Regulation of cell cycle progression by G alpha 12

Galpha12, the alpha-subunit of the heterotrimeric G protein, stimulates proliferation in many cell types. In addition, it has been shown that Galpha12 induces neoplastic transformation of fibroblast cell lines. While previous studies have established the critical role of Galpha 12 in proliferation, the mechanism by which Galpha12 interacts with the cell cycle machinery is poorly understood. My dissertation studies are focused on identifying the mechanism by which Galpha12 interfaces with the cell cycle machinery. To investigate the role of Galpha12 in the regulation of events associated with G1 to S-phase progression, NIH3T3 cells transformed by an activated mutant of Galpha12 (Galpha 12QL-NIH3T3) were analyzed for changes in G1/S-phase cyclins, cyclin dependent kinases (CDKs), and cyclin dependent kinase inhibitors (CKIs). Results from these studies indicated an increase in the levels of cyclin D1, cyclin E and cyclin A in Galpha12QL-NIH3T3 cells. A corresponding increase in the CDK4 and CDK2-kinase activity was observed in Galpha12QL-NIH3T3 cells. Since the activities of these kinases are regulated by the CKI p27 Kip1, the levels of p27Kip1 were determined in Galpha 12QL-NIH3T3 cells. The result from such analysis indicated a decrease in the levels of p27Kip1 in Galpha12-NIH3T3 cells. A decrease in the p27Kip1 levels could be associated with an increase in the levels of E3 ubiquitin ligase, Skp2, therefore, the levels of Skp2 were monitored in Galpha12QL-NIH3T3 cells. Results from this analysis indicated an increase in the levels of Skp2 in Galpha 12QL-NIH3T3 cells. To rule out the possibility that the increase in Skp2 levels was due to the transformed phenotype of these cells, the Skp2 response to Galpha12QL expression in NIH3T3 cells was analyzed by transient transfection studies. Results indicated that the transient expression of Galpha12QL stimulated the expression of Skp2 with a concomitant decrease in p27Kip1 levels, suggesting the observed increase in Skp2-levels is specific to signaling by Galpha12QL.;Since the regulation of Skp2-expression is critical for cells entering G1 to S-phase, the mechanism underlying the increase in Skp2 mediated by Galpha 12 levels was investigated. Results from this analysis indicated that Galpha12-stimulated increase in Skp2 is due to increased protein stability in Galpha12QL-NIH3T3 cells. Further studies indicated that the mechanism through which Galpha12 regulated Skp2 involved, c-Jun N-terminal kinase (JNK) and Rho-mediated signaling pathways, as the inhibition of these respective pathways down regulated Galpha12QL-mediated increase in Skp2 levels.;Previously it has been shown that Lysophosphatidic acid (LPA), a lipid growth factor that can activate Galpha12 via specific LPA-receptors, can stimulate the proliferation of 1321N1 astrocytoma cells. Therefore, it is of interest to determine (i) whether LPA can activate Galpha 12 in 1321N1 astrocytoma cells and (ii) if so, whether such LPA mediated activation of Galpha12 induces an increase in Skp2 levels. Results from this analysis indicated that LPA activated Galpha12 in 1321N1 astrocytoma cells, as indicated by a GST-TPR (GST-fused TPR domain of Ser/Thr phosphatase 5) binding assay. In addition, these studies indicated that LPA stimulated an increase in proliferation of 1321N1 astrocytoma cells confirming previous observations. Further analysis indicated that LPA stimulated an increase in Skp2 levels similar to the effect seen with the expression of Galpha12QL. In order to establish whether the observed LPA induced increase in Skp2 levels is mediated by Galpha12, the effect of expressing dominant negative construct of Galpha12, CT-12, on LPA induced Skp2 levels was investigated in 1321N1 astrocytoma cells. Results from this analysis indicated that expression of the CT-12 construct attenuated the LPA-mediated increase in Skp2 levels in 1321N1 astrocytoma cells. Furthermore, it was observed that the expression of CT-12 attenuated LPA mediated proliferation of 1321N1 astrocytoma cells. Thus, the results presented here identify Skp2 as a novel-signaling target of Galpha 12-mediated signaling in cell cycle.