Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms ␣, ␦, and in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC ␦. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC ␣, ␦, and were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA-and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC ␣ could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway.Tumor promotion by phorbol esters involves the selective amplification of cells previously mutated in an appropriate growth-stimulatory gene (3,17). Phorbol esters exert their effects on the protein kinase C (PKC) family of genes, which consists of genes that encode at least nine distinct isoforms that are responsive to tumor-promoting phorbol esters (9). Phorbol esters first activate phorbol ester-responsive PKC isoforms, but upon prolonged treatment, these isoforms are proteolytically degraded (16). Using a cell culture model system in which cells overexpressing c-Src were transformed by phorbol ester treatment, we recently demonstrated that the tumor-promoting effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on these cells was due to the depletion of PKC ␦ (7). These data suggested that PKC ␦ may function as a tumor suppressor. Consistent with this hypothesis, PKC ␦ was inactivated by tyrosine phosphorylation in cells transformed by v-Src (19) and v-Ras (2). Thus, regulation of PKC ␦ at the level of activity and expression may be a very important cell growth control mechanism.PKC ␣ has been reported to become ubiquitinated in response to bryostatin 1, an activator of PKC that prevents tumor promotion in mouse skin by TPA (6). The ubiquitin-proteasome pathway is a nonlysosomal degradation system that controls the timed destruction of cell cycle-regulatory proteins, including the tumor suppressor p53; the cyclin-dependent kinase inhibitor p27; the cyclins; the oncogene products c-Myc, c-Jun, and c-Fos; and the transcription factors NF-B and E2F (reviewed in reference 13). This pathway involves the covalent tagging of proteins with u...

Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced downregulation of the PKC␦ isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKC␦ as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKC␦ mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKC␦, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKC␦, which has an apparent tumor suppressor function.Carcinogenesis is a multistep process involving successive rounds of mutation (initiation) and selected amplification (promotion) of mutated cells. Eventually, mutated cells acquire an appropriate complement of genetic changes such that the cells divide without proper control, giving rise to a tumor (10). This process can be accelerated by stimulating the replication and amplification of mutated cells, increasing the numbers of partially transformed cells subject to further mutation to a more cancerous state. Substances that stimulate the division of incompletely transformed cells are known as tumor promoters, and while not inducing directly the genetic changes that ultimately result in a tumor, they can dramatically speed up the process (44).The best-studied class of tumor promoters are the phorbol esters, which exert their effects on protein kinase C (PKC). The PKC isoforms, of which there are no fewer than nine that are responsive to the tumor-promoting phorbol esters, are encoded by a multigene family. Upon phorbol ester treatment, PKC isoforms become associated with the cell membrane and active (27). However, upon prolonged phorbol ester treatment, PKCs are proteolytically degraded (43). The time course for PKC depletion upon phorbol ester treatment varies substantially for different cell types, from a few hours to a few days. Tumor promotion requires repeated long-term exposure to phorbol esters, suggesting that depletion rather than activation of PKC is important for tumor promotion. However, it has been pointed out that even though PKC is depleted by prolonged phorbol ester treatment, newly synthesized PKC would be brought to the membrane, where there would be a shortlived, but potentially significant, ph...

3Y1 rat fibroblasts overexpressing the epidermal growth factor (EGF) receptor (EGFR cells) become transformed when treated with EGF.A common response to oncogenic and mitogenic stimuli is elevated phospholipase D (PLD) activity. RalA, a small GTPase that functions as a downstream effector molecule of Ras, exists in a complex with PLD1. In the EGFR cells, EGF induced a Ras-dependent activation of RalA. The activation of PLD by EGF in these cells was dependent upon both Ras and RalA. In contrast, EGF-induced activation of Erk1, Erk2, and Jun kinase was dependent on Ras but independent of RalA, indicating divergent pathways activated by EGF and mediated by Ras. The transformed phenotype induced by EGF in the EGFR cells was dependent upon both Ras and RalA. Importantly, overexpression of wild-type RalA or an activated RalA mutant increased PLD activity in the absence of EGF and transformed the EGFR cells. Although overexpression of PLD1 is generally toxic to cells, the EGFR cells not only tolerated PLD1 overexpression but also became transformed in the absence of EGF. These data demonstrate that either RalA or PLD1 can cooperate with EGF receptor to transform cells.Overexpression of a tyrosine kinase is a common genetic defect in a variety of human tumors (21). The epidermal growth factor (EGF) receptor, which has an intrinsic tyrosine kinase that is activated in response to EGF, is frequently overexpressed in human breast and ovarian cancer (35). However, overexpression of a tyrosine kinase such as the EGF receptor is not sufficient for a fully transformed or cancerous phenotype. We recently demonstrated that downregulation of protein kinase C ␦ (PKC ␦) transforms 3Y1 rat fibroblasts overexpressing either c-Src (28) or the EGF receptor (19). The EGF receptor-overexpressing cells (EGFR cells) could also be transformed when treated with EGF (19), suggesting that EGF could accomplish what PKC ␦ downregulation accomplished. Interestingly, downregulation of PKC ␦ also caused an increase in phospholipase D (PLD) activity (19,38), which is commonly elevated in response to oncogenic and mitogenic stimuli (11,41). Both EGF-induced increases in PLD activity and EGFinduced transformation were dependent upon the ␣ isoform of PKC (19), suggesting that PLD may be an important component of the mitogenic and oncogenic properties of the EGF receptor.We demonstrated previously (30) that PLD1 associates directly with the small GTPase RalA, a downstream target of Ras (13). RalA is required for PLD activation in response to v-Src and v-Ras (22). RalA has also been implicated in cell transformation (1, 39), indicating a possible role for PLD in mitogenic signaling. In this paper, we report that both RalA and PLD1 can cooperate with an overexpressed EGF receptor to transform cells. MATERIALS AND METHODSCells and cell culture conditions. Rat 3Y1 cells or rat 3Y1 cells expressing the EGF receptor were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (HyClone) as described previously (28, 29). The EGF...