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.
Highlights
Defects in signal transduction cascades involved in the regulation of cell growth often lead to pathological conditions, including the development of neoplasms
PI and PC hydrolysis are induced by a variety of mitogens, PI hydrolysis is often transient while PC hydrolysis is usually sustained in the continuous presence of growth factors
The data in this paper demonstrate for the first time that the a subunit of the Go GTP-binding protein is involved in regulating cell growth and PC-PLC activity
Summary
(Received for publication, February 4, 1997, and in revised form, April 30, 1997). From the ‡Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the Departments of §Cell and ¶Pharmacological and Physiological Sciences, St. Subunits, which, in response to receptor activation, dissociate into free a subunits and bg dimers Both the GTP-bound a subunits and bg dimers have been shown to play roles in a variety mitogenic signal transduction cascades [1], including those involving induced lipid metabolism [2, 3]. Mutations in the Gi2a gene yield another oncogene (gip2) characterized by a substitution of amino acid 179 in Gi2a (R179C/H) which has been found in ovarian sex cord stromal tumors and adrenal cortical tumors [5] These data provide strong support for the notion that these G proteins are important components involved in the regulation of mitogenic signal transduction cascades and represent potential targets for oncogenic mutations in human tumors. The relationship between Goa, the transformed phenotype and the constitutive activation of PC-PLC is discussed
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