To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we employed several lines of investigation including biophysical, biochemical and cellular assays. A high-affinity nerve growth factor (NGF) receptor is thought to be a complex of two receptors, p75 and the receptor tyrosine kinase, TrkA. The existence of a gp75-TrkA complex was demonstrated by a copatching technique. p75 on the surface of intact cells is patched with an anti-p75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with an anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression system, which allows high level expression of wild-type and mutated NGF receptors. TrkA and p75 copatch in both the absence and presence of NGF. This association is specific, since p75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-β and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with p75. A chimeric receptor with TrkA transmembrane and intracellular domains shows partial copatching with p75. Deletion of the intracellular domain of p75 decreases but does not eliminate copatching. A point mutation that inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with p75. Hence, although interactions between the p75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role. To study what signal transduction mechanisms were activated by the two receptors to bring about differentiation and survival, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET-R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR) and the TrkA transmembrane and intracellular domains. EGF activated the ET-R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation, but enhanced the EGF-induced response, leading to differentiation of almost all of the cells. A mutated NGF, 3T-NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF-induced differentiation required at least nanomolar concentrations of NGF, consistent with the low-affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhances apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF appears to enhance survival through the phosphatidylinositol-3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET-R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase-3. An anti-p75 antibody blocked the survival effect of NGF. Brain-derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.

T cell development in the thymus produces multiple lineages of cells, including conventional naïve CD4+ and CD8+ T cells, regulatory T cells, and innate T cells. Innate T cells encompass γδ T cells, invariant natural killer (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and H2-M3-restricted cells (Berg, 2007). Although they are a minor subset of all thymocytes, innate T cells develop in the thymus and share characteristics of the innate and adaptive immune systems (Berg, 2007). These lymphocytes undergo antigen receptor rearrangement and are able to exert their effector function immediately upon ex vivo stimulation (Berg, 2007). However, in several strains of mice harboring mutations in T cell signaling proteins or transcriptional regulators, conventional CD8+ T cells develop as innate cells that share characteristics with memory T cells (Atherly et al., 2006b; Broussard et al., 2006; Fukuyama et al., 2009; Gordon et al., 2011; Verykokakis et al., 2010b; Weinreich et al., 2010). One of these signaling proteins, inducible T cell kinase (Itk) is a nonreceptor protein tyrosine kinase that signals downstream of the T cell receptor (TCR) (Berg et al., 2005). Upon TCR activation, Itk is activated and recruited to the TCR signaling complex, where Itk interacts with Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), linker for activation of T cells (LAT), and phospholipase C γ1 (PLCγ1) (Berg et al., 2005). Thus, in Itk-deficient mice, TCR signaling is disrupted, which results in mature CD4- CD8+ (CD8SP) thymocytes that are CD44high, CD62Lhigh, CD122+, and CXCR3+ and that express high levels of the transcription factor, Eomesodermin (Eomes) (Atherly et al., 2006b; Broussard et al., 2006; Weinreich et al., 2010). Recently, it was determined that the development of these innate CD8SP thymocytes in itk-/- mice is dependent on IL-4 produced in the thymic environment by a poorly characterized subset of CD3+ thymocytes expressing the transcriptional regulator, promyelocytic leukemia zinc finger (PLZF) (Gordon et al., 2011; Verykokakis et al., 2010b; Weinreich et al., 2010). Here we show that a sizeable proportion of mature CD4+ CD8- (CD4SP) thymocytes in itk-/- mice also develop as Eomesodermin+ innate T cells. These Eomes+ innate CD4+ T cells are CD44high, CD62Lhigh, CD122+, and CXCR3+ (Atherly et al., 2006b; Broussard et al., 2006; Dubois et al., 2006; Weinreich et al., 2010). Surprisingly, neither CD4SP nor CD8SP innate thymocytes in itk-/- mice are dependent on γδ T cells for their development as was previously hypothesized (Alonzo and Sant'Angelo, 2011). Instead, both subsets of innate itk-/- T cells require the presence of a novel PLZF-expressing, SAP-dependent thymocyte population that is essential for the conversion of conventional CD4+ and CD8+ T cells into Eomesodermin-expressing innate T cells with a memory phenotype. This novel subset of PLZF-expressing SAP-dependent innate T cells preferentially home to the spleen and mesenteric lymph nodes and have a restricted TCR repertoire. Thus, we have christened this subset as CD4+ PLZF + MAIT-like cells. We have characterized multiple subsets of innate T cells that expand in the absence of Itk. Therefore, we were interested in how innate T cells respond to infection. Although Itk KO mice have defects in cytolytic function and cytokine production during an acute infection, these mice are able to clear viral infections (Atherly et al., 2006a; Bachmann et al., 1997). Hence, we hypothesized that Itk-deficient memory CD8+ T cells would be able to provide protection upon a challenge infection. Conversely, we found this not to be true although Itk-deficient memory CD8+ T cells were present in similar frequencies and cell numbers as WT memory CD8+ T cells at 42 days post-infection. Furthermore, Itk-deficient memory CD8+ T cells were able to produce IFNγ and exert cytolytic function upon stimulation. Although the function of Itk-deficient memory CD8+ T cells appeared to be intact, we found that these cells were unable to expand in response to a challenge infection. Remarkably, conventional memory CD8+ T cells lacking Itk were able to expand and form protective memory responses upon challenge. Thus, the inability of Eomes+ innate CD8+ T cells to form protective memory responses does not appear to be intrinsic to cells deficient in Itk. This thesis is divided into six major chapters. The first chapter will provide an introduction to T cell development and the role of Itk in T cell development. Additionally, it will introduce a variety of innate T cell subsets that will be discussed throughout this thesis and will provide an overview of CD4+ and CD8 + T cell differentiation during infection. This section will explain the role of Itk in CD4+ helper T cell differentiation and describe how Itk-deficient CD8+ T cells respond to acute infection. The introduction will also discuss the generation of conventional memory CD8+ T cells. The second chapter will provide the details of the experimental procedures used in this thesis. The third chapter will describe the characterization and development of Eomes+ innate CD4+ T cells that develop in the absence of Itk. Additionally, this chapter will address the subset of PLZF+ innate T cells that induce the expression of Eomes in innate T cells. The fourth chapter will further characterize and explore the development of itk-/- CD4+ PLZF+ MAIT-like T cells. The fifth chapter will examine the role of Eomes + innate CD8+ T cells in protective memory responses. Chapters three through five will display work that is in preparation to be submitted to a peer-reviewed journal. The sixth chapter will discuss the results of this thesis and their implications.

No description available.

Introduction: Chronic myeloid leukaemia is a blood cancer which progresses from a chronic phase to an acute blast crisis if untreated. Disease progression and treatment resistance may be precipitated by the mutator action of BCR/ABL protein tyrosine kinase (PTK), but only few protein phosphosites involved in the DNA damage response have been investigated with respect to BCR/ABL action. Aim: The aim of this PhD project was to demonstrate that BCR/ABL PTK expression can affect the response to genotoxic stress signalling at the protein phosphorylation level. Methodology: Etoposide-induced DNA damage response has been studied in control and BCR/ABL PTK-expressing Ba/F3 cells using apoptosis and γH2AX assays. Quantitative phosphoproteomics was performed with iTRAQ peptide labelling to discover putative modulated phosphorylation sites. Absolute quantification (AQUA ) performed with selected reaction monitoring was used to validate discovery phosphoproteomics. The effect of genotoxic stress on the THO complex protein Thoc5/Fmip was studied using western blots. Results: The expression of BCR/ABL PTK induced γH2AX phosphorylation after etoposide exposure. This was associated with the modulation of H2AX tyrosine 142 phosphorylation, MDC1 (serines 595 and 1053) and Hemogen serine 380 phosphorylation among proteins regulated by both BCR/ABL PTK and etoposide. We identified that leukaemogenic PTKs mediate Thoc5/Fmip phosphorylation on tyrosine 225 via Src proto-oncogene and oxidative stress, while ATM and MEK1/2 may control its phosphorylation. Human CD34+ CD38- leukaemic stem cells showed pronounced level of THOC5/FMIP tyrosine phosphorylation. Expression of phosphomutant Thoc5/Fmip Y225F might reduce apoptosis mediated by etoposide and H2O2. Conclusion: BCR/ABL PTK can sustain, create, block and change the intensity of protein phosphorylation related to genotoxic stress. Modulation of H2AX, MDC1, Hemogen and Thoc5/Fmip post-translational modifications by BCR/ABL PTK might promote unfaithful DNA repair, genomic instability, anti-apoptotic signalling or abnormal cell differentiation, resulting in leukaemia progression.

<p>Protein tyrosine kinases play an important role in the regulation of various cellular processes such as</p><p>growth, differentiation and survival. GTK, a novel SRC-like cytoplasmic tyrosine kinase, was recently cloned from a mouse insulinoma cell line and the present work was conducted in order to find a biological function of GTK in insulin producing and neuronal cells. It was observed that kinase active GTK-mutants, expressed in RINm5F cells, transferred to the cell nucleus and increased the levels of the cell cycle regulatory protein p27^KIP1, reduced cell growth and stimulated glucagon mRNA expression. Furthermore, wild type GTK induces neurite outgrowth in the rat adrenal pheochromocytoma PC12 cell line, through activation of the RAP1-pathway, suggesting a role of GTK for cell differentiation. Studies using transgenic mice, expressing GTK under the control of the rat insulin 1 promoter, demonstrated a dual role of GTK for β-cell growth: Whereas GTK increases the β-cell mass and causes enhanced β-cell proliferation in response to partial pancreatectomy it also induced β-cell death in response to proinflammatory cytokines and impaired the glucose tolerance in mice treated with the β-cell toxin streptozotocin suggesting a possible role of GTK for β-cell destruction in Type 1 diabetes. We have also observed that GTK-transgenic islets and GTK-expressing RINm5F cells exhibit a reduced insulininduced activation of the insulin receptor substrate (IRS-1 and IRS-2)-pathways, partly due to an increased basal activity of these. GTK was found to associate with and phosphorylate the SH2 domain adapter protein SHB, which could explain many of the GTK-dependent effects both in vitro and in vivo. In summary, the present work suggests that the novel tyrosine kinase GTK is involved in various signal transduction pathways, regulating different cellular responses, such as proliferation, differentiation and survival.</p>

Protein tyrosine kinases play an important role in the regulation of various cellular processes such as growth, differentiation and survival. GTK, a novel SRC-like cytoplasmic tyrosine kinase, was recently cloned from a mouse insulinoma cell line and the present work was conducted in order to find a biological function of GTK in insulin producing and neuronal cells. It was observed that kinase active GTK-mutants, expressed in RINm5F cells, transferred to the cell nucleus and increased the levels of the cell cycle regulatory protein p27KIP1, reduced cell growth and stimulated glucagon mRNA expression. Furthermore, wild type GTK induces neurite outgrowth in the rat adrenal pheochromocytoma PC12 cell line, through activation of the RAP1-pathway, suggesting a role of GTK for cell differentiation. Studies using transgenic mice, expressing GTK under the control of the rat insulin 1 promoter, demonstrated a dual role of GTK for β-cell growth: Whereas GTK increases the β-cell mass and causes enhanced β-cell proliferation in response to partial pancreatectomy it also induced β-cell death in response to proinflammatory cytokines and impaired the glucose tolerance in mice treated with the β-cell toxin streptozotocin suggesting a possible role of GTK for β-cell destruction in Type 1 diabetes. We have also observed that GTK-transgenic islets and GTK-expressing RINm5F cells exhibit a reduced insulininduced activation of the insulin receptor substrate (IRS-1 and IRS-2)-pathways, partly due to an increased basal activity of these. GTK was found to associate with and phosphorylate the SH2 domain adapter protein SHB, which could explain many of the GTK-dependent effects both in vitro and in vivo. In summary, the present work suggests that the novel tyrosine kinase GTK is involved in various signal transduction pathways, regulating different cellular responses, such as proliferation, differentiation and survival.

Protein phosphorylation is an important post-translational modification of proteins, which can either activate or inhibit the function of a given protein. The enzymes, protein kinases and protein phosphatases catalyze the phosphorylation and dephosphorylation of target proteins, respectively. Protein kinases catalyze the transfer of γ-phosphate from ATP to serine, threonine or tyrosine residues in target proteins. They are traditionally classified as protein serine/threonine kinases and protein tyrosine kinases based on the amino acid to which they transfer the phosphate group. Protein tyrosine kinases play vital roles in numerous pathways that regulate growth, development and oncogenesis in animals. However, no protein tyrosine kinase has been cloned so far from plants. The sequence motif, CW(X)6RPXF of sub-domain XI is well conserved among biochemically characterized protein tyrosine kinases from human, rat, mice, worm, fruitfly and Dictyostelium. To seek plant genes encoding tyrosine kinase, we have performed extensive genome-wide analysis of Arabidopsis thaliana using the delineated tyrosine kinase from animal systems. Repetitive database mining with CW(X)6RPXF sequence motif revealed the presence of 57 different protein kinases that have tyrosine kinase motifs. Myosin light chain protein kinase was identified as false positive with this motif. Multiple sequence alignment of all the 57 kinases indicated the presence of twelve conserved sub-domains in their kinase catalytic domain. Out of the 12 sub-domains present in protein kinases, sub-domain VIb confers serine/threonine kinase Specificity and sub-domains VIII and XI confer tyrosine kinase specificity. All the 57 kinases were Verified to contain CW(X) 6RPXF in sub-domain XI. However, the catalytic domain of all the catalogued kinases contain KXXN motif in sub-domain VIb, which is indicative of serine/threonine Kinase specificity. None of the kinases had the tyrosine kinase consensus motif RAA or ARR in sub-domain VIb. Thus, the catalytic domains of all the identified Arabidopsis protein kinases have motifs for serine/threonine specificity in sub-domain VIb and tyrosine kinase motif in sub-domain XI. These results indicate that perhaps all the kinases belong to the dual-specificity kinase family. Hence, we have tentatively named these protein sequences as STY (serine/threonine/tyrosine) protein kinases. To examine the relationships of Arabidopsis STY protein kinases, a topographic cladogram was constructed. Casein kinase 1 was used as an outgroup to perceive the true class of STY protein kinase family. Neighbor joining tree was constructed with the full-length protein sequences following the alignments. Dendrogram of STY protein kinases suggested that the kinases are mainly clustered into four groups. Group I includes kinases related to ATN1-like kinases, peanut STY related kinases, soybean GmPK6-like kinases and ATMRK1-like kinases. Group II consists of MAP3K-like kinases, CTR1 and EDR1 related kinases. Group III includes protein kinases that harbor ankyrin domain repeat motifs. These kinases are related to Medicago sativa ankyrin kinase, MsAPK1. Group IV consists of light sensory kinases that are related to Ceratodon purpureus phytochrome kinase. C. purpureus light sensory kinase has both phytochrome and protein kinase domains. However, the protein kinases of group IV do not have a phytochrome domain. BLAST analysis was performed using CW(X)6RPXF motif against all the available plant sequences in the database. We retrieved 11 rice protein kinases and 14 Dictyostelium kinases. In addition, we obtained STY protein kinases from wheat, barley, soybean, tomato, beech and alfalfa. Dendrogram analysis indicated that the plant STY protein kinases are clustered in similar manner as observed for Arabidopsis. Large number of sequences were retrieved when the tyrosine kinase motif CW(X)6RPXF was used to perform BLAST analysis against all the known sequences from animals. As large numbers of protein tyrosine kinases are available in animals, we have used representative kinases of each family towards the construction of phylogenetic tree. The main difference between the animal and plant tyrosine kinases is in the consensus motif conferring the tyrosine and serine/threonine specificity in the sub-domain VIb. Animal tyrosine kinases have consensus ARR/RAA in sub-domain VIb and plant kinases have KXXN which is indicative of serine/threonine specificity. Expression analysis of Arabidopsis STY protein kinases was performed using Genevestigator online search tool Meta-Analyzer. Genes were grouped based on their relative expression levels during a specific growth stage, in a particular organ or following different environmental stresses. Various kinases are highly expressed in stamens and seeds while some kinases are expressed ubiquitously. A number of biotic and abiotic factors upregulated plant STY protein kinases. Gene expression data suggests the importance of STY protein kinases in plant growth and development. Genome-wide analysis is supported by phosphoproteomics in Arabidopsis seedlings. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, phosphoamino acid analysis and peptide mass fingerprinting. Alkaline treatment detected two proteins corresponding to 46 and 37.5 kD. Phosphoamino acids analysis confirmed their dual-specificity nature. MALDI mass spectrometry and peptide mass fingerprinting analysis identified these two proteins as ATN1 and peanut serine/threonine/tyrosine protein kinase like protein from Arabidopsis. To further support the in silico approach, we have overexpressed one of the identified Arabidopsis thaliana serine/threonine/tyrosine protein kinases (AtSTYPK) in E. coli. The recombinant kinase was induced with IPTG and purified by using nickel-nitrilotriacetic acid affinity chromatography. AtSTYPK exhibited a strong preference for manganese over magnesium for kinase activity. The autophosphorylation activity of AtSTYPK was inhibited by the addition of calcium to reaction buffer containing manganese. The rate of autophosphorylation reaction was linear with increasing time and protein concentration. The AtSTYPK phosphorylated histone H1 (type III-S), and myelin basic protein (MBP) in substrate phosphorylation reaction and it did not phosphorylate casein or enolase. To see whether calcium or magnesium inhibits phosphorylation of MBP, we have performed the reaction in the presence of combination of different metal ions. The MBP phosphorylation reaction is more efficient in the presence of Mg2++ Mn2+ than Ca2++ Mn2+ under the same conditions. The recombinant kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK harbors a manganese-dependent serine/threonine kinase domain, COG3642. H248 and S265 on COG3642 are conserved in AtSTYPK and the site-directed mutation of H248 to alanine resulted in loss of serine/threonine kinase activity, but the mutation of S265 to alanine showed a slight increase in its kinase activity. The protein kinase activity is regulated by various mechanisms that include autophosphorylation, protein phosphorylation by other kinases and by the action of regulatory domains or subunits. The role of tyrosine residues in the regulation of peanut dual-specificity kinase activity is well documented, but the importance of serine and threonine residues in the regulation of dual-specificity protein kinase is not studied so far. The kinase activity is generally regulated by phosphorylation of one or more residues within the kinase activation loop. The role of threonine residues in the kinase activation loop and the TEY motif of AtSTYPK were investigated in the present study. Four threonine residues in the activation loop and a T208 in the TEY sequence motif were converted to alanine to study their role in the regulation of kinase activity. The protein kinase activity was abolished when T208 and T293 of the activation loop were converted to alanine. Interestingly, the conversion of T284 in the activation loop to alanine resulted in an increase in both auto- and substrate phosphorylations. The mutation of T288 and T291 to alanine had no effect on kinase activity. There are eight serine residues in the kinase catalytic domain of AtSTYPK and surprisingly there is no serine residue in the kinase activation loop. So it is worthwhile to see how phosphorylation of serine residues regulates the dual-specificity protein kinase activity. The role of each serine residue was studied individually by substituting them with alanine. Serines at positions 215, 259, 269 and 315 regulate the kinase activity both in terms of autophosphorylation and substrate phosphorylation of myelin basic protein. The mutation of serine 265 to alanine resulted in slight increase in auto- and substrate phosphorylations, suggesting that it could be autoinhibitory in function. The other serine residues at positions 165, 181 and 360 did not show any change in the phosphorylation status when compared to wild-type AtSTYPK. In conclusion, this data suggests the importance of serine and threonine residues in the regulation of dual-specificity protein kinase activity and emphasizes the complexity of regulation of dual-specificity protein kinases in plants. To summarise, this study suggests that tyrosine phosphorylation in plants could be brought about only by dual-specificity protein kinases that phosphorylate on serine, threonine and tyrosine residues. This raises an interesting possibility that plants lack classical tyrosine kinases. The results provide a first report of manganese-dependent dual-specificity kinase from plant systems. This data also suggests that plant dual-specificity kinases undergo phosphorylation at multiple amino acid residues and their activity is regulated by various mechanisms, suggesting that they could be regulated by different mechanisms at different stages of plant growth and development. However, the role of dual-specificity kinases in planta has to be understood by mutant analysis in order to assign the physiological roles to these kinases. Further studies are needed to identify the upstream kinase(s) and downstream targets. Determination of physiological functions for dual-specificity protein kinases raises an important challenge in future in the area of plant signal transduction.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 192 pages. Includes vita. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.