- Studies over the last several years have focused on the activation of protein kinase C (PKC) members and their regulation by phosphorylation, membrane recruitment, and downregulation by proteolysis; however, not much is known about the regulatory mechanisms leading to PKC synthesis. Most cellular mRNAs have short 5′ untranslated regions (UTRs), sufficient for efficient initiation of translation by the cap-dependent ribosome scanning model. The 5′ UTRs of many mRNAs encoding oncogenes and cell cycle regulators are extraordinarily long and highly structured (32, 68). Cumbersome 5′ UTRs are often associated with complex translational control mechanisms mediated by upstream open reading frames (uORFs) or internal ribosome entry sites (IRESs). uORFs were shown to usually function as translation inhibitors of downstream ORFs (48). They are found in about 10% of eukaryotic mRNAs, but are common in the majority of oncogenes, with a rough estimation of about two-thirds (29). Among the PKC isoforms, the presence of a regulatory uORF was shown only for PKCɛ. This uORF suppressed the expression of PKCɛ in an in vitro system of rabbit reticulocyte lysates, but these effects could not be fully recapitulated in vivo in growing fibroblasts (50). It is generally accepted that mRNAs containing repressive uORFs may have an advantage during translation under stress conditions (25, 27). Exposure of cells to various types of stresses, such as UV irradiation, nutrient limitations, oxidative stress, hypoxia, and exposure to various drugs or toxins, induces specific cascades, which are mediated by four distinct protein kinases (15, 31), each of which directly phosphorylates Ser51 of eIF-2α. Phosphorylation on eIF-2α inhibits eIF-2B activity and thus the exchange of GDP to GTP on eIF2, leading to decreased levels of the ternary complex (Met-tRNAMet-eIF-2-GTP) and consequent inhibition of global protein synthesis (27). Under conditions whereby eIF-2α is phosphorylated and translation of most mRNAs is suppressed, the translation of a subclass of mRNAs is enhanced due to specific features including uORFs within the 5′ UTR. The first and best-studied example is the yeast transcriptional activator GCN4 mRNA, which contains four uORFs in its 5′ UTR, playing a role in translational control under conditions of amino acid starvation (26, 51). In mammalian cells, two regulatory uORFs were found in mRNAs encoding proteins involved in the stress response, such as the transcription factors ATF4 and ATF5 and the eIF-2 phosphatase GADD34 (44, 65, 67). PKC represents a family of phospholipid-dependent serine/threonine kinases that are key mediators in signal transduction pathways, involved in a wide variety of cellular processes, including cell proliferation, differentiation, and apoptosis (14, 64). Based on their primary structure, cofactors, and enzymatic properties, PKC members are divided into three subgroups: the conventional PKCs (α, βΙ, βΙΙ, and γ), the novel PKCs (δ, ɛ, η, and θ), and the atypical PKCs (ζ and λ) (47, 54). Understanding the cellular functions of PKCs is hampered by the fact that they represent a family of 10 members, which differ in their primary structures, biochemical properties, tissue distributions, and subcellular localizations (13, 52). Thus, elucidating the biological roles of individual PKC enzymes and the molecular mechanisms controlling their expression is crucial for understanding their specific contribution to tumor development, progression, or apoptosis. The novel PKCη isoform has a unique tissue distribution and is primarily expressed in epithelial tissues and cells undergoing high turnover (3). It is implicated in diverse cellular functions, including a role in terminal differentiation (16, 23, 43, 55, 56), proliferation (20, 28) and secretion (8, 19, 24). In some of these studies, the mechanism of action involved modulation of cell cycle components (20, 21, 30, 42, 43, 61). Recent studies suggest that PKCη has a special role in the response to stress and regulation of apoptosis (2, 46). It provides protection against apoptosis induced by the chemotherapeutic drugs camptothecin and doxorubicin in Hodgkin's lymphoma lines and breast adenocarcinoma MCF-7 cells (1, 59). In addition, PKCη expression was found to correlate with drug resistance and drug resistance-associated genes in patients with breast cancer (4), ovarian cancer (5), and acute myeloid leukemia blasts (6). Here we show that the expression of PKCη is regulated at the translational level both under normal growth conditions and during stress imposed by amino acid starvation. The human 5′ UTR of PKCη is unusually long (659 nucleotides [nt]), is GC rich, and contains two conserved small uORFs. Using a reporter gene system, we demonstrate that each of these two uORFs suppresses expression of PKCη in growing cells, thus maintaining its low basal expression levels. However, this suppression is relieved during amino acid starvation by leaky scanning causing its translational upregulation. Using wild-type and knockout GCN2 mouse embryonic fibroblasts (MEFs), we demonstrate that the GCN2 kinase is required for the stress-induced upregulation. This is the first report demonstrating that the uORFs of one of the PKC family members has a regulatory role under stress.