Reproduction de : Thèse de doctorat : Pharmacie. Chimie thérapeutique : Nantes : 2008. / Bibliogr.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Toledo, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Title from title page of PDF document. Bibliography: pages 64-70, 104-108, 121-158.

Thesis (Ph. D.)--Cornell University, May, 1998. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [78]-93).

Dissertation (Ph.D.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 138-150.

ABSTRACT Phosphorylation is a post-translational modification important for regulating protein activity and protein binding capacity. It is used in many different signaling pathways within the cell. Src Family Kinases and Protein Kinase A (PKA) are two prototyptical non-receptor tyrosine and serine/ threonine kinases, respectively, which are found in canonical signaling pathways. These two kinases are critical for signaling in essentially every cell of a multicellular organism, and are particularly important in development, cell migration and proliferation. Although both proteins have been intensely studied for many decades, an understanding of the molecular mechanisms which govern their regulation and the regulation that they effect on other proteins are still being elucidated. Fyn, like its related Src Family Kinase members, has previously been shown to be regulated by two tyrosine phosphorylation events at residues Y420 and Y531. Y420 is located in the kinase (Src Homology 1(SH1)) domain and it is a highly-characterized intermolecular autophosphorylation site that increases the activity of the kinase. Y531 is located near the C-terminus and is phosphorylated by C-terminal Src kinase (Csk). Phosphorylation of Y531 allows it to bind to R176 in the SH2 domain in an intramolecular fashion. In this conformation Fyn has only basal activity. Since these sites are essential for regulating the activity of the kinase, we hypothesized that four novel sites of tyrosine phosphorylation in Fyn could also importantly regulate the protein. Three of the novel sites lie in the SH2 domain, and one is located in the kinase domain. Mass spectrometry, in vitro kinase assays, as well as western blot analysis aided in uncovering that these novel Fyn phosphorylation sites fine tune the activity and substrate binding of the protein. PKA has been implicated in a multitude of signaling pathways and is particularly important in cell growth, proliferation, and migration. Fyn and PKA have classically been considered to be in separate signaling pathways. However, research over the past several decades has provided evidence that there is crosstalk that exists between the two pathways. The SFK Fyn and PKA can phosphorylate each other, thereby regulating each other's activity. Based on these data, we hypothesized the existence of downstream effectors of this relatively uncharacterized pathway. It was hypothesized that the presence of Fyn could lead to PKA activation and to differences in PKA binding partners. Through the use of co-immunoprecipitations, Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and quantitative mass spectrometry, many proteins were found to increase their binding to PKA in the presence of Fyn. Several proteins were selected and further biochemically validated. These data suggest that the presence of Fyn could allow for PKA to more importantly interact with discrete pools of proteins within the cell to effectuate its signal transduction. Together these studies provide understanding on critical and fundamental processes by which all cells function.

The non-receptor tyrosine kinase Src has been found to be overexpressed and activated in many human cancers, where it has been implicated in changes in cellular proliferation, adhesion, migration, apoptosis, angiogenesis, and tumour growth. In addition, several other members of the Src family have also been implicated in various cancer phenotypes. Our examination of a wide panel of colon, breast, and lung cancer cell lines revealed that not only Src, but also Yes, Fyn, Lyn, and Lck, were expressed at both the mRNA and protein levels in different combinations, and at varying levels, between cell lines. When examined for kinase activity, it was discovered that only a subset of the expressed Src family members had detectable kinase activity within a given cell line. To investigate the involvement of the Src family members in the proliferation, adhesion, migration, and colony forming ability of four selected cancer cell lines, both Src family kinase inhibitors, which inhibit the kinase activity of multiple Src family members, and RNA interference, which selectively decreases the expression of individual proteins, were used. It was found that the involvement of these proteins in all of the cellular processes investigated was cell line-specific, with the greatest effects observed in HT29 cells, which have relatively high Src protein levels and kinase activity. Furthermore, the consequences of Src family member inhibition were also inhibitor specific, as treatment with PP2 and SKI I generally had greater effects on the cellular processes examined than did treatment with SU6656 or SKI II. It was also found that the inhibition of multiple Src family kinases by at least one of the inhibitors generally resulted in greater effects on the cancer cell phenotypes investigated than were observed when the expression of Src, Fyn, or Yes was decreased using RNA interference. This suggests that multiple Src family members may need to be targeted in order to inhibit the increased proliferation, cell-matrix adhesion, migration, and colony forming ability exhibited by cancer cells. The identification of the cancer cell phenotypes in which particular Src family members are involved is of interest, as these proteins are attractive targets for cancer therapy.

The non-receptor tyrosine kinase Src has been found to be overexpressed and activated in many human cancers, where it has been implicated in changes in cellular proliferation, adhesion, migration, apoptosis, angiogenesis, and tumour growth. In addition, several other members of the Src family have also been implicated in various cancer phenotypes. Our examination of a wide panel of colon, breast, and lung cancer cell lines revealed that not only Src, but also Yes, Fyn, Lyn, and Lck, were expressed at both the mRNA and protein levels in different combinations, and at varying levels, between cell lines. When examined for kinase activity, it was discovered that only a subset of the expressed Src family members had detectable kinase activity within a given cell line. To investigate the involvement of the Src family members in the proliferation, adhesion, migration, and colony forming ability of four selected cancer cell lines, both Src family kinase inhibitors, which inhibit the kinase activity of multiple Src family members, and RNA interference, which selectively decreases the expression of individual proteins, were used. It was found that the involvement of these proteins in all of the cellular processes investigated was cell line-specific, with the greatest effects observed in HT29 cells, which have relatively high Src protein levels and kinase activity. Furthermore, the consequences of Src family member inhibition were also inhibitor specific, as treatment with PP2 and SKI I generally had greater effects on the cellular processes examined than did treatment with SU6656 or SKI II. It was also found that the inhibition of multiple Src family kinases by at least one of the inhibitors generally resulted in greater effects on the cancer cell phenotypes investigated than were observed when the expression of Src, Fyn, or Yes was decreased using RNA interference. This suggests that multiple Src family members may need to be targeted in order to inhibit the increased proliferation, cell-matrix adhesion, migration, and colony forming ability exhibited by cancer cells. The identification of the cancer cell phenotypes in which particular Src family members are involved is of interest, as these proteins are attractive targets for cancer therapy.

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury in both native kidneys and renal allografts. Previous studies in our lab have shown that a subset of Eph family receptor tyrosine kinases, including EphA2, is strongly and persistently upregulated in renal tubular cells in both in vitro and in vivo models of the renal IRI. Src kinases are necessary and sufficient for upregulation of EphA2. We have proposed that IRI-induced EphA2 upregulation may serve as a necessary step in renal tubular remodelling. / In this study, we have further defined the mechanism of Src kinase-induced EphA2 upregulation by identifying the -145/+137 EphA2 promoter region as the minimal region required for basal and Src kinase-induced activation of the promoter. Moreover, we have identified within this region, at position -45, a canonical cAMP response element (CRE) (Nowakowski et al.), which is essential for EphA2 promoter activation. However, we also found that the prototypical CRE-binding transcription factor, CREB, was not necessary for activation of the EphA2 promoter, suggesting that CREB-related or -unrelated transcription factors are responsible for EphA2 upregulation.