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Ablation of Go alpha-subunit results in a transformed phenotype and constitutively active
J Cheng1, J D Weber, J J Baldassare
1Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Abstract:
Modulation of the components involved in mitogenic signaling cascades is critical to the regulation of cell growth. GTP-binding proteins and the stimulation of phosphatidylcholine (PC) hydrolysis have been shown to play major roles in these cascades. One of the enzymes involved in PC hydrolysis, a PC-specific phospholipase C (PC-PLC) has received relatively little attention. In this paper we examined the role of a particular heterotrimeric GTP-binding protein, Go, in the regulation of cell growth and PC-PLC-mediated hydrolysis of PC in IIC9 fibroblasts. The Go alpha-subunit was ablated in IIC9 cells by stable expression of antisense RNA. These stably transfected cells acquired a transformed phenotype as indicated by: (a) the formation of multiple foci in monolayer cultures, (b) the acquisition of anchorage-independent growth in soft agar; and (c) an increased level of thymidine incorporation in the absence of added mitogens. These data implicate Goalpha as a novel tumor suppressor. Interestingly, PC-PLC activity was constitutively active in the Goalpha-ablated cells as evidenced by the chronically elevated levels of diacylglycerol and phosphorylcholine in the absence of growth factors. In contrast, basal activities of PC-phospholipase D, phospholipase A2, or phosphoinositol-PLC were not affected. These data demonstrate, for the first time, a role for Go in regulating cell growth and provide definitive evidence for the existence of a PC-PLC in eukaryotic cells. The data further indicate that a subunit of Go, is involved in regulating this enzyme.
Insights
Go alpha subunit acts as a tumor suppressor by regulating cell growth. Ablating Go alpha in fibroblasts leads to constitutive phosphatidylcholine-specific phospholipase C (PC-PLC) activity and a transformed phenotype.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Mitogenic signaling cascades regulate cell growth, involving GTP-binding proteins and phosphatidylcholine (PC) hydrolysis.
- PC-specific phospholipase C (PC-PLC) plays a role in PC hydrolysis but has been understudied.
- Heterotrimeric GTP-binding proteins, like Go, are implicated in cellular signaling pathways.
Purpose of the Study:
- To investigate the role of the Go alpha-subunit in regulating cell growth.
- To determine the involvement of Go in PC-PLC-mediated hydrolysis of PC.
- To elucidate the function of Go alpha in fibroblast cell lines.
Main Methods:
- Stable expression of antisense RNA to ablate the Go alpha-subunit in IIC9 fibroblasts.
- Assessment of cellular transformation through focus formation, anchorage-independent growth, and thymidine incorporation.
- Measurement of PC-PLC activity by quantifying diacylglycerol and phosphorylcholine levels.
Main Results:
- Go alpha-ablated IIC9 cells exhibited a transformed phenotype, including increased focus formation, anchorage-independent growth, and thymidine incorporation.
- PC-PLC activity was constitutively active in Go alpha-ablated cells, with elevated diacylglycerol and phosphorylcholine.
- Basal activities of other phospholipases (PC-PLD, PLA2, PI-PLC) remained unaffected.
Conclusions:
- The Go alpha-subunit acts as a novel tumor suppressor, regulating cell growth.
- Go alpha is involved in the regulation of PC-PLC activity.
- This study provides the first evidence for Go's role in cell growth regulation and confirms the existence of PC-PLC in eukaryotic cells.