Receptor protein-tyrosine kinases

Top PDF Receptor protein-tyrosine kinases:

The non-receptor protein tyrosine kinases Btk and Tec and their role in macrophages in response to infection / submitted by Afitap Derya Köprülü

The non-receptor protein tyrosine kinases Btk and Tec and their role in macrophages in response to infection / submitted by Afitap Derya Köprülü

Tyrosine kinases play crucial and essential roles in almost all biological processes and thus also in cells of the hematopoietic system. Among the various tyrosine kinase families, the Tec kinase family of non-receptor protein tyrosine kinases constitute the second largest family in the hematopoietic system. Tec family kinases (TFK) have been shown to be crucial regulators of antigen receptor signaling in lymphocytes. During the last years it became clear that TFKs are also important regulators of myeloid cell lineages such as monocytes/macrophages, dendritic cells, neutrophils and mast cells. TFKs have been shown to be involved in Toll-like receptor (TLR) signaling and in the regulation of cytokine production, which is central for both the length and magnitude of the inflammatory response. Therefore, TFKs constitute targets with huge therapeutic potential in chronic inflammatory diseases such as rheumatoid arthritis or in cardiovascular disease and cancer [1-3]. During my Ph.D. studies, I investigated the role of TFKs in macrophages in response to infection with Listeria monocytogenes (Lm) and analyzed their role in the regulation of cytokine expression upon activation with TLR ligands.
Mehr anzeigen

128 Mehr lesen

The ligand-specific co-receptor function of CD44 for receptor tyrosine kinases

The ligand-specific co-receptor function of CD44 for receptor tyrosine kinases

has been found to be important for proliferation and migration in primary as well as invasion and metastasis formation in several cancer cell lines. CD44v6 not only presents the ligand HGF to Met but features yet another molecular function, namely the mediation of downstream signaling by recruiting the ERM-protein ezrin. Since the co-receptor function of CD44v6 is specifically dependent on exon v6, the minimal v6 amino acid sequence required for Met activation was identified using linker scan mutations of the v6 sequence, replacing three amino acids at a time by alanines or glycine (in place of alanine) (Matzke et al, 2005). This linker scan analysis identified a sequence of three amino acids that is essential for the co-receptor function of CD44v6. Peptides containing the amino acid sequences EWQ in rat, GWQ in mouse and RWH in human, that are at least 5 amino acids in length can block the co- receptor function of CD44v6. This leads to an inhibition of Met-phosphorylation and signal transduction to its downstream targets Ras and Erk (Matzke et al, 2005). The CD44v6 peptide therefore inhibits the activation of Met with the same specificity as a CD44v6-antibody or a soluble ectodomain of CD44v6 (Matzke et al, 2005; Orian- Rousseau et al, 2002).
Mehr anzeigen

112 Mehr lesen

Tyrosine Kinases in Helicobacter pylori Infections and Gastric Cancer

Tyrosine Kinases in Helicobacter pylori Infections and Gastric Cancer

structural similarities with SFKs and are characterized by an N-terminal Src homology 2 (SH2) domain binding to phosphorylated tyrosine residues and a Src homology 3 (SH3) domain which preferentially binds to proline-rich protein sequences. The C-terminal tail of c-Abl consists of a DNA-binding domain, F- and G-actin-binding domains, nuclear localization signals (NLS), and nuclear export signals (NES), and is of high importance for the regulation of kinase activity and localization [ 90 ]. Abl kinases are activated by various factors, such as PDGFR and EGFR, substrate binding, oxidative stress, or bacterial pathogens [ 89 – 91 ]. H. pylori is included in the list since many studies have provided strong evidence showing that it utilizes c-Abl-CagA signaling for efficient colonization and pathogenesis [ 92 , 93 ]. The presence of H. pylori leads to a continuous activation of c-Abl and phosphorylation of the kinase on tyrosine residues 245 and 412 [ 60 , 61 , 64 ]. Moreover, kinase inhibition of c-Abl, silencing by RNA interference, or expression of a kinase-dead version cause a significant reduction in cell elongation and scattering and a decrease in late CagA phosphorylation upon H. pylori infection [ 60 , 61 ]. Furthermore, cross-talk between c-Abl and EGFR signaling has been observed. c-Abl-mediated phosphorylation of EGFR on tyrosine residue 1173 inhibits receptor endocytosis leading to an increase in EGFR surface expression in H. pylori infections [ 65 , 66 ]. This process has been shown to be dependent on CagA, but independent of CagA phosphorylation [ 65 ]. Additionally, EGFR protein turnover is slowed down by activated c-Abl via suppression of the ubiquitin ligase casitas B-lineage lymphoma (Cbl), which is involved in EGFR degradation [ 66 ] (Figure 1 C).
Mehr anzeigen

15 Mehr lesen

Expression patterns of EphA2 tyrosine kinase receptor in human digestive
system organs and tumors

Expression patterns of EphA2 tyrosine kinase receptor in human digestive system organs and tumors

EphA2 was first identified in 1990 as a result of screening an epithelial cell cDNA library with degenerate oligonucleotides designed to hybridize to highly conserved regions of protein tyrosine kinases [32]. It was initially referred to as eck (epithelial cell kinase) for its expression in the majority of epithelial cells. The human EphA2 gene which is located on chromosome 1p36, encodes a receptor tyrosine kinase of 976 amino acids with an apparent molecular weight of 130 kDa and has a 90 % amino acid sequence homology to mouse EphA2 [33]. The EphA2 extracellular regions include an N-terminal ligand binding domain, a cysteine-rich motif, and two fibronectin-like repeats. Intracellularly, the juxtamembrane domain contains two conserved tyrosines that undergo autophosphorylation and is followed by a tyrosine kinase domain. The COOH-terminal end of EphA2 serves as a docking site for interacting proteins that may mediate downstream single transduction processes and includes a sterile α motif and a PDZ protein domain-binding motif [34].
Mehr anzeigen

89 Mehr lesen

Transcriptional regulation of flotillins by the extracellularly regulated kinases and retinoid X receptor complexes

Transcriptional regulation of flotillins by the extracellularly regulated kinases and retinoid X receptor complexes

One major function of lipid rafts is to organize signaling partners into functional complexes [8]. Within rafts, flotillins have been suggested to provide platforms for the assembly of signaling molecules and thus function as regulators of several signal transduction pathways associated with membrane receptors, e.g. insulin [9], IgE receptor [10], IL-6/STAT3 signaling [11], G protein coupled receptor [12] and of the neurotrophin receptor TrkA. Furthermore, we have recently shown that flotillin-1 is crucial for receptor tyrosine kinase signaling through the epidermal growth factor (EGF) [13] and fibroblast growth factor (FGF) receptors [14] and thereby regulates the MAP kinase signaling [13]. In the case of all signal transduction pathways in which flotillins are known to be involved, the absence of flotillins leads to a severe impairment of the signaling cascade [10–13]. During EGF receptor (EGFR) signaling, the absence of flotillin-1 results in reduced phosphorylation of specific tyrosines in the
Mehr anzeigen

17 Mehr lesen

Functional consequences of interactions between the neural cell adhesion molecule NCAM, the receptor protein tyrosine kinase TrkB and the inwardly rectifying K+ channel Kir3.3 in Mus musculus (Linnaeus, 1758)

Functional consequences of interactions between the neural cell adhesion molecule NCAM, the receptor protein tyrosine kinase TrkB and the inwardly rectifying K+ channel Kir3.3 in Mus musculus (Linnaeus, 1758)

Reichardt, 2003). Hence, the corresponding protooncogene was named trk – tropomyosin- receptor kinase (Martin-Zanca et al., 1986a; Barbacid, 1994). TrkB is a glycoprotein of an apparent molecular weight of 145 kDa and was discovered as the second member of the family due to its high homology to the original Trk, which is now commonly referred to as TrkA (Klein et al., 1989; Middlemas et al., 1991). Specific patterns of expression within the nervous system suggested roles in neuronal development and function, but the Trk receptors were originally only a small percentage of the large number of orphan tyrosine kinases with high expression in the nervous system. The ligands binding and activating the Trk receptors are known as neurotrophins. Different neurotrophins show binding specificity for particular receptors – nerve growth factor (NGF) binds preferentially to TrkA; brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4) to TrkB; and neurotrophin 3 (NT3) to TrkC (Klein et al., 1991; Dechant et al., 1993; Kaplan and Miller, 2000; Huang and Reichardt, 2001). The discovery of the Trk receptors had a revolutionary impact on this field, because it provided essential tools for pursuing the signaling pathways controlled by neurotrophins.
Mehr anzeigen

129 Mehr lesen

Src kinases and Flt3: phosphorylation, interference with receptor maturation and mechanism of association

Src kinases and Flt3: phosphorylation, interference with receptor maturation and mechanism of association

As described in 3.3, Flt3 inducibly associates with Hck in HEK-293 cells. To investigate whether Flt3 associates with Hck in 32D cl. 3 cells, cells were starved in the absence of IL-3 for 12 hours and then stimulated with FL for 10 minutes to induce Flt3 autophosphorylation. Hck KR was immuno- precipitated from cellular lysates using Hck specific antibodies. Flt3 was detected in immuno-precipitates by immunobloting with Flt3 specific and phospho-tyrosine specific antibodies. As a control, HEK-293 lysates con- taining equal amounts of protein were used. Figure 3.19 (a) shows that no Flt3 was detected in Hck immuno-precipitates from 32D cl. 3 cells even when more sensitive phospho-tyrosine specific antibodies were used for de- tection. In contrast, Flt3 could be easily detected by both Flt3 and phospho- tyrosine specific antibodies in Hck KR immuno-precipitates from HEK-293 cell lysates containing equal amount of protein. No Flt3 could be detected in immuno-precipitates of wild type and hyperactive Hck from 32D cl. 3 lysates either (data not shown). The following possibilities could account for the lack of association between Hck and Flt3 in 32D cl. 3 cells: 1) interference of a protein expressed in 32D cl. 3, but not HEK-293 cells, which binds to Hck or Flt3 competitively preventing Hck-Flt3 interaction, 2) restriction of the interaction of human Hck and Flt3 to the cell lines of human origin, 3) high activity of phosphatases de-phosphorylating Hck binding sites on Flt3 in 32D cl. 3 cells as compared with HEK-293 cells, and 4) lower levels of over-expressed Hck in 32D cl. 3 cells than in HEK-293 cells. To test whether lower levels of Hck KR expression could account for the lack of association between Hck KR and Flt3 in 32D cl. 3 cells, HEK-293 cells were transfected with lower amounts of Hck KR and Hck KR RL as a negative control and association of the two proteins was analyzed by co- immunoprecipitation. Figure 3.19 (b) shows that FL inducible association between Flt3 and Hck KR could be detected in HEK-293 cells, even when significantly lower Hck KR amounts were expressed. Considering only a slight difference in the expression of Hck KR between 32D cl. 3 and HEK- 293 cells, these data suggest that lower Hck KR expression levels in 32D cl. 3 cells do not account for the lack of association between Hck and Flt3 in these cells.
Mehr anzeigen

153 Mehr lesen

Receptor Tyrosine Kinase Activation in Human Carcinoma Cells

Receptor Tyrosine Kinase Activation in Human Carcinoma Cells

Intensive research efforts focused on the elucidation of these signalling pathways, and as increasingly larger numbers of cell signalling components and pathways are being identified and studied it has become apparent that cellular signalling pathways are not isolated from each other as linear tracts but are highly interconnected to form complex signalling networks. Reversible protein phosphorylation has been identified as a key element in signal transduction processes (Cohen, 2002). Phosphorylation by the combined action of a protein kinase and a protein phosphatase can reversibly alter the activity of an enzyme, target proteins for degradation or increase its stability, alter its subcellular localization or change its affinity towards interacting proteins. One third of all mammalian proteins can be phosphorylated, and the sequencing of the human genome revealed that there are approximately 520 protein kinases and 130 protein phosphatases encoded by human genes (Blume-Jensen and Hunter, 2001; Manning et al., 2002). Both protein kinases and phosphatases can be subdivided into cellular and transmembrane molecules, and according to their substrate specificity into tyrosine, serine/threonine or dual specific kinases. In particular tyrosine phosphorylation has been early recognized as a major mechanism of transmembrane signalling (Cohen, 2002).
Mehr anzeigen

109 Mehr lesen

The interaction between tyrosine protein kinase receptor B (TrkB) and neural cell adhesion molecule NCAM in Mus musculus

The interaction between tyrosine protein kinase receptor B (TrkB) and neural cell adhesion molecule NCAM in Mus musculus

effects of neurotrophins on neurite growth have also been described (Griffin and Letour- neau, 1980). Furthermore, the involvement of α-2,8-linked PSA on NCAM-dependent neurite outgrowth after BDNF stimulation was documented by using the PSA-specific Endo-N enzyme, which splits PSA chains from NCAM. The inhibitory influence of BDNF was slightly reduced after Endo-N treatment (see 5.14, Fig. 29). Loss of PSA seems to lead to less inhibition of NCAM-dependent neurite outgrowth after BDNF stimulation. To ex- clude effects by endogenous BDNF, neurite outgrowth assays were performed in the presence of K252a, which specifically inhibits Trk tyrosine kinase activity when used at low concentrations (Tapley et al., 1992; Segal et al., 1995). Blockage of endogenous BDNF resulted in a minor but still significant inhibitory effect of BDNF on NCAM-dependent neurite outgrowth. The analysis of the neurite outgrowth assays confirmed the hypothesis of a functional interplay between NCAM and TrkB. Homophilic interaction takes place between NCAM molecules (Walmod et al., 2004) and leads to the stimulation of neurite outgrowth based on the activation of two pathways: Heterophilic interaction (between NCAM and FGFR) and activation of the FGFR first triggers the PLCγ-mediated pathway and then the Ras–MAPK pathway via activation of Fyn and FAK after preferential binding to NCAM140 (Beggs et al., 1997; Povlsen et al., 2003). Recently, PI3K also has been im- plicated in the NCAM-mediated signaling (Ditlevsen et al., 2003). To illuminate the functional role of the intracellular domains of both NCAM isoforms on neurite outgrowth, NCAM140-ID and NCAM180-ID (including the TMD) were chosen for overexpression studies in PC12 cells. When NCAM140-ID was overexpressed, NCAM-mediated neurite outgrowth was decreased because NCAM140-ID seems to compete with endogenous NCAM140 for Fyn and FAK, the activators of the Ras–MAPK pathway. This is in contrast to the overexpressed NCAM180-ID which stimulated neurite outgrowth but normally in- creased stabilization and cell adhesion due to a competition between cytoskeletal proteins (e.g. spectrin) and endogenous NCAM180 (Büttner et al., 2004). Based on the results of Büttner et al. (2004), a hypothetical model was developed to explain the inhibitory effect of BDNF on NCAM-mediated neurite outgrowth (see 5.14, Fig. 29). Receptor tyrosine kinases such as TrkB use signaling pathways similar to those utilized by NCAM, including common interaction partners such as the non-receptor tyrosine kinase Fyn (Beggs et al., 1997; Iwasaki et al., 1998). Fyn has been shown to be associated with TrkB-ID. This inter- action is dependent on autophosphorylation of TrkB and on BDNF induction (Iwasaki et
Mehr anzeigen

139 Mehr lesen

Functional characterization of non-receptor tyrosine kinase dependent signal transduction in acute lymphoblastic leukemia of childhood

Functional characterization of non-receptor tyrosine kinase dependent signal transduction in acute lymphoblastic leukemia of childhood

The aim of this study was to assess the expression of protein tyrosine kinases (PTK) in acute lymphoblastic leukemia (ALL) patient samples at the protein level. Xenotransplanted primary ALL blasts and ALL cell lines were used as model system for the functional analysis of the role of PTK in ALL. The analysis revealed that Lyn, a member of the Src family kinase (SFK), was prominently expressed in a subgroup of ALL patient samples. To further investigate the biological consequence of elevated Lyn expression in ALL cells, Nalm6 and CALL3 cells were used as a model which recapitulated the high and low Lyn expression profile observed in patient specimens, respectively. Lyn is known to be associated with pre-BCR after receptor crosslinking. Analysis of the functional role of Lyn upon shRNA mediated Lyn repression and pre-BCR crosslinking showed that phosphorylation of downstream signaling proteins was strikingly reduced or delayed in Nalm6 cells. In addition, cell proliferation was substantially reduced in Nalm6 Lyn-knockdown cells. Conversely, an increase in the tyrosine phosphorylation was found in CALL3 Lyn-knockdown cells. Membrane microdomain, called lipid rafts, were shown to concentrate and regulate SFK 111 . However, data of the Lyn localization in the plasma membrane indicates that, whereas Lyn was exclusively present within defined lipid rafts in CALL3 cells, the protein was aberrantly localized all over the membrane in Nalm6 cells. The Lyn mislocalization was likely independent of lipid rafts and it could enable Lyn to interact with other proteins located outside rafts structures and promote its activation. Ultimately, preliminary data suggests that overexpression of Lyn is implicated in resistance to tyrosine kinase inhibitor (TKI) treatment.
Mehr anzeigen

92 Mehr lesen

Effect of tyrosine autophosphorylation on catalytic activity and subcellular localisation of homeodomain-interacting protein kinases (HIPK)

Effect of tyrosine autophosphorylation on catalytic activity and subcellular localisation of homeodomain-interacting protein kinases (HIPK)

Whereas more and more putative targets of HIPK2 are being identified [15], enhancing our understanding of its function in cell death and cell survival, much less is known about the other members of the HIPK family. Due to their highly similar primary structures, HIPK1 and HIPK2 are assumed to have at least a certain degree of redundant activity [16]. Whereas HIPK1/2 double knockout mice die at embryonic day 12.5, either HIPK1 or HIPK2-deficient mice develop grossly normal but ex- hibit differences in apoptosis induction and eye develop- ment [17-20]. Although the cellular functions of HIPK1 are not very well defined, available evidence indicates a role in the regulation of apoptosis by interaction with nu- clear proteins [16,21]. HIPK3 has been characterized as a regulator of the androgen receptor and Runt-related tran- scription factor 2 (Runx2) [22,23]. Recently, HIPK3 −/− mice were shown to have impaired glucose-induced insu- lin secretion [24], and HIPK3 has been implicated in the pathogenesis of human type 2 diabetes [25]. Compared to HIPK2, HIPK1 and HIPK3 are less well characterized re- garding their molecular and cellular function, and it is un- clear to which degree these kinases fulfill redundant or divergent tasks. Almost nothing is known about HIPK4 except for its capacity to phosphorylate p53 at Ser9 [4].
Mehr anzeigen

11 Mehr lesen

Role of phospholipase d in g-protein coupled receptor function

Role of phospholipase d in g-protein coupled receptor function

1. Introduction Phospholipase D (PLD) is a widely distributed phospholipid-specific diesterase that hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. PLD is rapidly activated in response to extracellular stimuli, and the generation of PA is considered to mediate many biological functions attributed to PLD and to play an important role in the regulation of cell function and activity. This includes a wide array of cellular responses as calcium mobilization, secretion, superoxide production, endocytosis, exocytosis, vesicle trafficking, glucose transport, rearrangement of actin cytoskeleton, mitogenesis and apoptosis [1]. PLD superfamily members are widely distributed and found in viruses, bacteria, yeast, plants and animals. To date, more than 4000 PLD enzymes have been entered into the NCBI GenBank. The present review focuses on mammalian PLDs and their roles in the G-protein coupled receptor function. For the first time, the PLD activity in human tissue has been described [2]. Two mammalian PLD genes, PLD1 and PLD2, both with two splice variants have been identified [3–5]. In line with a role for PLD enzymes in different cellular tasks, PLD1 and PLD2 show a diverse subcellular distribution. PLD1 is found throughout the cell, but primarily localizes to intracellular compartments, including the Golgi apparatus, endosomes, and the perinuclear region [6–8]. PLD2 is almost exclusively present at the plasma membrane in lipid raft fractions [9]. Also, PLD1 is found in lipid rafts. The PLD activity appears to be present in nearly all cell types. PLD1 and PLD2 are both robustly expressed in heart, brain, and spleen. PLD1 exhibits low expression in peripheral blood leukocytes and synovial tissue [10], and PLD2 is poorly expressed in liver, skeletal muscle [11] and articular chondrocytes [12]. Both PLD enzymes have been shown to associate with membrane receptors including G-protein coupled receptors (GPCR), receptor tyrosine kinases or integrins, which all mediate signalling of PLD activation. GPCRs constitute a large group of membrane binding receptors known to modulate a wide range of biological responses, including cell growth, differentiation, migration, and inflammatory processes. Extracellular stimuli trigger dissociation of G Į and GȕȖ heterotrimeric G proteins. Uncoupled heterotrimer subunits elicit signalling cascades through downstream effector proteins. Many of these pathways elicit functional responses through signalling to PLD in multiple ways. On the other side, the PLD influences GPCR function in many respects. PLD-generated PA affects the GPCR function via modulation of vesicle trafficking, endocytosis and membrane receptor recycling.
Mehr anzeigen

17 Mehr lesen

Role of phospholipase d in g-protein coupled receptor function

Role of phospholipase d in g-protein coupled receptor function

1. Introduction Phospholipase D (PLD) is a widely distributed phospholipid-specific diesterase that hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. PLD is rapidly activated in response to extracellular stimuli, and the generation of PA is considered to mediate many biological functions attributed to PLD and to play an important role in the regulation of cell function and activity. This includes a wide array of cellular responses as calcium mobilization, secretion, superoxide production, endocytosis, exocytosis, vesicle trafficking, glucose transport, rearrangement of actin cytoskeleton, mitogenesis and apoptosis [1]. PLD superfamily members are widely distributed and found in viruses, bacteria, yeast, plants and animals. To date, more than 4000 PLD enzymes have been entered into the NCBI GenBank. The present review focuses on mammalian PLDs and their roles in the G-protein coupled receptor function. For the first time, the PLD activity in human tissue has been described [2]. Two mammalian PLD genes, PLD1 and PLD2, both with two splice variants have been identified [3–5]. In line with a role for PLD enzymes in different cellular tasks, PLD1 and PLD2 show a diverse subcellular distribution. PLD1 is found throughout the cell, but primarily localizes to intracellular compartments, including the Golgi apparatus, endosomes, and the perinuclear region [6–8]. PLD2 is almost exclusively present at the plasma membrane in lipid raft fractions [9]. Also, PLD1 is found in lipid rafts. The PLD activity appears to be present in nearly all cell types. PLD1 and PLD2 are both robustly expressed in heart, brain, and spleen. PLD1 exhibits low expression in peripheral blood leukocytes and synovial tissue [10], and PLD2 is poorly expressed in liver, skeletal muscle [11] and articular chondrocytes [12]. Both PLD enzymes have been shown to associate with membrane receptors including G-protein coupled receptors (GPCR), receptor tyrosine kinases or integrins, which all mediate signalling of PLD activation. GPCRs constitute a large group of membrane binding receptors known to modulate a wide range of biological responses, including cell growth, differentiation, migration, and inflammatory processes. Extracellular stimuli trigger dissociation of Gα and Gβγ heterotrimeric G proteins. Uncoupled heterotrimer subunits elicit signalling cascades through downstream effector proteins. Many of these pathways elicit functional responses through signalling to PLD in multiple ways. On the other side, the PLD influences GPCR function in many respects. PLD-generated PA affects the GPCR function via modulation of vesicle trafficking, endocytosis and membrane receptor recycling.
Mehr anzeigen

17 Mehr lesen

The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

HeLa cells confirmed that DCAF7 interacts with DYRK1A at physiological expression levels (Fig. 1c). Zebrafish DYRK1B has been reported to interact with the orthologous DCAF7 protein (also termed WDR68 20 ), suggesting that the binding interface between DCAF7 and class 1 DYRKs is conserved in evolution. To test this, we determined whether the DCAF7 and class 1 DYRK orthologs from various species where able to interact with each other. We found that human DCAF7 was co-immunoprecipitated with Xenopus DYRK1B (Fig. 1d). Furthermore, mouse DYRK1A, human DYRK1B and Xenopus DYRK1B bind to GFP-DCAF7 from zebrafish (Fig. 1e). These results show that the structural determinants required for the interaction of DYRK1 with DCAF7 are conserved in vertebrates.
Mehr anzeigen

15 Mehr lesen

The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

!!!"#$%&'(")*+,-).(#%./.)'(0*'%-, ! !"#$%&#'#" ( !"#$!%& ȁͼǣ͸;͸ͺͷȁ ǣͷͶǤͷͶ͹;Ȁ•”‡’͸;͸ͺͷ HIPK2 sequences, while the corresponding sequence in HIPK1 differs in several positions. Co-IP experiments showed that DCAF7 bound to a GFP fusion protein containing the first 135 amino acids of HIPK2, but not to a shorter construct comprising amino acids 1–114 (Fig. 3d). This result localizes the DCAF7 binding site of HIPK2 to a segment of 21 amino acids in the N-terminal region of HIPK2. Considering the difference in DCAF7 binding between HIPK1 and HIPK2, we speculated that the presence of a proline in the corresponding sequence of HIPK1 might disrupt the binding interface (Fig. 3c). Indeed, the substitution of Thr125 by proline abolished DCAF7 binding to HIPK2 (Fig. 3d). As with DYRK1A, DCAF7 binding did not depend on the catalytic activity of HIPK2, since the interaction was maintained by the kinase deficient HIPK2 D324N point mutant (Fig. 3d). The
Mehr anzeigen

29 Mehr lesen

G-Protein Coupled Receptor Targeting on Myeloid Cells in Atherosclerosis

G-Protein Coupled Receptor Targeting on Myeloid Cells in Atherosclerosis

RvE1, a metabolite of EPA (a type of polyunsaturated fatty acids), plays an important role in the return to tissue homeostasis ( Schwab et al., 2007 ; Gao et al., 2013 ; Hasturk et al., 2015 ) and is suggested to exhibit anti-inflammatory and pro-resolving effects via ChemR23 or leukotriene B4 receptor 1 (BLT1) ( Arita et al., 2005b ; Arita et al., 2007 ) (please also see section “Pro- resolving Lipid Mediators”). RvE1-dependent blockage of VSMC migration, a critical process in the progression of atherosclerosis, and switching into a protective anti-atherosclerotic phenotypic in VSMCs, confer an anti-inflammatory role of vascular ChemR23 signaling ( Ho et al., 2010 ). Moreover, RvE1 rescues impaired neutrophil phagocytosis, oxidized LDL uptake and phagocytosis of macrophages, promotes phagocytosis-induced neutrophil apoptosis ( El Kebir et al., 2012 ; Herrera et al., 2015 ; Artiach et al., 2018 ), and also attenuates APC functions targeting dendritic cell migration and reducing IL-12 production via ChemR23 ( Arita et al., 2005a ). Furthermore, RvE1 can restore inflammation induced mitochondrial dysfunction and reduce polymorphonuclear leukocyte infiltration in BLT1 dependent manner ( Arita et al., 2007 ; Mayer et al., 2019 ), along with ChemR23-mediated counter regulatory actions to mediate the resolution of inflammation. It is also evident that RvE1 suppresses inflammatory cytokine release, facilitating the healing process, and inhibits macrophage migration by activating ChemR23 in a ligation model of acute MI ( Liu et al., 2018 ). Moreover, supplementation of Apoe − /−
Mehr anzeigen

24 Mehr lesen

Die Rolle des low density lipoprotein receptor-related protein 1 im humanen Lipoproteinstoffwechsel

Die Rolle des low density lipoprotein receptor-related protein 1 im humanen Lipoproteinstoffwechsel

ein spezifisches Signal vortäuschen, z. B. im Sinne zweier Tyrosinkinase-Rezeptoren, die nach Ligandenbindung dimerisieren und den aktivierten Rezeptor bilden oder im Sinne eines noch nicht verstandenen Ko-Rezeptor-Signalmechanismus von LRP1 (Newton et al. 2005). Dagegen spricht jedoch die grundsätzlich schwache GFP-Interaktion, die in aller Regel nicht zu Artefakten führt (Zhang et al. 2002). Um dies für die konkrete Fragestellung an LRP1 abzusichern, wurden aus pEGFP-N1 durch gezielte Mutagenese Mutanten nach Zacharias et al. (2002) hergestellt, in denen die Dimerisierungs- oberfläche unterbrochen ist, und diese mit LRP1 analog zu LRP1-EGFP fusioniert. Da das daraus erhaltene Protein keinen mikroskopisch erkennbaren Unterschied in seiner Lokalisation aufwies (Daten nicht gezeigt), wurde eine Dimerisierung des EGFP-Anteils von LRP1-EGFP als Grund für die Beobachtung der beschriebenen LRP1-Vesikel ausgeschlossen. Ein weiterer Grund für ein Artefakt könnte die räumliche Ausdehnung des EGFP-Anteils sein, der möglicherweise ebenfalls die Bindung von Adaptor- Proteinen behindern könnte, die für die korrekte Sortierung des Rezeptors erforderlich sind. Trotz zahlreicher Versuche gelang es leider weder ein internes noch ein N- terminales Fusionsprotein zu erstellen. Solche Konstrukte hätten jedoch letztlich die Problematik nur verlagert, da aufgrund der Vielzahl von Liganden von LRP1 davon ausgegangen werden muß, daß die Konformation sowohl zwischen den beiden Untereinheiten als auch in den Ligandenbindungsdomänen von LRP1 Auswirkungen auf die Sortierung des Rezeptors hat. Unabhängig von der Position des EGFP-Anteils muß mit solchen Effekten immer gerechnet werden. Daher wurden fixierte, transfizierte und nicht-transfizierte Zellen ausgiebig miteinander verglichen, mit dem Ergebnis, daß die in lebenden Zellen in großen Mengen beobachteten, großen Vesikel von LRP1-EGFP in fixierten Präparaten nicht bzw. nur so selten nachzuweisen waren wie entsprechende Vesikel aus endogenem LRP1 (Abbildung 21 und Abbildung 22, S. 61). Da bekannt ist, daß große Vesikel während des Fixier- und Färbeprozesses kollabieren können (Bowers und Maser 1988), trifft dies mit großer Wahrscheinlichkeit auch auf die LRP1-EGFP- Vesikel zu (T. Kirchhausen, Boston, MA, persönliche Kommunikation). Gleiches gilt dann auch für die Vesikel aus endogenem LRP1, so daß im Umkehrschluß auch diese Vesikel in lebenden Zellen eine differenziertere Morphologie aufweisen als im fixierten Präparat. Ein letzter Beweis kann jedoch nicht erbracht werden, da einerseits bislang noch keine Daten zur Darstellung von LRP1 in lebenden Zellen veröffentlicht sind und andererseits mit den zur Verfügung stehenden Methoden endogenes LRP1 in lebenden Zellen nicht direkt beobachtet werden kann.
Mehr anzeigen

113 Mehr lesen

The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

The remarkable conservation of the DCAF7/DYRK interaction is highlighted by results showing that human DCAF7 can partially complement a defect in flower pigmentation in petunia an11 mutants 36 and that the Drosophila wap protein can rescue the defects in craniofacial development of zebrafish caused by WDR68 mutation 19 . Taking into consideration that DCAF7 was repeatedly co-purified with DYRK1A from various sources 14–17,37 , we hypothesize that DCAF7 functions as an important subunit of DYRK1A and other class 1 DYRKs. RACK1 (Receptor for Activated C Kinase 1) is a WD repeat scaffold protein that selectively interacts with PKC in the activated status 38 . In contrast, DCAF7 binds to DYRK1A and HIPK2 independently of kinase activity, although the serine residue (Ser97) within the DCAF7 binding motif is a well characterized autophos- phorylation site of DYRK1A 39 . In this way, DCAF7 appears to function as a hub for protein-protein interactions, like the WD repeat domain of LRRK2 (leucine-rich repeat kinase 2) 40 . In accordance with this idea, DCAF7 coordinates signalling complexes by acting as an adaptor for proteins such as the cytoskeletal regulator mDia1 14 and mitogen-activated protein kinase kinase kinase 1 (MEKK1) 18 . DCAF7 does not seem to act as an anchor- ing protein for its interacting kinases, because previous studies showed that DCAF7 is recruited to the nucleus by overexpressed DYRK1A or HIPK2 16,18 . Unexpectedly, our results show that the simultaneous overexpression of E1A and DCAF7 induced a massive redistribution of DYRK1A and E1A from the nucleus to the cytoplasm (Fig. 8c). The bipartite nuclear localisation sequences (NLS) of DYRK1A and E1A are located in close vicin- ity of the DCAF7 binding site (DYRK1A, Fig. 2a) or even overlap (E1A, ref. 41). It appears possible that steric hindrance by the interacting proteins in the DYRK1A/DCAF7/E1A complex shields the nuclear localisation sequences in DYRK1A and in E1A from interacting with the importin complex. The functional consequences of this redirection of DYRK1A and E1A to the cytoplasm remain to be elucidated.
Mehr anzeigen

29 Mehr lesen

Total internal reflection fluorescence microscopy (TIRFM) guided analysis of receptor tyrosine kinase (RTK) signaling / eingereicht von: Peter Lanzerstorfer

Total internal reflection fluorescence microscopy (TIRFM) guided analysis of receptor tyrosine kinase (RTK) signaling / eingereicht von: Peter Lanzerstorfer

These distinct roles of IRS proteins indicate that the specificity of insulin/IGF signaling and biological responses in part reflects the availability of IRS proteins to IR/IGF-IR. Many of the interactions between IR/IGF-IR and IRS have been inferred from the studies using yeast two-hybrid assay [5, 6, 34]. However, this technique is not well suited to investigate the protein-protein interaction quantitatively, because the detection is not based in the mammalian cell environment but in the yeast nucleus and has a high frequency of false-positives. On the other hand, their interaction has been analyzed by in vitro pull- down assay or co-immunoprecipitation assay [14, 35]. However, these data also do not reflect interaction in the live cell context. Here we studied the interaction properties of IRS proteins with IR/IGF-IR by the use of µ- patterned surfaces in combination with total internal reflection fluorescence (TIRF) microscopy. Live cell µ-patterning has been shown to be a suitable tool for detection and quantitation of protein-protein events in cell membranes [36-41]. It is a system to quantify interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in living cells. Cells are plated on µ- patterned surfaces functionalized with antibodies to the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey in cell membranes using TIRF microscopy. In our experiments the µ-patterning assay was utilized to enrich endogenous bait IR/IGF-IR in HeLa cells into microscopic domains and monitor the co-recruitment of fluorescent prey IRS proteins. We were able to confirm and quantitate the recruitment of different IRS
Mehr anzeigen

92 Mehr lesen

Investigations on the role of mitogen-activated protein kinases 11 and 17 in defense response of Arabidopsis thaliana

Investigations on the role of mitogen-activated protein kinases 11 and 17 in defense response of Arabidopsis thaliana

Additionally, an interaction of MPK1 and 2 with MKK3 was reported [Dóczi et al., 2007]. The role of AtMPK7 and 14 is still unclear, although Shi et al. [2010] revealed an involvement of Gossypium hirsutum (cotton) GhMPK7, the closest orthologue to AtMPK7, in pathogen defense. All MPK members of group D, MPK8, 9, and MPK15-20, have the TDY amino acid motif in the activation loop and are usually bigger than members of the other groups because of an extended C-terminal region [GroupMAPK, 2002]. Additionally, group D kinases lack the common docking (CD) domain which is important for interaction with other proteins [Tanoue and Nishida, 2003]. A recent report by Takahashi et al. [2011] shows an important role of MPK8 in the regulation of ROS homeostasis. MPK9, together with MPK12, are involved in the ROS signaling pathway in guard cells, as reported by Jammes et al. [2009]. MPK18 was found to affect microtubule stability [Walia et al., 2009]. Information on the biological function of MPK15–17, MPK19 and MPK20 is not available by now.
Mehr anzeigen

121 Mehr lesen

Show all 2793 documents...