Big mitogen-activated protein (MAP) kinase (BMK1), also known as ERK5, is a member of the MAP kinase family whose cellular activity is elevated in response to growth factors, oxidative stress, and hyperosmolar conditions. Previous studies have identified MEK5 as a cellular kinase directly regulating BMK1 activity; however, signaling molecules that directly regulate MEK5 activity have not yet been defined. Through utilization of a yeast two-hybrid screen, we have identified MEKK3 as a molecule that physically interacts with MEK5. This interaction appears to take place in mammalian cells as evidenced by the fact that cellular MEK5 and MEKK3 co-immunoprecipitate. In addition, we show that a dominant active form of MEKK3 stimulates BMK1 activity through MEK5. Moreover, we demonstrate that MEKK3 activity is required for growth factor mediated cellular activation of endogenous BMK1. Taken together, these results identify MEKK3 as a kinase that regulates the activity of MEK5 and BMK1 during growth factor-induced cellular stimulation.The mitogen-activated protein (MAP) 1 kinase cascades represent a primary mechanism by which cells transduce intracellular signals (1-3). These kinase cascades display a high degree of evolutionary conservation, as evidenced in a variety of eukaryotes ranging from yeast to mammals (2, 4). Three sequentially activated kinases make up the core of the MAP kinase module: a MAP kinase kinase kinase, or MEKK; a MAP kinase kinase, or MEK; and a MAP kinase (5, 6). In addition to delivering signals from extracellular stimuli to intended effectors, these kinase modules harmonize incoming signals from parallel signaling pathways and provide signal amplification as well as biological specificity. To date, four separate MAP kinases have been identified in mammalian cells and are known as ERK, JNK/SAPK, p38, and BMK1/ERK5 (7-13).BMK1/ERK5 represents the newest member of the mammalian MAP kinase family and was independently cloned by our laboratory and another group using different experimental approaches (7,11). We have previously demonstrated that BMK1 is activated by growth factors, oxidative stress, and hyperosmolar conditions (8,9,14). Upon activation, BMK1 stimulates the activity of myocyte enhancer factor 2C (MEF2C), a transcription factor that induces the expression of the proto-oncogene c-jun (9). Through the use of a dominant negative form of BMK1, we have demonstrated that BMK1 is required for growth factor-induced cell proliferation and cell cycle progression (8). Using the yeast two-hybrid system, MEK5 was identified by Zhou et al. (11) as the molecule responsible for regulating BMK1 activity. Subsequently, we have determined that MEK5 specifically activates BMK1 but not other mammalian MAP kinases in vivo (9). In addition, we have shown that MEK5 activity is required for the activation of BMK1 induced by extracellular stimuli (8, 9).The upstream kinase responsible for regulating MEK5 activity within the BMK1 signaling module has not yet been reported. In this regard, studies by English et a...

We demonstrated that peripheral T cell tolerance toward murine melanoma self-antigens gp100 and TRP-2 can be broken by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to minigenes encoding peptide epitopes gp100 25–33 and TRP-2 181–188 . These epitopes contain dominant anchor residues for MHC class I antigen alleles H-2D b and H-2K b , respectively. The DNA vaccine was delivered by oral gavage by using an attenuated strain of Salmonella typhimurium as carrier. Tumor-protective immunity was mediated by MHC class I antigen-restricted CD8 + T cells that secreted T H 1 cytokine IFN-γ and induced tumor rejection and growth suppression after a lethal challenge with B16G3.26 murine melanoma cells. Importantly, the protective immunity induced by this autologous DNA vaccine against murine melanoma cells was at least equal to that achieved through xenoimmunization with the human gp100 25–33 peptide, which differs in its three NH 2 -terminal amino acid residues from its murine counterpart and was previously reported to be clearly superior to an autologous vaccine in inducing protective immunity. The presence of ubiquitin upstream of the minigene proved to be essential for achieving this tumor-protective immunity, suggesting that effective antigen processing and presentation may make it possible to break peripheral T cell tolerance to a self-antigen. This vaccine design might prove useful for future rational designs of other recombinant DNA vaccines targeting tissue differentiation antigens expressed by tumors.

Activation of the mammalian mitogen-activated protein kinase known as BMK1 is required for growth factor-induced cell proliferation. To understand the mechanism by which BMK1 mediates this cellular response, this kinase was used as bait in a yeast two-hybrid-based library screening. Here, we report the identification of serum and glucocorticoid-inducible kinase (SGK) as a cellular protein that physically interacts with BMK1. During growth factor-induced cell stimulation, BMK1 activates SGK by phosphorylation at serine 78. This BMK1-mediated phosphorylation event is necessary for the activation of SGK and, more importantly, for cell proliferation induced by growth factors.Genetic and biochemical studies have identified the mitogenactivated protein (MAP) 1 kinases as central intracellular molecules that deliver signals from activated cell surface receptors to downstream regulatory proteins. These MAP kinases have been conserved in all eukaryotes, ranging from yeast to mammals, and have a universal role in controlling cell growth through the regulation of cell cycle progression (1-6). The rate of cell cycle progression is tightly regulated by both growth factors and stress-related stimuli, and MAP kinases deliver and integrate both types of these extracellular signals to the cell cycle machinery by modulating the phosphorylation state of intracellular proteins. The MAP kinases ERK1/2, JNK1, and p38 control cell cycle progression by regulating either the expression or the activity of key molecules required for G 1 to S phase transition. We have previously demonstrated that BMK1/ERK5, the newest member of the MAP kinase family (7-11), is required for growth factor-induced cell proliferation and cell cycle progression (10). Although we have established that the activity of BMK1 is required for the growth factormediated entry of cells into the S phase of the cell cycle (10), the downstream effector(s) of this process have not yet been reported.To investigate the mechanism by which BMK1 mediates the entry of cells into S phase, this kinase was used as bait in a yeast two-hybrid screening of a cDNA library. Here we report the identification of serum-and glucocorticoid-inducible protein kinase (SGK) as a molecule that physically interacts with BMK1. SGK is a serine/threonine protein kinase with significant sequence homology throughout its catalytic domain with protein kinase B, ribosomal protein S6 kinase, cAMP-dependent protein kinase, and members of the protein kinase C family (12). A variety of stimuli, including glucocorticoids, hydrogen peroxide, hyperosmotic stress, serum, and insulin-like growth factor, have been shown to induce both the cellular expression and kinase activity of SGK (12-16). Similar to BMK1, the activity of SGK is closely linked to the G 1 /S transition of the cell cycle (17). Here, we show that BMK1 activates SGK as a result of growth factor-induced cellular activation through the phosphorylation of serine 78. Moreover, we demonstrate that the BMK1-mediated phosphorylation of SGK is critical fo...