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Tau associates with protein tyrosine phosphatase SHP2Kim, Yohan 01 May 2017 (has links)
The microtubule-associated protein tau normally functions to bind to and stabilize microtubules. However, evidence now indicates that tau may also play a critical role in signaling pathways linked to neuronal development and neurodegeneration. The tau association with numerous signaling proteins such as tyrosine kinases, adaptor proteins, and scaffold proteins support this hypothesis. Phospho-Y18 tau was previously found in Alzheimer’s disease (AD) brain. Interestingly, this phosphorylation appeared to be regulated during neurodegeneration possibly by a tyrosine phosphatase(s). Identifying a candidate phosphatase, our lab found the association between tau and SHP2 in a neuronal cell line and dephosphorylation of phospho-Y18 by protein tyrosine phosphatase SHP2 in vitro. Since both tau and SHP2 play a critical role in NGF-induced signaling pathway, these findings raised the possibility that the tau-SHP2 association has a role in NGF signaling.
The aim of this dissertation research is to characterize the tau-SHP2 association and its role in neuronal signaling. Here, we provide evidence that tau phosphorylation is not required for SHP2 association but significantly enhances the interaction. The SHP2 binding region of tau napped to residues 256-273, which contain the microtubule binding repeat 1 of tau. Using in situ proximity ligation assay (PLA), we also showed the presence of endogenous tau-SHP2 and tau-activated SHP2 complexes in neuronal cells. The number of complexes was increased in the cells in response to NGF. Our PLA data also showed the localization of these complexes to actin ruffles. In NGF signaling, we showed that phosphorylation at T231 of tau was necessary for the increase in tau-SHP2 association. Lastly, we provide evidence that tau-SHP2 complexes are present in mouse primary neuronal cultures and mouse brain sections. Together, these findings show a role for tau phosphorylation in SHP2 binding and a potential role for tau-SHP2 interaction in neuronal signal transduction. Based on our findings, we speculate that there is a role for tau-SHP2 association during early brain development and in neurodegenerative disease.
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Requirement of integrin [alpha]5[beta]1 and tyrosine phosphorylation of SHC for prohb-EGF release by GPR30, a seven transmembrane receptor for estrogen /Quinn, Jeffrey Alan. January 2006 (has links)
Thesis (Ph. D.)--University of Rhode Island, 2006. / Typescript. Includes bibliographical references (leaves 104-121).
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Transcription Cofactor LBH is a Direct Target of the Oncogenic WNT Pathway with an Important Role in Breast CancerRieger, Megan Elizabeth 14 July 2010 (has links)
Limb-Bud and Heart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of functional TCF/LEF binding sites in the LBH locus and rapid beta-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/beta-catenin signaling is inhibited by Wnt7a, which in limb development signals through a non-canonical pathway involving Lmx1b. Furthermore, we show that Lbh is aberrantly overexpressed in mammary tumors of MMTV-Wnt1 transgenic mice and in aggressive basal-subtype human breast cancers that display Wnt/beta-catenin hyperactivation. Deregulation of LBH in human breast cancer appears to be Wnt/beta-catenin dependent as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. RNAi mediated knockdown of LBH in basal breast cancer cell lines resulted in loss of CD44high/CD24low tumor cells, luminal differentiation, reduced cell growth, reduced colony forming ability, and increased apoptosis, suggesting a novel pro-survival and stem cell maintenance function of LBH in breast cancer. Reciprocal overexpression studies in the basal breast carcinoma line BT549 resulted in increased tumorigenicity in vitro, suggesting that LBH overexpression is indeed oncogenic. Finally, we further characterized LBH protein expression patterns and post-transcriptional regulation. Collectively, this thesis demonstrates that LBH is a direct Wnt target gene in both development and basal breast cancer that promotes the undifferentiated phenotype and survival of basal breast tumor cells.
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Muc4, the Integral Membrane Modulator of ErbB2: The Effects of Muc4 Expression on ErbB2 and ErbB3 Phosphorylation, Receptor Levels and Sub-Cellular Localization In Breast Cancer Cells Treated With NeuregulinBoothe, Patricia 19 August 2010 (has links)
Muc4, a heterodimeric transmembrane mucin containing EGF-like domains, has been described as an ErbB2-binding protein which modulates signaling via the ErbB2-ErbB3 pathway. In Muc4-transfected MCF-7 cells, Muc4 expression resulted in alteration of both the time course and phosphorylation levels of NRG beta 1 induced phosphorylation and activation of both ErbB2 and ErbB3. Muc4 significantly enhanced the autophosphorylation of ErbB2 over the early (defined 0-30 min) and intermediate (30-120 min) NRG beta 1 treatment times at three sites, Y1248, Y1221 and Y1139. The sites displayed differential maximal phosphorylation times. At Y1248 and Y1139, maximal phosphorylation occurred entirely during the early treatment phase. However, Y1221/2 showed maximal phosphorylation during the intermediate phase with a smaller peak during the early phase. The ratio of phosphorylated ErbB3 and total receptor level was significantly enhanced (in cells that expressed Muc4 compared without Muc4) over both the early and intermediate NRG beta 1 treatment time at the Y1289 site. This motif is one of several similar ErbB3 motifs whose phosphorylation mediates the binding of PI3-kinase. This phospholipid kinase is a key modulator of numerous cellular pathways leading to proliferation, motility and survival. Aberrancies in the ErbB2-ErbB3 signaling pathway have been implicated in the aggressive behavior of tumor cells, and the identification and characterization of modulators of this pathway are being sought as targets of potential therapeutic interventions. Muc4 significantly enhanced activated ERK in the absence of NRG beta 1 treatment while a NRG beta 1 mediated activation of AKT was observed. At early NRG beta 1 treatment time phases, Muc4 co-localized with phosphorylated ErbB2 (pY1248) independent of NRG beta 1 treatment; co-localization of Muc4 and ErbB2 receptor (activated/receptor forms) was observed at the apical surface or around the cell surface membrane. These data provide evidence in the Muc4-transfected MCF-7 cells for the biological NRG beta 1 mediated ErbB2 and ErbB3 activation. Our data suggests that Muc4 affects steady state phosphorylation levels and duration of the phosphorylation signal of both ErbB receptors, and that NRG beta 1 might affect ErbB2 and ErbB3 signaling differently. Additionally, the results of the timing of phosphorylation studies suggest the possibility that temporal aspects of phosphorylation at different sites may determine the pathways activated preferentially in the subsequent signaling cascades.
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Characterization of NimA-related Kinase 10 (NEK10): A Role in Checkpoint ControlMoniz, Larissa 31 August 2010 (has links)
Deregulation of the cell cycle is a hallmark of neoplastic transformation and plays a central role in both the initiation and progression of cancer. Members of the NimA-related kinase (NEK) family of protein kinases are emerging as important players in regulation of the eukaryotic cell cycle during normal cell cycle progression and checkpoint activation in response to genotoxic stresses. The focus of this thesis is NEK10, a previously uncharacterized member of the NEK family. While little is known about the biology of NEK10, recent cancer genomics studies have identified NEK10 as a candidate susceptibility gene at chromosome 3p24 in cancer.
Work herein describes a role for NEK10 in the cellular response to ultraviolet (UV) irradiation. NEK10 was required for the activation of ERK1/2 signaling upon UV irradiation, but not in response to mitogens, such as the epidermal growth factor. NEK10 interacted with Raf and MEK and enhanced MEK activity through a novel mechanism involving MEK autoactivation. Significantly, appropriate maintenance of the G2/M checkpoint following UV irradiation required NEK10 expression and ERK1/2 activation. In support of a conserved role for NEK10 in the cellular response to UV irradiation, nekl-4, the NEK10 C.elegans homologue, affected embryonic sensitivity to UV-irradiation.
In search of regulatory inputs into NEK10, using mass spectrometry, our laboratory identified 19 distinct sites of NEK10 phosphorylation. Characterization of a number of these sites revealed a role for intermolecular autophosphorylation in achieving full NEK10 catalytic activity through activation loop phosphorylation on S684 and S688. Further, a C-terminal phosphorylation site on NEK10, S933, was found to be a 14-3-3 binding site, and was essential for NEK10 cytoplasmic to nuclear translocation following UV irradiation.
Taken together, my studies have discovered a role for NEK10 in the engagement of the G2/M cell cycle checkpoint and provided a mechanistic insight into the relationship between NEK10 and the Raf/MEK/ERK cascade, and the control of NEK10 subcellular localization. This work will serve as a foundation for future studies aimed at understanding the molecular mechanism of NEK10 action and its function in development and tumourigenesis.
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Characterization of NimA-related Kinase 10 (NEK10): A Role in Checkpoint ControlMoniz, Larissa 31 August 2010 (has links)
Deregulation of the cell cycle is a hallmark of neoplastic transformation and plays a central role in both the initiation and progression of cancer. Members of the NimA-related kinase (NEK) family of protein kinases are emerging as important players in regulation of the eukaryotic cell cycle during normal cell cycle progression and checkpoint activation in response to genotoxic stresses. The focus of this thesis is NEK10, a previously uncharacterized member of the NEK family. While little is known about the biology of NEK10, recent cancer genomics studies have identified NEK10 as a candidate susceptibility gene at chromosome 3p24 in cancer.
Work herein describes a role for NEK10 in the cellular response to ultraviolet (UV) irradiation. NEK10 was required for the activation of ERK1/2 signaling upon UV irradiation, but not in response to mitogens, such as the epidermal growth factor. NEK10 interacted with Raf and MEK and enhanced MEK activity through a novel mechanism involving MEK autoactivation. Significantly, appropriate maintenance of the G2/M checkpoint following UV irradiation required NEK10 expression and ERK1/2 activation. In support of a conserved role for NEK10 in the cellular response to UV irradiation, nekl-4, the NEK10 C.elegans homologue, affected embryonic sensitivity to UV-irradiation.
In search of regulatory inputs into NEK10, using mass spectrometry, our laboratory identified 19 distinct sites of NEK10 phosphorylation. Characterization of a number of these sites revealed a role for intermolecular autophosphorylation in achieving full NEK10 catalytic activity through activation loop phosphorylation on S684 and S688. Further, a C-terminal phosphorylation site on NEK10, S933, was found to be a 14-3-3 binding site, and was essential for NEK10 cytoplasmic to nuclear translocation following UV irradiation.
Taken together, my studies have discovered a role for NEK10 in the engagement of the G2/M cell cycle checkpoint and provided a mechanistic insight into the relationship between NEK10 and the Raf/MEK/ERK cascade, and the control of NEK10 subcellular localization. This work will serve as a foundation for future studies aimed at understanding the molecular mechanism of NEK10 action and its function in development and tumourigenesis.
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Phosphorylation sites of HPrNapper, Scott 01 January 1999 (has links)
The histidine-containing protein (HPr) is a central phosphotransfer component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) that transports carbohydrates across the cell membrane of bacteria. There are two HPr phosphorylation events investigated in this thesis. Firstly, BPr from Gram-positive species may undergo a regulatory phosphorylation of an absolutely conserved Ser46 residue. There are numerous metabolic consequences to this phosphorylation, including inducer exclusion and expulsion, inhibition of PTS sugar uptake and catabolite repression. While HPr from Gram-negative sources cannot undergo phosphorylation of Ser46 'in vivo' or ' in vitro' it is possible to mimic the phosphorylation through the Ser46Asp mutation. To determine the structural consequences of the mutation the crystallographic structure of the 'E. coli'. Ser46Asp HPr was determined at 1.5 Å resolution. The structure revealed that no significant structural rearrangements are induced by the mutation and the inability to accept phosphotransfer from Enzyme I is due to electrostatic disruption of the interaction of these proteins. Phosphorylation of an absolutely conserved His15 for the purpose of phosphotransfer represents the second phosphorylation event to be investigated. The absolute requirement for histidine at the 15 position was investigated through mutagenesis. The mutation of His15Asp of 'E. coli' HPr was able to accept a phosphoryl group from Enzyme I and further transfer the phosphoryl group to Enzyme IIAglc. None of the other mutations of the fifteen position were able to be phosphorylated. The His15Asp mutant had a Vmax of 0.1% and a ten-fold increase in Kin with respect to wild type HPr. As a consequence of the phosphorylation of His15Asp HPr a third protein species of higher pI than the original protein was identified. This high pI species seemed to share numerous similarities to succinimides which are known to be involved in deamidation. The inability to detect the known degradation products of succinimides suggested that the high pI species may involve isoimide formation. Isoimides have been proposed, but never experimentally demonstrated in proteins. A mechanism through which the phosphoacyl intermediate may catalyze isoimide formation is proposed. In addition the potential involvement of isoimide formation as a mechanism in physiological regulatory signaling is discussed.
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Phosphorylation Bar Codes Induce Distinct Conformations and Functionalities of beta-ArrestinNobles, Kelly Nicole January 2010 (has links)
<p>Seven transmembrane spanning receptors (7TMRs), or G-protein coupled receptors (GPCRs), represent the largest and most ubiquitous of the several families of plasma membrane receptors and regulate virtually all known physiological processes in humans. The classical paradigm of signal transduction in response to 7TMR stimulation involves an agonist-induced conformational change of the receptor which leads to interaction with and dissociation of the heterotrimeric G-protein into independent Galpha and Gbeta;gamma signaling subunits. Following their activation, 7TMRs are phosphorylated by G-protein coupled receptor kinases (GRKs) and subsequently recruit beta-arrestins. beta-arrestins are multifunctional adaptor proteins which not only desensitize G-protein signals, but also facilitate receptor internalization and mediate numerous signaling pathways on their own. As beta-arrestins universally interact with members of the 7TMR superfamily, we (1) developed an in vitro model system to assess conformational changes that occur in beta-arrestins in response to phosphorylation and (2) to map the sites of phosphorylation on the beta2 adrenergic receptor by different GRKs which would determine the conformation(s) assumed by beta-arrestin and thereby, in turn, instruct its functional capabilities. </p><p>We determined conformational changes in beta-arrestin1 in vitro using limited tryptic proteolysis and MALDI-TOF MS analysis in the presence of a phosphopeptides derived from the C-terminus of the V2 vasopressin receptor (V2Rpp or V2R4p) or the corresponding unphosphorylated peptide (V2Rnp). Upon V2Rpp binding, we show that the previously shielded R393 becomes accessible, which indicates release of the C-terminus. Moreover, we have shown that R285 becomes more accessible and this residue is located in a region of β-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1. We also show a functional consequence of the release of beta-arrestin1's C-terminus by enhanced clathrin binding. In addition, we have shown marked protection of beta-arrestin1's N-domain in the presence of V2Rpp; consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. Using a differentially phsophorylated V2R peptide (V2R4p), we show that beta-arrestin1 is able to adopt distinct conformations in response to different phosphorylation patterns. Futhermore, a striking difference is observed in the conformation of V2Rpp-bound beta-arrestin1 when compared to beta-arrestin2, namely the flexibility of the inter-domain hinge region. These data represent the first direct evidence that the beta-arrestin1 conformation is differentially instructed by phosphorylation patterns and that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.</p><p>Phosphorylation of 7TMRs by GRKs plays essential roles in regulation of receptor function by promoting interactions of the receptors with beta-arrestins. We hypothesized that different GRKs phosphorylate distinct sets of sites thereby establishing a "bar code." In order to test this hypothesis, we monitored the phosphorylation events of the beta2AR upon stimulation with a classical full agonist, isoproterenol, or a beta-arrestin "biased" agonist, carvedilol, in the presence of a full complement of GRKs or when individual GRKs (2 or 6) were depleted by siRNA. We demonstrate that at least thirteen sites on the beta2AR show changes in phosphorylation in response to the agonist isoproterenol. Of these, phosphorylation increased 10 to more than 300 fold in 12 (S261, S262, S345, S346, S355, S356, T360, S364, S396, S401, S407 AND S411) and decreased 50% in one (S246). Depletion of GRK2 or 6 by siRNA indicates that S355, 356 are GRK6 sites whereas the remainder are GRK2 sites. Phosphorylation of GRK2 sites inhibits that of GRK6 sites. Carvedilol, a beta-arrestin biased agonist, promotes phosphorylation of only the GRK6 sites S355, 356. In HEK293 cells, GRK2 phosphorylation is found to be the major positive regulator of receptor internalization; to contribute to receptor desensitization; and to inhibit beta-arrestin mediated ERK activation. Phosphorylation of the two GRK6 sites contributes to receptor desensitization and internalization and is required for beta-arrestin mediated ERK activation. These data indicate that different ligands promote distinct patterns of receptor phosphorylation which dictate different patterns of beta-arrestin mediated function.</p> / Dissertation
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The subunit exchange rate of the cyanobacterial circadian clock component kaic is independent of phosphorylation stateIhms, Elihu Carl 15 May 2009 (has links)
The study of the in vitro circadian oscillator of the cyanobacterium
Synechococcus elongatus has uncovered a complex interplay of its three protein
components. Synchronization of the clock's central oscillatory component, KaiC, has
been thought to be achieved through subunit shuffling at specific intervals during the
clock’s period. By utilizing an established fluorescence-based analysis on completely
phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has
shown that shuffling rates are largely unaffected by phosphorylation state. These
findings conflict with previous reports and hence revise our understanding of this
oscillator.
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The subunit exchange rate of the cyanobacterial circadian clock component KaiC is independent of phosphorylation stateIhms, Elihu Carl 10 October 2008 (has links)
The study of the in vitro circadian oscillator of the cyanobacterium
Synechococcus elongatus has uncovered a complex interplay of its three protein
components. Synchronization of the clock's central oscillatory component, KaiC, has
been thought to be achieved through subunit shuffling at specific intervals during the
clock's period. By utilizing an established fluorescence-based analysis on completely
phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has
shown that shuffling rates are largely unaffected by phosphorylation state. These
findings conflict with previous reports and hence revise our understanding of this
oscillator.
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