The bmi-1 and myc oncogenes collaborate strongly in murine lymphomagenesis, but the basis for this collaboration was not understood. We recently identified the ink4a-ARF tumor suppressor locus as a critical downstream target of the Polycomb-group transcriptional repressor Bmi-1. Others have shown that part of Myc's ability to induce apoptosis depends on induction of p19arf. Here we demonstrate that down-regulation of ink4a-ARF by Bmi-1 underlies its ability to cooperate with Myc in tumorigenesis. Heterozygosity for bmi-1 inhibits lymphomagenesis in Eµ-myc mice by enhancing c-Myc-induced apoptosis. We observe increased apoptosis in bmi-1 −/− lymphoid organs, which can be rescued by deletion of ink4a-ARF or overexpression of bcl2. Furthermore, Bmi-1 collaborates with Myc in enhancing proliferation and transformation of primary embryo fibroblasts (MEFs) in an ink4a-ARF dependent manner, by prohibiting Myc-mediated induction of p19arf and apoptosis. We observe strong collaboration between the Eµ-myc transgene and heterozygosity for ink4a-ARF, which is accompanied by loss of the wild-type ink4a-ARF allele and formation of highly aggressive B-cell lymphomas. Together, these results reinforce the critical role of Bmi-1 as a dose-dependent regulator of ink4a-ARF, which on its turn acts to prevent tumorigenesis on activation of oncogenes such as c-myc.

MAP kinases phosphorylate specific groups of substrate proteins. Here we show that the amino acid sequence FXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to substrates in multiple protein families. FXFP and the D box, a different docking site, form a modular recognition system, as they can function independently or in combination. FXFP is specific for ERK, whereas the D box mediates binding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities of ERK and JNK result from recognition of shared and unique docking sites. These findings enabled us to predict new ERK substrates and design peptide inhibitors of ERK that functioned in vitro and in vivo. Mitogen-activated protein (MAP) kinases are components of signaling cascades that regulate normal development and pathological processes such as oncogenesis. MAP kinases were identified during biochemical searches for serine/threonine-specific protein kinases stimulated by growth factors in vertebrate cells (for review, see Sturgill and Wu 1991). MAP kinases were also identified in screens for mutations that affect intercellular signaling in yeast, worms, and flies (for review, see Ferrell 1996). Together, these investigations revealed that MAP kinases function in many cell types, are regulated by a diverse group of extracellular stimuli, and mediate a wide variety of cellular responses. MAP kinases can be divided into subfamilies based on specific conserved residues, particularly a TXY motif in the activation loop (Ferrell 1996). The three best-characterized subfamilies in vertebrates are named extracellular-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK, also called stress-activated protein kinase), and p38. There are probably several additional vertebrate MAP kinase subfamilies, since Saccharomyces cerevisiae contains six different MAP kinases (Madhani and Fink 1998). Here we use the name MAP kinase to refer to all members of the family, and the names ERK, JNK, and p38 to refer to members of those subfamilies.MAP kinases function in modules composed of three protein kinases (for review, see Marshall 1994). MAP kinase kinase kinases, such as Raf-1, phosphorylate and thereby activate MAP kinase kinases, such as MEK (MAP kinase kinase or ERK kinase). MAP kinase kinases are serine/threonine and tyrosine-specific protein kinases that phosphorylate the TXY motif and thereby activate MAP kinases. In general, MAP kinases in different subfamilies are members of separate modules and are regulated by distinct extracellular stimuli (for review, see Whitmarsh and Davis 1996). For example, ERK is activated strongly by receptor tyrosine kinases (RTK) such as the epidermal growth factor receptor, whereas JNK is activated strongly by stress stimuli such as ultraviolet light. Several of the signaling pathways leading from extracellular stimuli to the activation of a MAP kinase module are well defined, whereas others have yet to be characterized in detail. Whereas the upstream signaling events that r...

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Mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK) are important signaling proteins that phosphorylate (S/T)P sites in many different protein substrates. ERK binding to substrate proteins is mediated by docking sites including the FXFP motif and the D-domain. We characterized the sequence of amino acids that can constitute the FXFP motif using peptide and protein substrates. Substitutions of the phenylalanines at positions 1 and 3 had significant effects, indicating that these phenylalanines provide substantial binding affinity, whereas substitutions of the residues at positions 2 and 4 had less effect. The FXFP and D-domain docking sites were analyzed in a variety of positions and arrangements in the proteins ELK-1 and KSR-1. Our results indicate that the FXFP and D-domain docking sites form a flexible, modular system that has two functions. First, the affinity of a substrate for ERK can be regulated by the number, type, position, and arrangement of docking sites. Second, in substrates with multiple potential phosphorylation sites, docking sites can direct phosphorylation of specific (S/T)P residues. In particular, the FQFP motif of ELK-1 is necessary and sufficient to direct phosphorylation of serine 383, whereas the D-domain directs phosphorylation of other (S/T)P sites in ELK-1. The MAP1 kinase superfamily is composed of several subfamilies including ERK, c-Jun N-terminal kinase, and p38 (1, 2). These MAP kinases can be activated by a remarkably diverse set of stimuli that function through a variety of signaling pathways. MAP kinase activation is regulated by two upstream protein kinases; a MAP kinase kinase kinase, such as Raf, phosphorylates and thereby activates a MAP kinase kinase (3, 4). The MAP kinase kinase that regulates ERK is called MEK (MAP kinase kinase or ERK kinase). MEK is a dual specificity protein kinase that phosphorylates a threonine and tyrosine in a TXY motif of ERK, resulting in a significant increase in ERK kinase activity. ERK is inactivated by dual specificity phosphatases that dephosphorylate the TXY motif. Many different stimuli can activate the protein kinase cascade that activates ERK. One extensively characterized activation pathway is initiated by a secreted growth factor, such as epidermal growth factor, that leads to the activation of a receptor tyrosine kinase, Ras, and Raf (5).Signaling pathways that include ERK mediate a remarkably diverse set of responses during the development and homeostasis of organisms such as Caenorhabditis elegans, Drosophila, and vertebrates. The mechanisms that enable highly conserved signaling pathways to elicit cell type-specific responses are being actively investigated, yet remain poorly understood. In principle, any protein in a signaling cascade could function differently in different cell types and, thus, contribute to a specific response. However, ERK is likely to play an important role in generating cell type-specific responses (6). By contrast to Raf and MEK, whose only well documented physiologic...

The LIN-1 ETS transcription factor inhibits vulval cell fates during Caenorhabditis elegans development. We demonstrate that LIN-1 interacts with UBC-9, a small ubiquitin-related modifier (SUMO) conjugating enzyme. This interaction is mediated by two consensus sumoylation motifs in LIN-1. Biochemical studies showed that LIN-1 is covalently modified by SUMO-1. ubc-9 and smo-1, the gene encoding SUMO-1, inhibit vulval cell fates and function at the level of lin-1, indicating that sumoylation promotes LIN-1 inhibition of vulval cell fates. Sumoylation of LIN-1 promoted transcriptional repression and mediated an interaction with MEP-1, a protein previously shown to associate with the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies showed that mep-1 inhibits vulval cell fates and functions at the level of lin-1. We propose that sumoylation of LIN-1 mediates an interaction with MEP-1 that contributes to transcriptional repression of genes that promote vulval cell fates. These studies identify a molecular mechanism for SUMO-mediated transcriptional repression.