p15Ink4b is a critical tumour suppressor in the absence of p16Ink4a

The CDKN2b-CDKN2a locus on chromosome 9p21 in human (chromosome 4 in mouse) is frequently lost in cancer. The locus encodes three cell cycle inhibitory proteins: p15INK4b encoded by CDKN2b, p16INK4a encoded by CDKN2a and p14ARF (p19Arf in mice) encoded by an alternative reading frame of CDKN2a (ref. 1). Whereas the tumour suppressor functions for p16INK4a and p14ARF have been firmly established, the role of p15INK4b remains ambiguous. However, many 9p21 deletions also remove CDKN2b, so we hypothesized a synergistic effect of the combined deficiency for p15INK4b, p14ARF and p16INK4a. Here we report that mice deficient for all three open reading frames (Cdkn2ab-/-) are more tumour-prone and develop a wider spectrum of tumours than Cdkn2a mutant mice, with a preponderance of skin tumours and soft tissue sarcomas (for example, mesothelioma) frequently composed of mixed cell types and often showing biphasic differentiation. Cdkn2ab-/- mouse embryonic fibroblasts (MEFs) are substantially more sensitive to oncogenic transformation than Cdkn2a mutant MEFs. Under conditions of stress, p15Ink4b protein levels are significantly elevated in MEFs deficient for p16Ink4a. Our data indicate that p15Ink4b can fulfil a critical backup function for p16Ink4a and provide an explanation for the frequent loss of the complete CDKN2b-CDKN2a locus in human tumours.     

HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSalpha (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSbeta (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3 ) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6 -deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3. Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.     

Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase

The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.     

Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.

Inheritance of one defective BRCA2 allele predisposes humans to breast cancer. To establish a mouse model for BRCA2-associated breast cancer, we generated mouse conditional mutants with BRCA2 and/or p53 inactivated in various epithelial tissues, including mammary-gland epithelium. Although no tumors arose in mice carrying conditional Brca2 alleles, mammary and skin tumors developed frequently in females carrying conditional Brca2 and Trp53 alleles. The presence of one wildtype Brca2 allele resulted in a markedly delayed tumor formation; loss of the wildtype Brca2 allele occurred in a subset of these tumors. Our results show that inactivation of BRCA2 and of p53 combine to mediate mammary tumorigenesis, and indicate that disruption of the p53 pathway is pivotal in BRCA2-associated breast cancer.     

Requirement for a functional Rb-1 gene in murine development.

Human retinoblastomas can occur both as hereditary and as sporadic cases. Knudson's proposal that they result from two mutational events, of which one is present in the germ line in hereditary cases, has been confirmed by more recent molecular analysis, which has shown both events to involve loss or mutational inactivation of the same gene, RB-1 (ref. 2). RB-1 heterozygosity also predisposes to osteosarcoma, and RB-1 allele losses are seen in sporadic lung, breast, prostate and bladder carcinomas. RB-1 is expressed in most, if not all, tissues and codes for a nuclear phosphoprotein which becomes hypophosphorylated in the G0 growth arrest state and in the G1 phase of the cell cycle. To gain a further insight into the role of RB-1 we and other groups have generated mice carrying an inactivated allele of the homologous gene, Rb-1 (ref. 10), by gene targeting. We report here that young heterozygous mice do not appear abnormal and do not develop retinoblastoma at a detectable frequency. However, homozygous mutant embryos fail to reach term and show a number of abnormalities in neural and haematopoietic development. Broadly similar results are reported by the other groups.     

A genome-wide association meta-analysis identifies new childhood obesity loci

Multiple genetic variants have been associated with adult obesity and a few with severe obesity in childhood; however, less progress has been made in establishing genetic influences on common early-onset obesity. We performed a North American, Australian and European collaborative meta-analysis of 14 studies consisting of 5,530 cases (≥95th percentile of body mass index (BMI)) and 8,318 controls (<50th percentile of BMI) of European ancestry. Taking forward the eight newly discovered signals yielding association with P < 5 × 10(-6) in nine independent data sets (2,818 cases and 4,083 controls), we observed two loci that yielded genome-wide significant combined P values near OLFM4 at 13q14 (rs9568856; P = 1.82 × 10(-9); odds ratio (OR) = 1.22) and within HOXB5 at 17q21 (rs9299; P = 3.54 × 10(-9); OR = 1.14). Both loci continued to show association when two extreme childhood obesity cohorts were included (2,214 cases and 2,674 controls). These two loci also yielded directionally consistent associations in a previous meta-analysis of adult BMI(1).     

Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase

Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.