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.

Mitogen activated protein kinase (MAPK) signaling consists of a phosphorylation cascade leading to the phosphorylation of an effector that can translocate to the nucleus and regulate transcription. MAPK signaling can be activated by diverse stimuli such as osmotic stress and hormones. Additionally, MAPK signaling can be activated by receptor tyrosine kinase (RTK) signaling in response to a growth factor binding to the RTK. It has been shown that heterodimerization of the RTKs human epidermal growth factor receptor 2 (HER2) with HER3 is highly correlated to tumor growth and metastasis in breast cancer. Previous research showed that in breast cancer cells, Mitogen Three Kinase 1 (MTK1) is a protein kinase that functions in association with the HER3 receptor upon heregulin (HRG) stimulation. Here, MTK1 has been shown to form a complex in breast cancer cells consisting of Grb2, Shc, GIT1 and ERK1/2. Furthermore, MTK1 was shown to induce extracellular acidification due to lactate secretion and cell migration. A shift from aerobic metabolism to glycolysis in cancer cells was first observed by Otto Warburg. Since then, increased glycolysis and the resulting lactate secretion have been described in multiple types of cancers and is recognized as one of the hallmarks of cancer. This dissertation aims to establish the function of MTK1 in HRG induced RTK signaling in breast cancer cells. To accomplish this goal, we identified proteins of known function that interact with MTK1 to infer the role of MTK1. Applying this strategy, we provide evidence for the first time of mitochondrial regulation of oxidative phosphorylation by MTK1, independent of glycolytic regulation. In addition, our results demonstrate that GIT1 regulates glycolysis-mediated lactic acidosis (higher extracellular acidification caused by increased lactic acid efflux) in MCF-7 cells, independent of mitochondrial function. For the first time, our research shows that the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) associates with MTK1 and GIT1 in MCF-7 cells. Furthermore, it is demonstrated that PGK1 is regulated by HRG stimulation through both phosphorylation of PGK1 and its dissociation from the MTK1 complex. The protein complex consisting of MTK1, GIT1, and PGK1 favors the conversion of pyruvate to lactate resulting rather than oxidative phosphorylation of pyruvate inside the mitochondria. The resulting lactate is secreted and drives extracellular acidification, a hallmark of cancer.

Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6-/- mice injected with biotinylated-fibrinogen, showed lower accumulation of the modified protein than did WT mice. Resting and activated αIIbβ3 levels, measured by FACS, were unchanged in Arf6-/- platelets. Arf6-/- platelets had normal agonist-induced aggregation and ATP release; however, they showed faster clot retraction and enhanced spreading, which appears due to altered αIIbβ3 trafficking since myosin light chain phosphorylation and Rac1 activation, in response to thrombin, were unaffected. Arf6-/- mice showed no hemostasis defect in tail-bleeding or FeCl3–induced carotid injury assays. These data suggest a role for Arf6 in integrin αIIbβ3 trafficking in platelets. Additionally, the regulation of Arf6 in platelets was also investigated, focusing on integrin αIIbβ3 outside-in signaling which was suggested to be responsible for the second wave of Arf6-GTP loss. G protein-coupled receptor kinase-interacting protein 1 (GIT1), a GTPase-activating protein (GAP) toward Arf6, is suggested to be involved in αIIbβ3 downstream signaling. I found that GIT1, complex with β-PIX, was translocated to the detergent-insoluble pellet upon human platelet activation, a process that is blocked by RGDS and myrArf6 peptide treatment. Moreover, tyrosine-phosphorylation of GIT1 was impaired by treatment with both peptides or with actin polymerization inhibitors. GIT1’s role in platelets was further studied using platelet-specific, GIT1 knockout mice. GIT1-/- platelets failed to show any defect, including clot retraction or fibrinogen storage. Unlike human platelets, GIT1 expression levels were much lower in mouse platelets, suggesting that GIT2 may be the functionally relevant Arf6-GAP in mouse platelets. The data in this dissertation identify that Arf6 mediates fibrinogen storage, implying its role in integrin αIIbβ3 trafficking in platelets.