Class of 2015 Abstract / Objectives: To develop an assay to permit the identification of compounds that can inhibit the interaction between GIT1 and the amino-terminal SH3 domain (SH3-N) of GRB2. Methods: The GIT1 protein was expressed in Sf9 insect cells and purified using Talon resin beads. The SH3-N domain of GRB2 was expressed in the E. coli strain, BL21(DE3)pLysS, and purified using glutathione resin beads. The SH3-N domain was fluorescently tagged on cysteine 32 using Cyanine 3 maleimide. The fluorescence of the assay was measured by using a plate reader with excitation wavelength of 555 nm and emission wavelength of 570 nm. Results: The GIT1 protein was expressed in Sf9 cells and purified using the Talon beads. The SH3-N domain of GRB2 was expressed in BL21 cells and purified from the glutathione resin beads. The SH3-N domain was cleaved from GST by using thrombin, which was engineered into the GST fusion protein and were fluorescently labeled using Cyanine 3 maleimide. Conclusions: The fluorescence polarization assay that will detect the interaction between GIT1 and the SH3-N domain of GRB2 is still under development, but it has progressed towards completion since both components of the assay are in hand.

A preorganised amino acid derivative containing a cyclopropyl constraint was designed to orient an amino acid into its bound conformation. This constrained mimic was determined by ITC to be equally potent to the native Phe derivative. It was found that a more favorable enthalpy of binding was compensated by an equally unfavorable entropy compared to the native ligand. In order to properly ascertain the effects of the cyclopropane constraint, a flexible control containing the same number of heavy atoms was synthesized and tested, and it was found to be at least 200 fold less potent than the constrained analog. However, without structural data of the flexible control, it is difficult to infer if the differences in ligand binding affinity arose from the ligand constraint or some other unknown complexity to binding. We studied the thermodynamic and structural effects of modifying alkyl chains of n-alka(e)nol and phenylalka(e)nol binders to MUP-I by both the removal of a rotor via deletion of a methylene unit and restriction of a rotor via the installation of an internal olefin. In general, we observed that a similar thermodynamic signature accompanies modifications for both the n-alka(e)nol and phenylalka(e)nol ligands: A favorable T[delta][delta]Sºo̳b̳s̳ is compensated by an unfavorable T[delta][delta]Hºo̳b̳s̳ such that T[delta][delta]Gºo̳b̳s̳ for both removal of a methylene and insertion of an internal olefin are unfavorable and equipotent, respectively. The insertion of an internal olefin into an alkyl chain led to significantly more favorable entropies than does methylene removal, yet enthalpy-entropy compensation leads to nearly equipotent binding energetics. However, we did find a strong correlation between [delta]Ho̳b̳s̳° and buried apolar Connolly Surface Area (CSA). The intrinsic free energies of introducing an internal olefin into the n-alkanols and phenylalkanols differ markedly from the observed data. It was observed that intrinsic affinities are more favorable than the observable because a favorable T[delta][delta]S⁰i̳n̳t̳ dominates an unfavorable [delta][delta]Hºi̳n̳t̳. Also, we discovered that the intrinsic entropies of inserting an internal olefin are nearly double that of removing a methylene group, suggesting that the insertion of an internal olefin results in the restriction of more C-C rotors. We have shown through ITC analysis that the added substituents probed in this study provided binding increases to our Grb2 SH2 ligands as expected, but that the thermodynamic driving force of binding affinities depended greatly upon the specific nature and flexible mobility of the ligands in the binding pocket. Through a combination of X-ray and ITC studies it was shown that ligands containing rigid and aromatic functional groups bound with a higher [delta]H° than the more flexible alkyl ligands, and that this effect correlates well with more direct vdW contacts made in the pocket. Finally, we described a case study where a strict adherence to the Topliss operational schemes led to an expedient development of novel MUP-I binding analogs. The validity of the schemes was also depicted through the synthesis and testing of ligands that were correctly predicted to be weaker/equipotent to the starting ligand. Of important note is that the degree to which the schemes led to affinity boost depended greatly on the starting potency of the initial compound. / text

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo. They exhibit unlimited proliferation in culture and have the ability to differentiate into all three germ layers of the developing organism, a property defined as pluripotency. Previously it was reported that growth factor-bound protein 2 (Grb2) is required for differentiation of the epiblast, the embryonic tissue that harbours the pluripotent founder cells of the foetus. GRB2 is an adapter protein involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in response to extracellular signals. It has also been implicated in the activation of the phosphoinositol-3-kinase (PI3K) pathway in response to fibroblast growth factor (FGF) signaling. The work presented in this thesis examines the role of Grb2 in ES cells and describes previously unreported contributions of this adaptor protein in regulating ES cell growth and differentiation. It has been previously been shown by others that Grb2 deficient (Grb2-/-) cells grow relatively normally in ES growth medium containing serum. However, in serum free conditions (N2B27 medium) in this project, proliferation of Grb2-/- cells is reduced compared with wild type and “restored” Grb2-/- cells stably expressing a Grb2 cDNA mini gene. Under serum free conditions, Grb2-/- cells grow in tight, refractive colonies. Nanog expression was uniformly upregulated, in contrast to the heterogeneous pattern reported in serum-based medium. Colony expansion on the substratum appears to be compromised, although there is no apparent defect in the initial attachment of Grb2-/- cells. Cell cycle analysis indicates that the slower growth of Grb2-/- cells in serum free medium could be due to lengthening of the G1 phase of the ES cell cycle. In an attempt to identify the signalling deficiency responsible for the growth defect of Grb2-/- cells, MAPK activation was restored by two methods, PMA a ligand that bypasses the requirement for Grb2, and Raf-ER, a conditionally regulated component of the MAPK pathway that acts downstream of Grb2 in the MAPK pathway. Although both approaches increased MAPK signalling they were unable to rescue the growth defect. This suggests that MAPK is not required or alone is not sufficient. Inhibition of Glycogen synthase kinase 3 β (GSK3 β ) is known to augment growth of ES cells under MAPK inhibition. Surprisingly, GSK3 β inhibition did not enhance Grb2-/- cell growth. Under GSK3 β inhibition, Grb2-/- ES cells fail to thrive. It is hypothesised that under these conditions cells undergo hyper-self-renewal at the cost of growth. Grb2-/- ES cells are reported to exhibit limited differentiation potential. To examine the potency of Grb2-/- cells, these cells were subjected to embryoid body (EB) and monolayer differentiation. Analysis of EBs showed a loss of Gata4, Gata6 and endoderm marker gene expression. However, markers of ectoderm (Sox1, Pax6, MAP2), the late epiblast/nascent mesoderm (Brachyury) and markers associated with gastrulation (Twist and Snail) were expressed. Outgrowths of morphologically and immunohistochemically identifiable neuronal cells confirmed differentiation of ectodermal cell types, indicating Grb2 is not required for neuronal differentiation. However, beating cardiomyocytes could not be identified in Grb2-/- EBs, though readily found in restored Grb2-/- cells expressing the Grb2 cDNA. This suggests that there is an essential role for Grb2 in the mesoderm/cardiomyocyte differentiation pathway. This may be due to a defect in GATA factor expression since these factors are essential for cardiogenesis. In serum-free monolayer differentiation, Grb2-/- cells formed neuronal cells. Additional inhibition of the MAPK pathway using a small chemical inhibitor failed to prevent this differentiation. However, biochemical analysis of the cells indicates that this occurs when ERK activation is very low, indicating differentiation was not MAPK-independent. Grb2 mediates FGF-MAPK induced exit from the naïve ground state. These data suggest a Grb2-independent pathway can also facilitate this transition. Grb2 is dispensable for differentiation in to some lineages. However as differentiation of Grb2-/- ES cells is restricted, this indicates Grb2 is required for true pluripotency.

A series of phosphotripeptide ligands for the Grb2 SH2 domain was designed and synthesized, each of which derived from the minimal consensus sequence required for binding: Ac-pYXN. The binding affinity and related thermodynamic parameters were determined by isothermal titration calorimetry. Both the size and connectivity of the side-chain was varied. The consequences of incorporating α,α-disubstitution at the pY+1 residue on binding thermodynamics were evaluated, as were the effects of constraining the side-chains in a ring. The series was evaluated from a number of perspectives: (1) increasing size of the pY+1 residue by utilizing various amino acid types: monoalkyl, dialkyl, or cycloalkyl; (2) comparisons between ligands with the same number of carbons (scission control); and (3) by comparing ligands incorporating cyclic pY+1 residues with those incorporating α,α-dialkyl residues with one fewer methylene group (excision control). Inconsistencies in the thermodynamic consequence of constraining the backbone were observed within this set of ligands, which reveal the limitations of our understanding of protein-ligand interactions. Aspects of both the classical and non-classical hydrophobic effect were observed, but the occurance of one over the other could not be explained. / text

We investigated the role of Shc¡BPYK2¡BGrb2-sos binding complex¡BRas and Raf proteins at the rostral ventrolateral medulla (RVLM), the origin of sympathetic neurogenic vasomotor tone, in mevinphos (Mev) intoxication. Adult Sprague-Dawley rats anesthetized by sodium pentobarbital (45 mg/kg) and maintained by propofol (20-25 mg/kg/hr) were used. Bilateral microinjection of Mev (10 nmol) into the RVLM elicited two distinct phases of cardiovascular responses, designated Phase I (sympathoexcitatory) and Phase II (sympathoinhibitory) Mev intoxication. Pretreatment with microinjection of a phospho-Shc-tyrosine 317, phospho-PYK2-tyrosine 402, phospho-PYK2-tyrosine 579/580 antibody (1:20), Grb2-sos complex inhibitor (SH3b-p), Ras specific inhibitors (manumycin A or FTA) or Raf specific inhibitor (GW5074) into the bilateral RVLM blunted the magnitude of the Mev-elicited sympathoexcitatory cardiovascular effect without affecting the duration. The Mev-elicited sympathoinhibitory cardiovascular effect was not influenced. Our results suggest that signaling pathways that involve Shc, PYK2, Grb2-sos complex, Ras or Raf protein in the RVLM participate in the sympathoexcitatory phase of Mev intoxication.

Le gène ShcA code pour trois isoformes de protéines adaptatrices (p46, p52 et p66Shc), possédant entre eux une forte homologie structurelle. Les études antérieures ont attribué un rôle essentiel à l'isoforme p52Shc dans l'activation de la voie mitogénique des MAPK, ainsi que dans la transformation cellulaire en aval des récepteurs de type tyrosine kinase (RTK). L'activation des MAPK fait intervenir une liaison directe de p52Shc aux RTK suivant leur activation, ce qui permet la phosphorylation sur tyrosines de Shc et ainsi, le recrutement de la protéine adaptatrice Grb2. Pendant longtemps, ces fonctions ont été attribuées aux trois isoformes des protéines Shc. Cependant, il est maintenant évident que Pisciforme p66Shc joue des rôles distincts de p52Shc. En effet, l'expression de p66Shc n'induit pas la transformation cellulaire et inhibe l'activité des MAPK en aval des RTK. D'ailleurs, p66Shc induit l'apoptose en réponse aux stress oxydatifs, par le biais de la phosphorylation d'une sérine située dans son domaine unique en N-terminal. Bien que ces caractéristiques proposent un pouvoir anti-tumorigénique à p66Shc, son rôle dans la progression du cancer demeure controversé. Mes travaux de recherche démontrent que p66Shc est constitutivement associé au récepteur dii facteur de croissance des hépatocytes, le RTK Met, et diminue fortement l'interaction entre le récepteur Met actif et Grb2. Nos études in cellulo et in vivo dans un modèle de cellules épithéliales intestinales transformées par Tpr-Met (Tpr-Met-IEC-6), une forme oncogénique du récepteur Met, révèlent que l'expression de p66Shc induit l'apoptose à l'atteinte de la confluence et diminue la croissance tumorale, dépendamment de sa phosphorylation sur sérine. En revanche, p66Shc augmente la capacité de ces cellules à survivre sans ancrage à la matrice extracellulaire et favorise la formation de métastases. En conclusion, nous démontrons qu'une protéine Shc s'associe à un RTK indépendamment de son activation. Il s'agit de la première évidence que les modes d'interaction de p66Shc avec les RTK different de ceux caractérisés pour p52Shc. De plus, nos études permettent d'illustrer la dualité fonctionnelle de p66Shc, où son expression diminue la capacité tumorigénique des cellules en adhésion, mais augmente leur potentiel métastatique lorsqu'elles sont en suspension, dénotant le rôle controversé de p66Shc dans le cancer. Cette nouvelle compréhension des rôles distincts associés à p66Shc selon le contexte de la cellule cancéreuse permet de cibler différentes étapes du développement du cancer.

CD4+ T cells are critical in the fight against parasitic, bacterial, and viral infections, but are also involved in many autoimmune and pathological disorders. Ligation of the T Cell Receptor (TCR) is the primary signal required for T cell activation proliferation, differentiation and cytokine release. Upon TCR activation, several kinases and adaptor proteins are assembled at the TCR/linker for activation of T cells (LAT) signaling complexes, a process indispensable for optimal signal transduction. One important group of proteins recruited to the TCR/LAT complexes is the GRB2 family of adaptors. Due to their role in mediating signaling complexes, the GRB2 family of adaptors are critical for development, proliferation, and survival of diverse cell types. These proteins have been linked to the initiation and progression of numerous pathological conditions including diabetes, asthma/allergy, and solid and hematopoietic malignancies. Therefore, it is essential to characterize and understand the complete functions of these proteins for the generation of safe and efficient targeting treatments for diseases mediated by these proteins. In T cells, GRB2 and its homologs, GADS and GRAP, are crucial for the propagation of signaling pathways through the TCR and adaptor protein LAT. These proteins recruit distinct sets of proline-rich ligands to LAT thereby inducing multiple signaling pathways such as MAP kinase activation, calcium influx and cellular adhesion. However, the role of GRB2 family members in controlling TCR and LAT mediated signaling in mature human T cells is not completely understood. Moreover, the relative role of GRB2 family members in the extent and timing of the recruitment of SH3 domain ligands to the LAT complex is unknown. Our hypothesis is that these proteins recruit distinct sets of ligands to the LAT complex that can drive differential downstream signaling events. As presented in CHAPTER III, we developed microRNA and shRNA targeting viral vectors to effectively inhibit the expression of GRB2 and GADS in human CD4+ T cells to examine the role of these adaptors in mature human T cells. We also established optimized protocols for high efficacy retro or lentiviral transduction of human T cell lines, activated and "hard-to-transduce" non-activated primary human CD4+ T cells. In CHAPTER IV, we demonstrate the requirement for GRB2 in TCR-induced IL-2 and IFN-γ release. The defects in cytokine release in the absence of GRB2 were attributed to diminished formation of LAT signaling microclusters, which resulted in reduced MAP kinase activation, calcium flux and PLC-γ1 recruitment to LAT signaling clusters. Overall, the data presented in this chapter demonstrate that the ability of GRB2 to facilitate protein clustering is as important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes. In CHAPTER V, we describe the role for GADS in mediating TCR-induced IL-2 and IFN-γ production. GADS was critical for the recruitment of SLP-76 and PLC-γ1 to the LAT complex and subsequent calcium influx. We also show, in contrast to the current paradigm, that recruitment of GADS/SLP-76 complexes to LAT is not required for TCR-mediated adhesion and cytoskeletal arrangement. Overall, our studies reveal novel mechanisms for the role of GRB2 family members in TCR-mediated signaling. They also provide insight into the mechanisms that regulate growth factor, cytokine and insulin receptors. Importantly, studies presented in this thesis will help us understand the mechanisms of T cell activation and highlight potential new therapies for T cell-mediated diseases, including leukemia, lymphomas, autoimmune disorders and cardiovascular disease.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 155-195.

No description available.

Activated protein tyrosine kinases, which have been implicated in the genesis of a number of human cancers, rely on a variety of protein-protein interactions to transmit their proliferative signals within the cell. These interactions are often mediated by Src homology 2 and 3 (SH2 and SH3) domains. A class of proteins which are mainly composed of such domains, termed adaptor proteins, has been identified. The Growth factor receptor bound proteins Grb2 and Grb7 are SH2 domain adaptor proteins which have been shown to associate directly or in complex with many tyrosine kinases, including the c-ErbB-2/Neu receptor tyrosine kinase. While overexpression of either protein alone in rat fibroblasts is not transforming, human breast cancer cell lines exhibit Grb2 and Grb7 gene amplification, and mRNA and protein overexpression. The role of Grb2 in polyomavirus middle T antigen-mediated mammary tumorigenesis has been examined utilizing gene targeting and transgenic approaches. Initial characterization of the progeny of matings involving Grb2+/mice and MMTV/middle T transgenic mice indicated that delayed tumor kinetics may be the result of Grb2 dosage differences between mT+;Grb2+/-and mT+;Grb2+/+ animals. Transgenic animals expressing a dominant negative version of Grb2 in the mammary epithelium have been generated to explore an alternate method for disrupting signaling from middle T antigen. The role of Grb2 and Grb7 in Neu-mediated mammary tumorigenesis is also being examined. Both MMTV/Grb2 and MMTV/Grb7 transgenic mice that express the transgene in the mammary epithelium have been identified by ribonuclease protection analysis. Matings involving these strains and MMTV/neu mice should aid in determining the effects of overexpressing Grb2 or Grb7 on Neu-mediated mammary tumorigenesis. / Thesis / Master of Science (MS)