Cyclin D1 is a proto-oncogene that functions by inactivation of the retinoblastoma tumor suppressor protein, RB. A common polymorphism in the cyclin D1 gene is associated with the production of an alternate transcript of cyclin D1, termed cyclin D1b. Both the polymorphism and the variant transcript are associated with increased risk for multiple cancers and the severity of a given cancer; however, the underlying activities of cyclin D1b have not been elucidated relative to the canonical cyclin D1a. Because cyclin D1b does not possess the threonine 286 phosphorylation site required for nuclear export and regulated degradation, it has been hypothesized to encode a stable nuclear protein that would constitutively inactivate the RB pathway. Surprisingly, we find that cyclin D1b protein does not inappropriately accumulate in cells and exhibits stability comparable to cyclin D1a. As expected, the cyclin D1b protein was constitutively localized in the nucleus, whereas cyclin D1a was exported to the cytoplasm in S-phase. Despite enhanced nuclear localization, we find that cyclin D1b is a poor catalyst of RB phosphorylation/inactivation. However, cyclin D1b potently induced cellular transformation in contrast to cyclin D1a. In summary, we demonstrate that cyclin D1b specifically disrupts contact inhibition in a manner distinct from cyclin D1a. These data reveal novel roles for D-type cyclins in tumorigenesis.Cyclin D1 is an essential regulator of cell cycle progression, and aberrant induction of cyclin D1 activity is well established in human tumorigenesis (1-7). Initially, cyclin D1 was identified as the PRAD1 oncogene by mapping the sites of amplification in parathyroid adenomas to 11q13 (1,8). Subsequently, deregulation of cyclin D1 has been observed to occur in a variety of cancer types (9). For example, the BCL-1 translocation in centrocytic lymphoma deregulates the expression of cyclin D1 (8). In animal models, cyclin D1 has also been shown to exhibit oncogenic activity when overexpressed in specific tissues (10). Lastly, cyclin D1 activity is critical for tumor formation induced by other oncogenes (e.g. Ras), as mice deficient in cyclin D1 are resistant to tumorigenesis (11,12). Because of the intimate connections between cyclin D1 and oncogenesis, extensive analyses have focused on the mechanisms through which cyclin D1 contributes to human cancer.Cyclin D1 exerts its effects on cellular proliferation by integrating external signals (e.g. mitogens) with the cell cycle machinery (3-7, 13-15). Given this critical role, cyclin D1 action is highly regulated. Cyclin D1 transcription is stimulated as a delayed early response to mitogenic signaling cascades (4, 16). Additionally, protein stability is regulated through the glycogen synthase kinase 3␤ signal transduction pathway to coordinately enhance accumulation of cyclin D1 protein (17, 18). Once synthesized, cyclin D1 interacts with and activates the G 1 cyclin-dependent kinases (CDK), 1 CDK4 and CDK6 (19,20). This interaction is assisted through the action of both m...

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DNA-damage evokes cell cycle checkpoints, which function to maintain genomic integrity. The retinoblastoma tumor suppressor (RB) and mismatch repair complexes are known to contribute to the appropriate cellular response to specific types of DNA damage. However, the signaling pathways through which these proteins impact the cell cycle machinery have not been explicitly determined. RB-deficient murine embryo fibroblasts continued a high degree of DNA replication following the induction of cisplatin damage, but were inhibited for G 2 /M progression. This damage led to RB dephosphorylation/activation and subsequent RBdependent attenuation of cyclin A and CDK2 activity. In both Rb؉/؉ and Rb؊/؊ cells, cyclin D1 expression was attenuated following DNA damage. As cyclin D1 is a critical determinant of RB phosphorylation and cell cycle progression, we probed the pathway through which cyclin D1 degradation occurs in response to DNA damage. We found that attenuation of endogenous cyclin D1 is dependent on multiple mismatch repair proteins. We demonstrate that the mismatch repair-dependent attenuation of endogenous cyclin D1 is critical for attenuation of CDK2 activity and induction of cell cycle checkpoints. Together, these studies couple the activity of the retinoblastoma and mismatch repair tumor suppressor pathways through the degradation of cyclin D1 and dual attenuation of CDK2 activity.DNA damage induces checkpoints to prevent damaged cells from progressing deleteriously through the cell cycle (1-5). It is postulated that genetic damage is sensed by specific proteins which initiate signal transduction pathways to inhibit cell cycle progression. Cell cycle transitions are driven by the coordinated activity of cyclin-dependent kinase (CDK) 1 cyclin complexes. Mitogens stimulate the expression of cyclin D and the subsequent activation of CDK4-cyclin D complexes (6). These cyclin D-associated complexes initiate the phosphorylation of RB, which disrupts RB-mediated transcriptional repression of specific target genes allowing progression through G 1 (7-9). It is believed that the targets for RB are encompassed by a host of E2F-regulated genes, including metabolic enzymes and cyclins E and A (10, 11). Since the activity of these cyclins is required for cell cycle progression, RB phosphorylation/inactivation is requisite for passage into S-phase. Subsequent activation of CDC2-cyclin B complexes is required for mitotic entry. Importantly, numerous participants in checkpoint processes are implicated in tumor development/progression.In fact, RB is a critical cell cycle regulator that has recently been shown to participate in the cellular response to DNA damage (12)(13)(14). Environmental stresses such as DNA damage prevent RB phosphorylation, thus leading to RB-dependent cessation of cell cycle progression. For example, RB is dephosphorylated/activated when primary fibroblasts are exposed to ionizing radiation, and this event triggers cell cycle arrest (13). Our laboratory has previously shown that the role of RB is also conser...

Protein tyrosine phosphorylation is an integral part of cytokine-induced proliferation and differentiation of hematopoietic cells. The authors previously reported cloning and characterization of the receptor tyrosine kinase Tif, also termed Tyro3. Using the yeast 2-hybrid technology, they recently identified that the p85 subunit of phosphatidylinositol 3-kinase (PI3 kinase) interacted with the cytoplasmic domain of Tyro3. On treatment with epidermal growth factor (EGF), NIH3T3 cells expressed EGFR/Tyro3 (a fusion receptor with the extracellular domain from epidermal growth factor receptor and the transmembrane and cytoplasmic domains from Tyro3), and EGFR/Tyro3 was rapidly phosphorylated on tyrosine residues. The interaction between Tyro3 and p85 was also confirmed by glutathione S-transferase (GST) pull-down experiments. Co-immunoprecipitation followed by Western blot analysis revealed that PI3 kinase was associated with and phosphorylated by the activated Tyro3. Tyro3-associated PI3 kinase exhibited an enhanced kinase activity. In addition, EGF treatment of EGFR/Tyro3-expressing cells led to enhanced phosphorylation of Akt, a downstream component of PI3 kinase. Treatment of NIH3T3 cells expressing a full length of rat Tyro-3, but not NIH3T3 cells, with protein S also resulted in phosphorylation of Akt. Soft agar colony assays showed that the addition of EGF to EGFR/Tyro3-transfected cells, but not to the parental NIH3T3 cells, resulted in a concentration-dependent increase in the formation of anchorage-independent colonies. Tyro3-mediated transformation of NIH3T3 cells was significantly blocked by wortmannin, a PI3 kinase-specific inhibitor. Results of these combined studies strongly suggested that the oncogenic transforming ability of Tyro3 was mediated at least in part by the PI3 kinase pathway.

The retinoblastoma (RB) and p16ink4a tumor suppressors are believed to function in a linear pathway that is functionally inactivated in a large fraction of human cancers. Recent studies have shown that RB plays a critical role in regulating S phase as a means for suppressing aberrant proliferation and controlling genome stability. Here, we demonstrate a novel role for p16ink4a in replication control that is distinct from that of RB. Specifically, p16ink4a disrupts prereplication complex assembly by inhibiting mini-chromosome maintenance (MCM) protein loading in G 1 , while RB was found to disrupt replication in S phase through attenuation of PCNA function. This influence of p16ink4a on the prereplication complex was dependent on the presence of RB and the downregulation of cyclin-dependent kinase (CDK) activity. Strikingly, the inhibition of CDK2 activity was not sufficient to prevent the loading of MCM proteins onto chromatin, which supports a model wherein the composite action of multiple G 1 CDK complexes regulates prereplication complex assembly. Additionally, p16ink4a attenuated the levels of the assembly factors Cdt1 and Cdc6. The enforced expression of these two licensing factors was sufficient to restore the assembly of the prereplication complex yet failed to promote S-phase progression due to the continued absence of PCNA function. Combined, these data reveal that RB and p16ink4a function through distinct pathways to inhibit the replication machinery and provide evidence that stepwise regulation of CDK activity interfaces with the replication machinery at two discrete execution points.Due to their profound influence on tumorigenesis, substantial effort has been directed at determining the function of the p16ink4a and retinoblastoma (RB) tumor suppressors in cell cycle control. Both tumor suppressors are inactivated in human tumors, as achieved by genetic mutation, gene silencing, or functional inactivation (6,53,63,64). Biochemical and genetic evidence demonstrate a functional connection between the two tumor suppressors as part of a growth inhibitory network that is disrupted in the majority of cancers.p16ink4a is a cyclin-dependent kinase (CDK) inhibitor, which arrests cells in G 1 (38,48) and is dependent on inhibition of CDK4 and CDK2-associated activity. The influence of p16ink4a on CDK4 is mediated by direct binding and involves the disruption of CDK4/cyclin D interactions (57,63). This function of p16ink4a is required for its ability to inhibit cell cycle progression and is disrupted in tumors which express elevated levels of CDK4 (thus titrating p16ink4a) or in tumors that harbor specific CDK4 mutations that compromise p16ink4a association (28,70). In addition, p16ink4a-mediated disruption of the CDK4/cyclin D complex releases p27Kip1 and p21Cip1, leading to inhibition of CDK2 complexes (31,65). This action is similarly required for p16ink4a-mediated cell cycle inhibition and therefore differentiates the function of p16ink4a from the genetic loss of CDK4 alone. A principle target of CDK4 and CD...

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According to the known primary sequences of three neurotoxins active on small conductance Ca 2 -activated potassium channels from the scorpion Buthus martensi Karsch, their corresponding cDNAs were cloned and sequenced using 3P P-and 5P P-RACE. All of them encoded a signal peptide composed of 28 residues and a mature toxin of 29, 28 and 33 residues, respectively. Their cDNA deduced sequences were totally consistent with those determined, and the C-terminal amidation of one neurotoxin was confirmed. The genomic DNAs of these three toxins were also amplified by PCR, cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptide at the same 3 310 position upstream from the mature toxin, consisting of 94, 78 and 87 bp, respectively. z 1999 Federation of European Biochemical Societies.

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