T cell-specific transcription factor (TCF-1) and lymphoid enhancer factor 1 (LEF-1) have been implicated exclusively in the regulation of T cell-specific genes. The only adult tissue other than thymus known to express these factors is spleen and lymph node, which contain low levels of LEF-1 and no TCF-1. We noticed that genes involved in hair-specific gene expression possess LEF-I/TCF-1 consensus motifs located in similar positions relative to their TATA box. We show that of the two factors only LEF-1 is expressed in hair follicles; it can be cloned in both splice forms from human skin keratinocytes and it can bind to these sites in the hair promoters. We show that LEF-1 mRNA is present in pluripotent ectoderm, and it is up-regulated in a highly restricted pattern just before the formation of underlying mesenchymal condensates and commitment of overlying ectodermal cells to invaginate and become hair follicles. New waves of ectodermal LEF-1 spots appear concomitant with new waves of follicle morphogenesis. To test whether LEF-1 patterning might be functionally important for hair patterning and morphogenesis, we used transgenic technology to alter the patterning and timing of LEF-1 over the surface ectoderm. Striking abnormalities arose in the positioning and orientation of hair follicles, leaving a marked disruption of this normally uniform patterning. This provides the first direct evidence that ectodermal cues are critical in establishing these developmental processes, which at later stages are known to be influenced by underlying mesenchyme. Remarkably, elevated LEF-1 in the lip furrow epithelium of developing transgenic animals triggered these cells to invaginate, sometimes leading to the inappropriate adoption of hair follicle and tooth cell fates. Collectively, our findings demonstrate that ectodermal expression of LEF-1 plays a central role in gene expression, pattern formation, and other developmental processes involving epithelial-mesenchymal associations.

SUMMARY Current models imply that the FERM domain protein Merlin, encoded by the tumor suppressor NF2, inhibits mitogenic signaling at or near the plasma membrane. Here, we show that the closed, growth inhibitory form of Merlin accumulates in the nucleus, binds to the E3 ubiquitin ligase CRL4DCAF1, and suppresses its activity. Depletion of DCAF1 blocks the promitogenic effect of inactivation of Merlin. Conversely, enforced expression of a Merlin-insensitive mutant of DCAF1 counteracts the antimitogenic effect of Merlin. Re-expression of Merlin and silencing of DCAF1 induce a similar, tumor-suppressive program of gene expression. Tumor-derived mutations invariably disrupt Merlin’s ability to interact with or inhibit CRL4DCAF1. Finally, depletion of DCAF1 inhibits the hyperproliferation of Schwannoma cells from NF2 patients and suppresses the oncogenic potential of Merlin-deficient tumor cell lines. We propose that Merlin suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4DCAF1

The CUL4-DDB1 ubiquitin ligase regulates cell proliferation, survival, DNA repair, and genomic integrity through targeted ubiquitination of key regulators, yet the substrate receptors that dictate the specificity of this ubiquitination machinery have been largely unknown. Recent work identified a family of DDB1 and CUL4-associated factors (DCAFs) as substrate receptors, implicating a broad spectrum of cellular processes regulated by CUL4-DDB1.

Although there is a binding site on the proteasome for the polyubiquitin chains attached to degradation substrates by the ubiquitination machinery, it is currently unclear whether in vivo the activities of the ubiquitination machinery and the proteasome are coupled. Here we show that two human homologs of the yeast ubiquitin-like Dsk2 protein, hPLIC-1 and hPLIC-2, physically associate with both proteasomes and ubiquitin ligases in large complexes. Overexpression of hPLIC proteins interferes with the in vivo degradation of two unrelated ubiquitin-dependent proteasome substrates, p53 and IkappaBalpha, but not a ubiquitin-independent substrate. Our findings raise the possibility that the hPLIC proteins, and possibly related ubiquitin-like family members, may functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.

Cyclin-dependent kinase 5 (Cdk5) was originally isolated by its close homology to the human CDC2 gene, which is a key regulator of cell cycle progression. However, unlike other Cdks, the activity of Cdk5 is required in post-mitotic neurons. The neuronal-specific p35 protein, which shares no homology to cyclins, was identified by virtue of its association and activation of Cdk5. Gene targeting studies in mice have shown that the p35/Cdk5 kinase is required for the proper neuronal migration and development of the mammalian cortex. We have investigated the regulation of the p35/Cdk5 kinase. Here we show that p35, the activator of Cdk5, is a short-lived protein with a half-life (t 1/2 ) of 20 to 30 min. Specific proteasome inhibitors such as lactacystin greatly stabilize p35 in vivo. Ubiquitination of p35 can be readily demonstrated in vitro and in vivo. Inhibition of Cdk5 activity by a specific Cdk inhibitor, roscovitine, or by overexpression of a dominant negative mutant of Cdk5 increases the stability of p35 by 2-to 3-fold. Furthermore, phosphorylation mutants of p35 also stabilize p35 2-to 3-fold. Together, these observations demonstrate that the p35/Cdk5 kinase can be subject to rapid turnover in vivo and suggest that phosphorylation of p35 upon Cdk5 kinase activation plays a autoregulatory role in p35 degradation mediated by ubiquitin-mediated proteolysis.

The DDB1-Cul4A ubiquitin ligase complex promotes protein ubiquitination in diverse cellular functions and is reprogrammed by the V proteins of paramyxoviruses to degrade STATs and block interferon signaling. Here we report the crystal structures of DDB1 alone and in complex with the simian virus 5 V protein. The DDB1 structure reveals an intertwined three-propeller cluster, which contains two tightly coupled beta propellers with a large pocket in between and a third beta propeller flexibly attached on the side. The rigid double-propeller fold of DDB1 is targeted by the viral V protein, which inserts an entire helix into the double-propeller pocket, whereas the third propeller domain docks DDB1 to the N terminus of the Cul4A scaffold. Together, these results not only provide structural insights into how the virus hijacks the DDB1-Cul4A ubiquitin ligase but also establish a structural framework for understanding the multiple functions of DDB1 in the uniquely assembled cullin-RING E3 machinery.

The S. cerevisiae SCFCdc4p ubiquitin-protein ligase complex promotes cell cycle transitions through degradation of cell cycle regulators. To investigate SCFCdc4p regulation in vivo, we examined the stability of individual SCFCdc4p components. Whereas Cdc53p and Skp1p were stable, Cdc4p, the F box-containing component responsible for substrate recognition, was short lived and subject to SCF-mediated ubiquitination. Grr1p, another F box component of SCF complexes, was also ubiquitinated. A stable truncated Cdc4pF-beta-gal hybrid protein capable of binding Skp1p and entering into an SCF complex interfered with proteolysis of SCF targets and inhibited cell proliferation. The finding that the F box-containing SCF components are unstable suggests a mechanism of regulating SCF function through ubiquitination and proteolysis of F box components.

SUMMARY It is intuitively obvious that the ability of a cell to repair DNA damage is saturable, either by limitation of enzymatic activities, the time allotted to achieve their function, or both. However, very little is known regarding the mechanisms that establish such a threshold. Here we demonstrated that the CUL4A ubiquitin ligase restricts the cellular repair capacity by orchestrating the concerted actions of nucleotide excision repair (NER) and the DNA damage-responsive G1/S checkpoint through selective degradation of the DDB2 and XPC DNA damage sensors and the p21/CIP1/WAF1 checkpoint effector. We generated Cul4a conditional knockout mice and observed that skin-specific Cul4a ablation dramatically increased resistance to UV-induced skin carcinogenesis. Our findings reveal that wild-type cells do not operate at their full DNA repair potential, underscore the critical role of CUL4A in establishing the cellular DNA repair threshold, and highlight the potential augmentation of cellular repair proficiency by pharmacological CUL4A inhibition.