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...