In S. cerevisiae, the G1/S transition requires Cdc4p, Cdc34p, Cdc53p, Skp1p, and the Cln/Cdc28p cyclin-dependent kinase (Cdk). These proteins are thought to promote the proteolytic inactivation of the S-phase Cdk inhibitor Sic1p. We show here that Cdc4p, Cdc53p, and Skp1p assemble into a ubiquitin ligase complex named SCFCdc4p. When mixed together, SCFCdc4p subunits, E1 enzyme, the E2 enzyme Cdc34p, and ubiquitin are sufficient to reconstitute ubiquitination of Cdk-phosphorylated Sic1p. Phosphorylated Sic1p substrate is specifically targeted for ubiquitination by binding to a Cdc4p/Skp1p subcomplex. Taken together, these data illuminate the molecular basis for the G1/S transition in budding yeast and suggest a general mechanism for phosphorylation-targeted ubiquitination in eukaryotes.

. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1⌬, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca2+influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4α-phorbol 12,13-didecanoate (4-αPDD), a TRPV4 ligand, increased intracellular Ca2+level only when Ca2+was present in the extracellular solution. The 4-αPDD-induced Ca2+response was inhibited by ruthenium red (1 μM), a known TRPV4 inhibitor, but not by capsazepine (1 μM), a TRPV1 antagonist, indicating that 4-αPDD-induced Ca2+response is mediated by TRPV4. Verapamil (10 μM), an L-type voltage-gated Ca2+channel inhibitor, had no effect on the 4-αPDD-induced Ca2+response, excluding the involvement of L-type Ca2+channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca2+channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca2+channel inhibitor nifedipine (1 μM) or by the TRPV1 inhibitor capsazepine (1 μM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins-ySKP1, CDC53 (Cullin), and the F-box protein CDC4-that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, we isolated hSKP1 in a two-hybrid screen with hCUL1, the closest human homologue of CDC53. Here, we show that hCUL1 associates with hSKP1 in vivo and directly interacts with both hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-like particle. Moreover, hCUL1 complements the growth defect of yeast cdc53 ts mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators͞components of hCUL1-based SCF complexes, to determine whether the hCUL2-hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins.The irreversible nature of proteolysis makes it well suited to serve as a regulatory switch for controlling unidirectional processes. This principle is clearly evident in the organization of the cell division cycle, where initiation of DNA replication, chromosome segregation, and exit from mitosis are triggered by the destruction of key regulatory proteins (1-3).Proteins typically are marked for proteolytic degradation by attachment of multiubiquitin chains. This process is initiated by a ubiquitin-activating enzyme (E1), which activates ubiquitin by adenylation and becomes linked to it via a thiolester bond. Ubiquitin then is transferred to a ubiquitin-conjugating enzyme, E2. Whereas E2s can attach ubiquitin directly to lysine residues in a substrate, most physiological ubiquitination reactions probably require a ubiquitin ligase, or E3 (4). E3s have been implicated in substrate recognition and, in one case, transfer of ubiquitin from E2 to a substrate via an E3-ubiquitin-thiolester intermediate (5). Once the substrate is multiubiquitinated, it then is recognized and degraded by the 26S proteasome.