We show here that c‐Myc antagonizes the cyclin‐dependent kinase (CDK) inhibitor p27Kip1. p27 expressed from recombinant retroviruses in Rat1 cells associated with and inhibited cyclin E/CDK2 complexes, induced accumulation of the pRb and p130 proteins in their hypophosphorylated forms, and arrested cells in G1. Prior expression of c‐Myc prevented inactivation of cyclin E/CDK2 as well as dephosphorylation of pRb and p130, and allowed continuous cell proliferation in the presence of p27. This effect did not require ubiquitin‐mediated degradation of p27. Myc altered neither the susceptibility of cyclin E/CDK2 to inhibition by p27, nor the intrinsic CDK‐inhibitory activity of p27, but induced sequestration of p27 in a form unable to bind cyclin E/CDK2. Neither Myc itself nor other G1‐cyclin/CDK complexes were directly responsible for p27 sequestration. Retroviral expression of G1 cyclins (D1–3, E or A) or of the Cdc25A phosphatase did not overcome p27‐induced arrest. Growth rescue by Myc required dimerization with Max, DNA binding and an intact transcriptional activation domain, as previously shown for cellular transformation. We propose that this activity is mediated by the product of an as yet unknown Myc‐Max target gene(s) and represents an essential aspect of Myc's mitogenic and oncogenic functions.

Ectopic expression of the c-Myc oncoprotein prevents cell cycle arrest in response to growth-inhibitory signals, differentiation stimuli, or mitogen withdrawal. Moreover, Myc activation in quiescent cells is sufficient to induce cell cycle entry in the absence of growth factors. Thus, Myc transduces a potent mitogenic stimulus but, concomitantly, induces apoptosis in the absence of survival factors. We review here recent progress in our understanding of the molecular mechanisms linking Myc activity to cell cycle control. Myc is a positive regulator of G1-specific cyclin-dependent kinases (CDKs) and, in particular, of cyclin E/CDK2 complexes. Cyclin D/CDK4 and CDK6 may conceivably also be activated by Myc, but the circumstances in which this occurs remain to be explored. Myc acts via at least three distinct pathways which can enhance CDK function: (1) functional inactivation of the CDK inhibitor p27Kip1 and probably also of p21Cip1 and p57Kip2, (2) induction of the CDK-activating phosphatase Cdc25A and (3) - in an ill understood and most likely indirect way - deregulation of cyclin E expression. Constitutive expression of either Myc or cyclin E can prevent growth arrest by p16INK4a (an inhibitor of cyclin D/CDK4, but not of cyclin E/CDK2). In cells, p16INK4a inhibits phosphorylation, and thus induces activation of the Retinoblastoma-family proteins (pRb, p107 and p130). Surprisingly, this effect of p16 is not altered in the presence of Myc or cyclin E. Thus, Myc and cyclin E/CDK2 activity unlink activation of p16 and pRb from growth arrest. Finally, Myc may itself be a functional target of cyclin D/CDK4 through its direct interaction with p107. We discuss how the effects of Myc on cell cycle control may relate to its oncogenic activity, and in particular to its ability to cooperate with activated Ras oncoproteins.

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.The ED1 gene encodes a protein, ectodysplasin-A (EDA), 1 recently recognized to be a member of the tumor necrosis factor (TNF) superfamily of ligands. Mutations within the ED1 gene cause an X-linked recessive disorder, hypohidrotic or anhidrotic ectodermal dysplasia (ED1, XLHED) (Mendelian inheritance in man 305100), involving abnormal morphogenesis of teeth, hair, and eccrine sweat glands. Various splice forms of the ED1 transcript have been detected, but two isoforms differing only by two amino acids, EDA-A1 (391 aa) and EDA-A2 (389 aa), contain a TNF homology domain (1-3). EDA is a type II transmembrane protein with a small N-terminal intracellular domain and a larger C-terminal extracellular domain containing a (Gly-X-Y) 19 collagen-like repeat with a single interruption and a C-terminal TNF homology domain (Fig. 1A). The TNF homology domain is similar to other members of the TNF family, consisting of 10 predicted anti-parallel ␤-sheets linked by variable loops (Fig. 1A). TNF family ligands homotrimerize to form a pear-shaped quaternary structure able to bind a receptor molecule at each monomer-monomer interface (4, 5). The closest EDA homologues in the TNF family are BAFF/ BLyS, APRIL, and TWEAK, although none of them contains collagen-like repeats (6 -9). All four ligands contain consensus sequences for proteolytic cleavage by furin within their extracellular domain. In the case of EDA, two overlapping consensus sites are located between the transmembrane and the collagenlike domains (Fig. 1A). EDA-A1, but not EDA-A2, has been shown to specifically bind to EDAR, a member of the TNF receptor superfamily that, like most members of the TNF receptor family, activates the NF-B and c-Jun N-terminal kinase pathways (3, 10). Mutations in DL (EDAR), the human homologue of the murine downless locus, produce an identical phenotype to loss of function of EDA (11,12)...

Retroviral expression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4a) in rodent fibroblasts induces dephosphorylation of pRb, p107 and p130 and leads to G1 arrest. Prior expression of cyclin E allows S-phase entry and long-term proliferation in the presence of p16. Cyclin E prevents neither the dephosphorylation of pRb family proteins, nor their association with E2F proteins in response to p16. Thus, cyclin E can bypass the p16/pRb growth-inhibitory pathway downstream of pRb activation. Retroviruses expressing E2F-1, -2 or -3 also prevent p16-induced growth arrest but are ineffective against the cyclin E-CDK2 inhibitor p27(Kip1), suggesting that E2F cannot substitute for cyclin E activity. Thus, cyclin E possesses an E2F-independent function required to enter S-phase. However, cyclin E may not simply bypass E2F function in the presence of p16, since it restores expression of E2F-regulated genes such as cyclin A or CDC2. Finally, c-Myc bypasses the p16/pRb pathway with effects indistinguishable from those of cyclin E. We suggest that this effect of Myc is mediated by its action upstream of cyclin E-CDK2, and occurs via the neutralization of p27(Kip1) family proteins, rather than induction of Cdc25A. Our data imply that oncogenic activation of c-Myc, and possibly also of cyclin E, mimics loss of the p16/pRb pathway during oncogenesis.

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