THESIS
2008
xvii, 135 leaves : ill. (some col.) ; 30 cm
Abstract
Gα-interacting protein (GAIP) is a regulator of G protein signaling (RGS), which acts as a GTPase activating protein (GAP) for Gα
i subunits and is implicated in growth factor signaling and the regulation of autophagy. In an attempt to gain additional insight on its functions, GAIP was stably expressed in mammalian cell lines and its effect on cell proliferation then examined. Overexpression of GAIP stimulated the growth of human embryonic kidney (HEK) 293, PC12, Caco-2 and NIH3T3 cells. Also, GAIP conferred upon HEK293 cells the ability to grow in a serum-independent manner, and the conditioned medium collected from 293/GAIP cells enhanced the growth of the parental cells in a dose-dependent manner. However, GAIP failed to induce neoplastic transformation in NIH3T3 cells as determined...[
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Gα-interacting protein (GAIP) is a regulator of G protein signaling (RGS), which acts as a GTPase activating protein (GAP) for Gα
i subunits and is implicated in growth factor signaling and the regulation of autophagy. In an attempt to gain additional insight on its functions, GAIP was stably expressed in mammalian cell lines and its effect on cell proliferation then examined. Overexpression of GAIP stimulated the growth of human embryonic kidney (HEK) 293, PC12, Caco-2 and NIH3T3 cells. Also, GAIP conferred upon HEK293 cells the ability to grow in a serum-independent manner, and the conditioned medium collected from 293/GAIP cells enhanced the growth of the parental cells in a dose-dependent manner. However, GAIP failed to induce neoplastic transformation in NIH3T3 cells as determined by focus formation and soft agar assays. Non-metastatsis suppressor, NM23 was identified in the conditioned medium prepared from 293/GAIP cells. Surprisingly, GAIP significantly suppressed the serum-induced mitogenic response along the Ras/Raf/MEK/ERK. The endogenous Ras/Raf/MEK/ERK signaling pathway in 293/GAIP cells was intact as treatment of 293/GAIP cells with forskolin or phorbol-12-myristate-13-acetate (PMA) induced ERK1/2 phosphorylation. Similar observations on serum- and specific stimulator-elevated p38 MAPK and JNK phosphorylations in 293/GAIP cells were obtained. Disruption of ERK and p38 MAPK cascades with specific inhibitors displayed no effect on the proliferation of HEK293 cells. In contrast, GAIP augmented serum-stimulated PTEN/PDK/Akt and Rb phosphorylations and upregulated the protein levels of cyclin D1/3, p27
Kip1, and Cdk6 in 293/GAIP cells. Consistent with an elevated Akt1 activity and upregulation of Cdk6, increased levels of phosphorylated Bad and c-Raf as well as diminished expressions of tuberin, INK4a/b and p21
Cip1 were detected in 293/GAIP cells. The PDZ binding motif of GAIP is critical for the proliferation of 293/GAIP. Also, preliminary results demonstrated that GAIP was able to increase the level of light chain 3 (LC3) and Beclin 1, cellular markers of autophagy. The present study demonstrates that GAIP, in addition to acting as a GAP, is able to regulate cell proliferation in a Ras-independent manner via the Akt pathway.
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