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Dissociation of Inositol Trisphosphate from Diacylglycerol Production in Rous Sarcoma Virus-Transformed Fibroblasts Timothy J

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Dissociation of Inositol Trisphosphate from Diacylglycerol Production in Rous Sarcoma Virus-Transformed Fibroblasts Timothy J Dissociation of Inositol Trisphosphate from Diacylglycerol Production in Rous Sarcoma Virus-transformed Fibroblasts Timothy J. Martins, Yoshikazu Sugimoto, and R. L. Erikson Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138 Abstract. The metabolism of phosphatidylinositol (PI) changes intermediate between those of transformed and and related intermediates was studied in uninfected nontransformed cells. NY72-4 CEF exhibited no in- and Rous sarcoma virus-(RSV) infected chicken em- crease in phosphoinositol concentrations before 8 h in- bryo fibroblasts (CEFs). Cells infected with wild-type cubation at 35°C, indicating that the transformation- Downloaded from http://rupress.org/jcb/article-pdf/108/2/683/1057290/683.pdf by guest on 26 September 2021 RSV exhibited twofold increases in steady-state con- specific changes in inositol metabolism were a delayed centrations of inositol trisphosphate (IP3) and inositol event. Furthermore, inositol turnover was not activated bisphosphate (IP2) as compared to uninfected CEFs. In during this time. In contrast to the case of inositol me- addition, increased concentrations of IP3 and IP2 were tabolism, significant increases in diacylglycerol (DAG) observed in CEFs infected with the RSV temperature- concentrations were observed within 15-30 min after sensitive transformation mutant NY72-4 when main- shift of NY72-4 CEFs to 35°C. tained at the permissive temperature (35°C) for >24 h. These findings suggest that (a) the major changes in Slight increases were observed in the amounts of ino- inositol metabolism are specific for RSV-transformed sitol lipids in RSV-transformed cells. Phosphoinositol cells; (b) transformation-specific changes in phos- metabolic changes were related to transformation and phoinositol content in RSV-infected CEFs are not an not to viral infection since CEFs infected with NY72- early effect of the expression of pp60v-s~; and (c) in- 4, maintained at the nonpermissive temperature creases in the DAG content of transformed cells occur (41.5°C), revealed amounts of phosphoinositols similar before changes in inositol metabolism, indicating that to that of uninfected cells. CEFs infected with a DAG may be derived from other lipid sources. transformation-defective virus exhibited PI metabolic HE transforming gene product of Rous sarcoma virus specific serine phosphotransferase has generated much in- (RSV)', pp60v-s% exhibits tyrosine-specific protein terest since it is one of the earliest responding enzymatic ac- T kinase activity, a property it shares with a number of tivities after cellular stimulation by several oncogenes and oncogene products and growth factor receptors (for review growth factors (9, 22, 28). However, the link between the see 25). However, the molecular events leading from tyro- proliferative stimuli including tyrosine phosphotransferase sine phosphotransferase activity to cellular mitogenesis re- activity and the identity and transduction of the mitogenic main obscure since phosphorylation of substrates identified signal responsible for the stimulation of the $6 kinase activ- appears to be incidental (29, 30). ity have remained elusive. Attempting to unravel the steps culminating in mitogene- The components of the phosphatidylinositol (PI) meta- sis, investigators have concentrated on the early biochemical bolic cycle appear to be signal transducers used by a number events that follow proliferative stimuli. Recently, several of hormones and growth factors including c~-adrenergic studies have implicated the importance of protein kinase ac- agents (50), thyrotopin-releasing hormone (21, 24), vaso- tivities specific to serine/threonine residues after mitogenic pressin (11), muscarinic cholinergic agents (11), bombesin stimulation of cells. For example, a ribosomal protein $6- (12), and platelet-derived growth factor (6). Agents that stimulate PI turnover can potentially generate two intracellu- lar second messengers; namely, diacylglycerol (DAG) and Dr. Sugimoto's current address is Division of Chemotherapy, Pharmaceuti- inositol-l,4,5-trisphosphate (IP3) (3). DAG in the presence cal Institute, School of Medicine, Keio University, Shinjuku, Tokyo, Japan. of calcium ion can influence the phosphorylation state of a number of intracellular proteins by activating protein kinase 1. Abbreviations used in this paper: CEF, chicken embryo fibroblast; DAG, C (3). Protein kinase C is of particular significance since 12- diacylglycerol; IP, IP2, and IP3, inositol mono-, bis-, and trisphosphate; O-tetradecanoylphorbol 13-acetate, a known mitogen for PA, phosphatidic acid; PI, phosphatidylinositol; PIP and PIP2, phosphati- dylinositol mono- and bisphosphate; RSV, Rous sarcoma virus; SRA, some cells, can activate the kinase by substituting for its en- Schmidt-Ruppin strain A of RSV. dogenous effector, DAG (3, 38). Likewise, IP3 is of interest © The Rockefeller University Press, 0021-9525/89/02/683/9 $2.00 The Journal of Cell Biology, Volume 108, February 1989 683-691 683 since it is thought to be involved in the release of Ca2÷ from Cellular Labeling with myo4,2-pH]inositol internal cellular stores after its binding to specific receptors For these experiments, 60-ram culture dishes (Costar) were seeded at either (6, 8, 26, 46, 47). Koch and Diringer (33) showed that trans- 0.25 or 1.0 x llYi cells per dish in 3 ml inositol-free DME (prepared ac- formed ceils exhibit increased PI turnover when compared cording to Gibco Laboratories, Grand Island, NY, but myo-inositol was to their nontransformed parent cells. These studies were ex- eliminated from the vitamin mixture) supplemented with 5% calf serum. tended to include transformation by RSV as well (19). More After 5 h, 1 ml of inositol-freeDME containing 5 % calf serum and 5 #Ci/mi myo-l,2-[3H]inositol was added to each dish. After 3 d of culture at 41.50C, recently, several investigations have demonstrated that treat- the cells were confluent, and the medium was then replaced with inositol- ment of quiescent cells with growth factors or serum results free DME containing 0.5% calf serum. The cells were maintained for in rapid changes in PI turnover. Since RSV-transformed cells 16-24 h in low serum before initiation of temperature shift or serum stimu- display a number of properties similar to those of growth fac- lation experiments. Cells could be maintained for at least three passages in tor-stimulated cells, initial investigations concentrated on inositol-free DME with no detrimental effects. To estimate the relative turn- over of phosphoinositols in CEFs, some plates of cells were incubated for the in vitro activity of key inositol-metabolizing enzymes. 30 rain with l0 mM LiC1 just before cell lysis. Lithium ion prevents inositol These studies indicated that transformation by RSV and monophosphate hydrolysis by inhibiting phosphomonoesterase (4, 21). related oncogenes resulted in elevated PI kinase activity (31, Hence, changes in inositol monophosphate concentration after lithium treat- 36, 48, 49), phospholipase C activity, PI turnover (27), and ment of cells should reflect the relative activity state of the PI metabolic elevation of DAG (51). Although the activities of different cycle. inositol-metabolizing enzymes were quantified in vitro, few Cellular Labeling with [32P]PO, data regarding the in vivo steady-state concentrations of phosphoinositides in RSV-transformed cells were available. Conditions for labeling with [32p]Po4 were identical to those described Furthermore, no data was available with regard to the tem- above for inositol labeling except that unmodified DME and 10 #Ci/ml Downloaded from http://rupress.org/jcb/article-pdf/108/2/683/1057290/683.pdf by guest on 26 September 2021 [32PlPO4 was used in place of inositol-free DME and [3H]inositol, respec- poral relationship of phosphoinositide metabolism and ex- tively. pression of pp60 v-sr~, the transforming protein. Therefore, the quantitation of phosphatidylinositol metabolic intermedi- Synthesis of myo-P2Plinositol-l,4,5-trisphosphate ates was performed in uninfected and RSV-infected chicken Preparation of myo-[a2p]inositol-l,4,5-trisphosphate was essentially as de- embryo fibroblasts (CEFs). scribed by Dowries et al. (20). After the release of inositol phosphates from The data reported below indicate that indeed, transformed erythrocyte membranes with Ca2+, the resulting supernatant was chro- cells exhibited altered amounts of phosphoinositols when matographed on AG-1-X8-formate columns, and desalted by lyophilization compared to their nontransformed counterparts. However, (16). Thin layer electrophoresis on cellulose sheets in 100 mM sodium oxa- experiments using temperature-sensitive, transformation- late, pH 1.5, showed that the radiolabeled compound migrated with authen- tic IP3 generated by partial acid hydrolysis of phytic acid. Contamination defective RSV-infected fibroblasts indicated that the change with [32p]PO4 never exceeded 5% of the total radioactivity. in inositol metabolism was a relatively late event. In fact, nontransforming mutant RSV-infected CEFs revealed some Hydrolysis of Phytic Acid PI metabolite changes that were similar to their transformed counterparts. In contrast, rapid increases in DAG concen- Partial acid hydrolysis of phytate can generate inositol phosphates contain- ing 1-5 mol PO4/mol of inositol (44). (It should be noted that inositol tration were observed after expression of pp60 v-src and phosphates generated in this way contain mixed forms of IP, IP2, IP3, IP4,
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