Mutation of Ser1137/Ser1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding as shown by GST-14-3-3 pulldown experiments and co-immunoprecipitation assays. IRS2 protein degradation was demonstrated by the application of cycloheximide and an increased IRS2 protein stability was observed when HEK293 cells stably expressing IRS2 or primary hepatocytes were incubated with forskolin. This reduced IRS2 protein degradation was dependent on the presence of Ser1137/Ser1138, since stimulation with forskolin did not increase protein stability of the double Ala1137/Ala1138 mutant. To conclude, Ser1137/Ser1138 are presented as novel cAMP-dependent phosphorylation sites on IRS2 and their importance in 14-3-3 binding and IRS2 protein stability is demonstrated. This represents a novel mechanism for the cAMP- dependent upregulation of IRS2 protein levels that can be of importance for hepatic metabolism and ß-cell survival.
Plant ammonium transporter (AMT) integration in regulatory networks
(2016) Straub, Tatsiana; Ludewig, Uwe
Ammonium is a ubiquitous key nutrient in agricultural soils and the preferred nitrogen source for plants. However, excessive ammonium accumulation represses plant growth and development. Ammonium is taken up by plant cells via high-affinity ammonium transporters (AMTs). Six AMT genes were identified in Arabidopsis, which are separated in two distinct clades, five AMT1s and one AMT2. In the plasma membrane, AMT proteins form homo- and heterotrimers with extra-cytoplasmic N-termini and cytoplasmic C-termini. In addition to transcriptional and post-transcriptional control of AMTs by ammonium, phosphorylation in the C-terminus serves as a rapid allosteric switch of the AMT activity and prevents further internal ammonium accumulation. In a physiological screen, a kinase (CIPK23) was identified, which directly regulates ammonium transport activity under high-NH4+ conditions. Interestingly, CIPK23 is already known to regulate nitrate and potassium uptake in roots. Lesion of the CIPK23 gene significantly increased ammonium uptake, but caused growth inhibition. As expected, cipk23 plants were also limited in potassium accumulation, but high potassium availability failed to rescue the cipk23 phenotype. Furthermore, cipk23 plants were more susceptible to methylammonium (MeA), a non-metabolizable analogue of ammonium. The sensitivity to MeA was lost upon genetic suppression of AMT1 genes in the cipk23 background. The data suggest that CIPK23 directly phosphorylates AMT1s in a complex with CBL1 (calcineurin B-like protein) and thereby regulates transport activity. The expression of the CIPK23 and the CBL1 genes were ammonium-dependent and increased when N-starved plants were resupplied with ammonium. Furthermore, cbl1 mutants had enhanced NH4+ accumulation; this phenocopies the larger ammonium uptake in the cipk23 loss-of-function mutant. In vivo experiments demonstrated bimolecular interaction between CIPK23, AMT1;1, and AMT1;2, but not with AMT2;1, suggesting direct phosphorylation of AMT1-type ammonium transporters by CIPK23. However, Western blot analysis with the cipk23 mutant suggested that the loss of the kinase was not sufficient to completely abolish AMT1;1 and AMT1;2 phosphorylation, indicating several independent pathways to regulate ammonium transport activity in AMT trimers. The data identify complex post-translational regulation of ammonium transporters via the CBL1–CIPK23 pathway, which ensures reduction of AMT1 activity and suppression of ammonium uptake under high external NH4+ concentrations.
Regulation of ammonium transport in Arabidopsis thaliana
(2022) Ganz, Pascal; Ludewig, Uwe
The overarching question of this thesis deals with how plants ensure the selective uptake of ammonium while maintaining ion and pH homeostasis. A key component of this is ammonium transporters (AMTs) with high affinity towards their substrate, which are at the same time part of a multilayered protection system against uncontrolled ammonium influx. Conserved protein sequences inside the transporter were analyzed as well as the regulatory system based on post-translational modification of the transporter.
Transporters mediating ammonium uptake in plants and their regulation by the abiotic stress signaling pathway
(2023) Porras Murillo, Romano; Ludewig, Uwe
Nitrogen nutrition refers to the uptake, assimilation, and utilization of ammonium, nitrate, and organic nitrogen sources. Ammonium is energetically a more cost-effective nitrogen source than nitrate but can be toxic for plants, and its use by plants is regulated at different levels. Ammonium transporters (AMTs) take up ammonium and are localized primarily in plant roots, working as trimers in the plasma membrane. Under high external ammonium concentrations, phosphorylation in AMTs C-termini shuts down transport to avoid toxicity. This phosphorylation is performed by CIPK23, a kinase shown to be inhibited by Clade A PP2Cs. This study aimed to characterize AMTs from wheat and analyze their transcriptional response to ammonium. Another aim was to determine the role of clade A PP2Cs and PYR/PYL receptor proteins for abscisic acid in ammonium nutrition. Chapter I describes the physiological responses of winter wheat to different nitrogen sources and ammonium concentrations. The plants mainly used root morphological responses to adapt to differences in the nitrogen source. High external concentrations of ammonium reduced plant growth, while these conditions induced the expression of TaAMT1;1 and TaAMT1;2. In Chapter II, we studied the capacity of TaAMT2s to transport ammonium and their transcriptional responsiveness to ammonium nutrition. From the six TaAMT2s, only TaAMT2;1 could transport ammonium in a yeast complementation line. Besides, its expression in roots is lower under ammonium than under nitrate. The expression pattern among the remaining TaAMT2s (TaAMT2;2-TaAMT2;6) is similar, with higher expression under ammonium, in both roots and leaves, compared to nitrate. Chapter III focused on the role of the PP2C phosphatase ABI1 (ABA-insensitive 1) in ammonium nutrition and the effect of external ammonium concentrations on ABA concentrations. Ammonium increased ABA concentrations in roots by activating ABA-GE, meaning ammonium toxicity could be sensed as abiotic stress through ABA. Without ammonium, ABI1 dephosphorylates AMTs and inhibits CIPK23; with ammonium, ABA-PYR/PYL complex-mediated inhibition of ABI1 releases CIPK23 to phosphorylate AMTs and avoids ammonium toxicity. Finally, in Chapter IV, we studied the role of AIP1 and its ammonium-dependent regulator, PYL8, in nitrogen nutrition. We described the function of AIP1, which was redundant to ABI1 in AMT regulation. Based on ammonium-dependent root architecture changes, and higher auxin accumulation in pyl8-1 root tips compared to the wild type, we suggest that PYL8 is involved in root-phenotype modulation in an ammonium-dependent manner.