Mouse knockouts of Cdk2 and Cdk4 have demonstrated that, individually, these genes are not essential for viability. To investigate whether there is functional redundancy, we have generated double knockout (DKO) mice. Cdk2-/- Cdk4-/- DKOs die during embryogenesis around E15 as a result of heart defects. We observed a gradual decrease of Retinoblastoma protein (Rb) phosphorylation and reduced expression of E2F-target genes, like Cdc2 and cyclin A2, during embryogenesis and in embryonic fibroblasts (MEFs). DKO MEFs are characterized by a decreased proliferation rate, impaired S phase entry, and premature senescence. HPV-E7-mediated inactivation of Rb restored normal expression of E2F-inducible genes, senescence, and proliferation in DKO MEFs. In contrast, loss of p27 did not rescue Cdk2-/- Cdk4-/- phenotypes. Our results demonstrate that Cdk2 and Cdk4 cooperate to phosphorylate Rb in vivo and to couple the G1/S phase transition to mitosis via E2F-dependent regulation of gene expression.

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Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder characterized by cutaneous fibrofolliculomas, pulmonary cysts, and kidney malignancies. Affected individuals carry germ line mutations in folliculin (FLCN), a tumor suppressor gene that becomes biallelically inactivated in kidney tumors by second-hit mutations. Similar to other factors implicated in kidney cancer, FLCN has been shown to modulate activation of mammalian target of rapamycin (mTOR). However, its precise in vivo function is largely unknown because germ line deletion of Flcn results in early embryonic lethality in animal models. Here, we describe mice deficient in the newly characterized folliculin-interacting protein 1 (Fnip1). In contrast to Flcn, Fnip1 ؊/؊ mice develop normally, are not susceptible to kidney neoplasia, but display a striking pro-B cell block that is entirely independent of mTOR activity. We show that this developmental arrest results from rapid caspaseinduced pre-B cell death, and that a Bcl2 transgene reconstitutes mature B-cell populations, respectively. We also demonstrate that conditional deletion of Flcn recapitulates the pro-B cell arrest of Fnip1 ؊/؊ mice. Our studies thus demonstrate that the FLCN-FNIP complex deregulated in BHD syndrome is absolutely required for B-cell differentiation, and that it functions through both mTORdependent and independent pathways. (Blood. 2012;120(6):1254-1261) IntroductionHuman Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant hereditary cancer syndrome characterized by the development of cutaneous fibrofolliculoma, multiple pulmonary cysts, spontaneous pneumothorax, and increased risk of renal cell neoplasia. 1,2 Affected individuals carry germ line mutations in FLCN, a recently characterized gene that encodes a 64-kDa protein, folliculin. 2 In kidney tumors, the FLCN wild-type (WT) allele is deleted by somatic mutation or loss of heterozygosity, [3][4][5] supporting the idea that FLCN is a classic tumor suppressor gene that follows the Knudson 2-hit hypothesis. 6 FLCN lacks predicted functional domains or homology to known proteins; however, it is also highly conserved across species indicating that it might play an essential role in development. 2 In agreement with this notion, homozygous loss of Flcn in mice causes visceral endoderm defects and embryonic lethality at E5.5-E6.5. 5 Although its precise in vivo function is unknown, folliculin may regulate cellular energy sensing by controlling mTOR signaling. [7][8][9][10] For instance, loss of Flcn in mouse kidneys leads to cellular hyperproliferation and overactivation of mTORC1, mTORC2, and MAPK pathways, whereas rapamycin treatment partially rescues this phenotype. 5,[11][12][13][14] In this context, FLCN is not unlike the 6 additional genes that have so far been implicated in kidney cancer (VHL, MET, TSC1, TSC2, FH, and SDH), in that they all interact with metabolic pathways that respond to stress or nutrient stimulation (reviewed by Linehan et al 15 ). However, mounting evidence indicates that folliculin may also reg...