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.[Keywords: SMG-1; eRF; Upf; phosphorylation; EJC; NMD] Supplemental material is available at http://www.genesdev.org.

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKC and PKC) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKC in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKC (KD or ⌬NKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKC, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone-or hyperosmolarity-induced glucose uptake, were inhibited by KD or ⌬NKD in a dosedependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKC was ϳ50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKC mutant that lacks the pseudosubstrate domain (⌬PD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of ⌬PD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKC. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKC pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.Phosphoinositide (PI) 3-kinase, a lipid kinase composed of an SRC homology 2 (SH2) domain-containing regulatory subunit and a 110-kDa catalytic subunit, catalyzes phosphorylation of the D3 position of PIs (46,48). This enzyme was first identified complexed with SRC kinase and the middle T antigen of polyomavirus and was later found to associate with various tyrosine-phosphorylated proteins in response to stimulation of cells with growth factors or cytokines (46, 48). Activation of PI 3-kinase, either by targeting of the enzyme to the plasma membrane (27) or as a consequence of direct interaction between the SH2 domain of the regulatory subunit and phosphorylated tyrosine residues present within specific motifs (5), results in the triggering of various important biological actions. Thus, with the use of either a dominant negative protein that blocks the interaction between PI 3-kinase and tyrosine-phosphorylated proteins (21,33,41) or pharmacological inhibitors of the enzyme, such as wortmannin or LY294002 (...

Overexpression of a TPA‐insensitive PKC member, an atypical protein kinase C (aPKClambda), results in an enhancement of the transcriptional activation of TPA response element (TRE) in cells stimulated with epidermal growth factor (EGF) or platelet‐derived growth factor (PDGF). EGF or PDGF also caused a transient increase in the in vivo phosphorylation level and a change in the intracellular localization of aPKClambda from the nucleus to the cytosol, indicating the activation of aPKClambda in response to this growth factor stimulation. These immediate signal‐dependent changes in aKPClambda were observed for a PDGF receptor add‐back mutant (Y40/51) that possesses only two of the five major autophosphorylation sites and binds PI3‐kinase, and were inhibited by wortmannin, an inhibitor of PI3‐kinase. Furthermore, an N‐terminal fragment of the catalytic subunit of PI3‐kinase, p110alpha, inhibited aPKClambda‐dependent activation of TRE in Y40/51 cells stimulated with PDGF. Overexpression of p110alpha resulted in an enhancement of TRE expression in response to PDGF and the regulatory domain of aPKClambda inhibited this TRE activation in Y40/51 cells. These results provide the first in vivo evidence supporting the presence of a novel signalling pathway from receptor tyrosine kinases to aPKClambda through PI3‐kinase.

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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...