A previously uncharacterized gene, DBC2 (deleted in breast cancer), was cloned from a homozygously deleted region at human chromosome 8p21. DBC2 contains a highly conserved RAS domain and two putative protein interacting domains. Our analyses indicate that DBC2 is the best candidate tumor suppressor gene from this region. It lies within the epicenter of the deletions and is homozygously deleted in 3.5% (7͞200) of breast tumors. Mutation analysis of DBC2 led to discovery of two instances of somatic missense mutations in breast tumor specimens, whereas no missense mutations were found in other candidates from the region. Unlike other genes in the region, expression of DBC2 is often extinguished in breast cancer cells or tissues. Moreover, our functional analysis revealed that DBC2 expression in breast cancer cells lacking DBC2 transcripts causes growth inhibition. By contrast, expression of a somatic mutant discovered in a breast cancer specimen does not suppress the growth of breast cancer cells.

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-box subunit of the SCF specifically recruiting a given substrate to the SCF core. Here we identify NIPA (nuclear interaction partner of ALK) as a human F-box-containing protein that defines an SCF-type E3 ligase (SCF(NIPA)) controlling mitotic entry. Assembly of this SCF complex is regulated by cell-cycle-dependent phosphorylation of NIPA, which restricts substrate ubiquitination activity to interphase. We show nuclear cyclin B1 to be a substrate of SCF(NIPA). Inactivation of NIPA by RNAi results in nuclear accumulation of cyclin B1 in interphase, activation of cyclin B1-Cdk1 kinase activity, and premature mitotic entry. Thus, SCF(NIPA)-based ubiquitination may regulate S-phase completion and mitotic entry in the mammalian cell cycle.

Anaplastic large-cell lymphoma is a subtype of nonHodgkin lymphomas characterized by the expression of CD30. More than half of these lymphomas carry a chromosomal translocation t(2;5) leading to expression of the oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). NPM-ALK is capable of transforming fibroblasts and lymphocytes in vitro and of causing lymphomas in mice. Previously, we and others demonstrated phospholipase C-␥ and phosphatidylinositol 3-kinase as crucial downstream signaling mediators of NPM-ALK-induced oncogenicity. In this study, we used an ALK fusion protein as bait in a yeast twohybrid screen identifying NIPA (nuclear interacting partner of ALK) as a novel downstream target of NPM-ALK. NIPA encodes a 60-kDa protein that is expressed in a broad range of human tissues and contains a classical nuclear translocation signal in its C terminus, which directs its nuclear localization. NIPA interacts with NPM-ALK and other ALK fusions in a tyrosine kinasedependent manner and is phosphorylated in NPM-ALKexpressing cells on tyrosine and serine residues with serine 354 as a major phosphorylation site. Overexpression of NIPA in Ba/F3 cells was able to protect from apoptosis induced by IL-3 withdrawal. Mutations of the nuclear translocation signal or the Ser-354 phosphorylation site impaired the antiapoptotic function of NIPA. In NPM-ALK-transformed Ba/F3 cells, apoptosis triggered by wortmannin treatment was enhanced by overexpression of putative dominant-negative NIPA mutants. These results implicate an antiapoptotic role for NIPA in NPM-ALK-mediated signaling events.

Nuclear interaction partner of ALK (NIPA) is an F-box-containing protein that defines a nuclear skp1 cullin F-box (SCF)-type ubiquitin E3 ligase (SCF NIPA ) implicated in the regulation of mitotic entry. The SCF NIPA complex targets nuclear cyclin B1 for ubiquitination in interphase, whereas phosphorylation of NIPA in late G 2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1. Here, we identify the region of NIPA that mediates binding to its substrate cyclin B1. In addition to the recently described serine residue 354, we specify 2 new residues, Ser-359 and Ser-395, implicated in the phosphorylation process at G 2 /M within this region. Moreover, we found cyclin B1/Cdk1 to phosphorylate NIPA at Ser-395 in mitosis. Mutation of both Ser-359 and Ser-395 impaired effective inactivation of the SCF NIPA complex, resulting in reduced levels of mitotic cyclin B1. These data are compatible with a process of sequential NIPA phosphorylation where cyclin B1/Cdk1 amplifies phosphorylation of NIPA once an initial phosphorylation event has dissociated the SCF NIPA complex. Thus, cyclin B1/Cdk1 may contribute to the regulation of its own abundance in early mitosis.The SCF 3 family of E3 ubiquitin ligases essentially regulates the abundance of key cell cycle regulatory proteins. The F-box protein is the only variable component of the SCF complex that mediates substrate binding and thus determines specificity of the respective SCF complex (1-4). To bind the respective ubiquitylation target, each F-box protein contains a protein interaction domain. Depending on the type of the interaction domain, F-box proteins have been classified into three major classes; two classes of F-box proteins contain WD40 repeats and leucine-rich repeats (5, 6), whereas a third class contains other types of protein interaction domains or no canonical interaction domain (reviewed in Ref. (4). These classes of F-box proteins have been termed FBXWs, FBXLs and FBXOs, respectively (4, 7).We previously reported the cloning of NIPA (nuclear interaction partner of ALK) and subsequently characterized NIPA as an F-box-containing protein that defines an ubiquitin E3 ligase (SCF NIPA ) that targets nuclear cyclin B1 in interphase, thus contributing to the timing of mitotic entry (8 -10). The oscillating activity of the SCF NIPA complex is governed by cell cycle-dependent inhibitory phosphorylation of NIPA in late G 2 phase that dissociates NIPA from the SCF core. This phosphorylation event was found to occur, at least in part, on Ser-354 (9). The identity of the kinase(s) responsible for NIPA phosphorylation at G 2 /M and during mitosis has so far remained elusive.The substrate of NIPA, cyclin B1, together with its associated kinase Cdk1, form the maturation-promoting factor that essentially regulates the transition from G 2 phase into mitosis. Although mitotic entry requires activity of cyclin B1/Cdk1, mitotic exit requires its destruction. At mitotic entry, regulation of the maturation-promoting factor occurs at two distinct levels:...

SUMMARYNIPA (nuclear interaction partner of ALK) is an F-box-like protein that monitors the timing of mitotic entry. Constitutively active NIPA delays mitotic entry by preventing accumulation of nuclear cyclin B1. Here, we have investigated the consequences of Nipa inactivation by using a conditional knockout strategy. Nipa-deficient animals are viable but show a lower birth rate and reduced body weight. Furthermore, Nipa-deficient males are sterile owing to a block of spermatogenesis during meiotic prophase. Whereas Nipa -/-mouse embryonic fibroblasts show no severe phenotype, Nipa -/-spermatocytes arrest during stage IV of the epithelial cycle with subsequent TUNEL-positive apoptosis resulting from improper synapsis, defects in the repair of DNA double-stranded breaks and synaptonemal complex formation. Moreover, we show nuclear accumulation of cyclin B1 with a subsequent premature increase in G 2 /M kinase activity in Nipa -/-spermatocytes. Together, these results reveal a novel role for NIPA in meiosis.