Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNA containing premature termination codons (PTCs). In mammalian cells, recognition of PTCs requires translation and depends on the presence on the mRNA with the splicing-dependent exon junction complex (EJC). While it is known that a key event in the triggering of NMD is phosphorylation of the trans-acting factor, Upf1, by SMG-1, the relationship between Upf1 phosphorylation and PTC recognition remains undetermined. Here we show that SMG-1 binds to the mRNA-associated components of the EJC, Upf2, Upf3b, eIF4A3, Magoh, and Y14. Further, we describe a novel complex that contains the NMD factors SMG-1 and Upf1, and the translation termination release factors eRF1 and eRF3 (SURF). Importantly, an association between SURF and the EJC is required for SMG-1-mediated Upf1 phosphorylation and NMD. Thus, the SMG-1-mediated phosphorylation of Upf1 occurs on the association of SURF with EJC, which provides the link between the EJC and recognition of PTCs and triggers NMD.

Ten years ago, par-1 and par-3 were cloned as two of the six par genes essential for the asymmetric division of the Caenorhabditis elegans zygote. PAR-1 is a protein kinase, whereas PAR-3 is a PDZ-domain-containing scaffold protein. Work over the past decade has shown that they are part of an evolutionarily conserved PAR-aPKC system involved in cell polarity in various biological contexts. Recent progress has illustrated the common principle that the PAR-aPKC system is the molecular machinery that converts initial polarity cues in the establishment of complementary membrane domains along the polarity axis. In most cases, this is achieved by mutually antagonistic interactions between the aPKC-PAR-3-PAR-6 complex and PAR-1 or PAR2 located opposite. However, accumulating evidence has also revealed that mechanisms by which the asymmetrically localized components of the PAR-aPKC system are linked with other cellular machinery for developing polarity are divergent depending on the cell type.

Although the involvement of protein kinase C (PKC) in the activation of the mitogen-activated protein (MAP) kinase pathway has been implicated through experiments using 12-O-tetradecanoylphorbol-13-acetate (TPA), there has been no direct demonstration that PKC activates the MAP kinase pathway. A Raf-dependent intact cell assay system for monitoring the activation of MAPK/ERK kinase (MEK) and extracellular signal-related kinase (ERK) permitted us to evaluate the role of PKC isotypes in MAP kinase activation. Treatment of cells with TPA or epidermal growth factor resulted in the activation of MEK and ERK. The activation of the MAP kinase pathway triggered by epidermal growth factor was completely inhibited by dominant-negative Ras (RasN17), whereas the activation triggered by TPA was not, consistent with previous observations. The introduction of an activated point mutant of PKCδ, but not PKCα or PKCϵ, resulted in the activation of the MAP kinase pathway. The activation of MEK and ERK by an activated form of PKCδ requires the presence of c-Raf and is independent of RasN17. These results demonstrate that activation of PKCδ is sufficient for the activation of MEK and ERK and that the pathway operates in a manner dependent on c-Raf and independent of Ras.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin‐binding EGF‐like growth factor (HB‐EGF) that exists in a membrane‐anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCδ binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin‐γ/ADAM9, a member of the metalloprotease–disintegrin family. Furthermore, the presence of constitutively active PKCδ or MDC9 results in the shedding of the ectodomain of proHB‐EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase‐negative PKCδ, suppress the TPA‐induced shedding of the ectodomain. These results suggest that MDC9 and PKCδ are involved in the stimulus‐coupled shedding of the proHB‐EGF ectodomain.

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCζ and PKCλ have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype–specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKCλ to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.