The G protein-coupled receptor (GPCR)-like molecule Smoothened (Smo) undergoes dynamic intracellular trafficking modulated by the microtubule associated kinase GRK2 and recruitment of β-arrestin. Of this trafficking, especially the translocation of Smo into primary cilia and back to the cytoplasm is essential for the activation of Hedgehog (Hh) signaling in vertebrates. The complete mechanism of this bidirectional transport, however, is not completely understood. Here we demonstrate that Growth Arrest Specific 8 (Gas8), a microtubule associated subunit of the Dynein Regulatory Complex (DRC), interacts with Smo to modulate this process. Gas8 knockdown in ciliated cells reduces Smo signaling activity and ciliary localization whereas overexpression stimulates Smo activity in a GRK2-dependent manner. The C terminus of Gas8 is important for both Gas8 interaction with Smo and facilitating Smo signaling. In zebrafish, knocking down Gas8 results in attenuated Hh transcriptional responses and impaired early muscle development. These effects can be reversed by the co-injection of Gas8 mRNA or by constitutive activation of the downstream Gli transcription factors. Furthermore, Gas8 and GRK2 display a synergistic effect on zebrafish early muscle development and some effects of GRK2 knockdown can be rescued by Gas8 mRNA. Interestingly, Gas8 does not interfere with cilia assembly, as the primary cilia architecture is unchanged upon Gas8 knock down or heterologous expression. This is in contrast to cells stably expressing both GRK2 and Smo, in which cilia are significantly elongated. These results identify Gas8 as a positive regulator of Hh signaling that cooperates with GRK2 to control Smo signaling.

Smoothened, a heptahelical membrane protein, functions as the transducer of Hedgehog signaling. The kinases that modulate Smoothened have been thoroughly analyzed in flies. However, little is known about how phosphorylation affects Smoothened in vertebrates, mainly, because the residues, where Smoothened is phosphorylated are not conserved from Drosophila to vertebrates. Given its molecular architecture, Smoothened signaling is likely to be regulated in a manner analogous to G protein-coupled receptors (GPCRs). Previously, it has been shown, that arrestins and GPCR kinases, (GRKs) not only desensitize G protein-dependent receptor signaling but also function as triggers for GPCR trafficking and formation of signaling complexes. Here we describe that a GRK contributes to Smoothened-mediated signaling in vertebrates. Knockdown of the zebrafish homolog of mammalian GRK2/3 results in lowered Hedgehog transcriptional responses, impaired muscle development, and neural patterning. Results obtained in zebrafish are corroborated both in cell culture, where zGRK2/3 phosphorylates Smoothened and promotes Smoothened signal transduction and in mice where deletion of GRK2 interferes with neural tube patterning. Together, these data suggest that a GRK functions as a vertebrate kinase for Smoothened, promoting Hedgehog signal transduction during early development.

The oncogenic process often leads to a loss of normal telomere length control, usually as a result of activation of telomerase. Nevertheless, there are also telomerase-independent events that involve a Rad50-dependent recombination mechanism to maintain telomere length. Previous work has implicated the Rb family of proteins in the control of telomere length, and we now demonstrate that the p130 member of the Rb family is critical for telomere length control. p130 interacts specifically with the RINT-1 protein, previously identified as a Rad50-interacting protein. We further show that RINT-1 is essential for telomere length control. We propose that p130, forming a complex with Rad50 through RINT-1, blocks telomerase-independent telomere lengthening in normal cells. Given previous work implicating E2F in the control of telomerase gene expression, these results thus point to complementary roles for the Rb/E2F pathway in the control of telomere length.

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

The E2F4 and E2F5 proteins specifically associate with the Rb-related p130 protein in quiescent cells to repress transcription of various genes encoding proteins important for cell growth. A series of reports has provided evidence that Rb-mediated repression involves both histone deacetylase (HDAC)-dependent and HDAC-independent events. Our previous results suggest that one such mechanism for Rb-mediated repression, independent of recruitment of HDAC, involves the recruitment of the COOH-terminal binding protein (CtBP) corepressor, a protein now recognized to play a widespread role in transcriptional repression. We now find that CtBP can interact with the histone acetyltransferase, cyclic AMP-responsive element-binding protein (CREB) binding protein, and inhibit its ability to acetylate histone. This inhibition is dependent on a NH2-terminal region of CtBP that is also required for transcription repression. These results thus suggest two complementary mechanisms for E2F/p130-mediated repression that have in common the control of histone acetylation at target promoters.

Huntingtin is a protein of unknown function that contains a polyglutamine tract, which is expanded in patients with Huntington's disease (HD). We investigated the localization and a potential function for huntingtin in the nucleus. In human fibroblasts from normal and HD patients, huntingtin localized diffusely in the nucleus and in subnuclear compartments identified as speckles, promyelocytic leukemia protein bodies, and nucleoli. Huntingtin-positive nuclear bodies redistributed after treatment with sodium butyrate. By Western blot, purified nuclei had low levels of full-length huntingtin compared with the cytoplasm but contained high levels of N- and C-terminal huntingtin fragments, which tightly bound the nuclear matrix. Full-length huntingtin co-immunoprecipitated with the transcriptional corepressor C-terminal binding protein, and polyglutamine expansion in huntingtin reduced this interaction. Full-length wild-type and mutant huntingtin repressed transcription when targeted to DNA. Truncated N-terminal mutant huntingtin repressed transcription, whereas the corresponding wild-type fragment did not repress transcription. We speculate that wild-type huntingtin may function in the nucleus in the assembly of nuclear matrix-bound protein complexes involved with transcriptional repression and RNA processing. Proteolysis of mutant huntingtin may alter nuclear functions by disrupting protein complexes and inappropriately repressing transcription in HD.

: Transcription repression of E2F target genes has been shown to be critical for the maintenance of cellular quiescence and growth control. While it is clear that E2F-Rb and E2F-pl3O complexes promote this transcriptional regulation, the mechanism of action of these complexes remains unclear. The work supported by this grant aims to elucidate the mechanisms of transcriptional repression through E2F-pl3O complexes. Previous results indicated that while repression through pl3O involves recruitment of histone deacetylase, other mechanisms that are histone deacetylase independent are also involved. We have found that pl3O can recruit the CtBP co-repressor protein through interaction with the CtIP protein. We have mapped the domain of CtBP that is important for its co-repressor function (PID domain) and data suggest that CtBP functions as a repressor in part by inactivating the histone acetyltransferase activity of the transcription co-activator, CBP.

Previous work has demonstrated the critical role for transcription repression in quiescent cells through the action of E2F-Rb or E2F-p130 complexes. Recent studies have shown that at least one mechanism for this repression involves the recruitment of histone deacetylase. Nevertheless, these studies also suggest that other events likely contribute to E2F/Rb-mediated repression. Using a yeast two-hybrid screen to identify proteins that specifically interact with the Rb-related p130 protein, we demonstrate that p130, as well as Rb, interacts with a protein known as CtIP. This interaction depends on the p130 pocket domain, which is important for repression activity, as well as an LXCXE sequence within CtIP, a motif previously shown to mediate interactions of viral proteins with Rb. CtIP interacts with CtBP, a protein named for its ability to interact with the C-terminal sequences of adenovirus E1A. Recent work has demonstrated that the Drosophila homologue of CtBP is a transcriptional corepressor for Hairy, Knirps, and Snail. We now show that both CtIP and CtBP can efficiently repress transcription when recruited to a promoter by the Gal4 DNA binding domain, thereby identifying them as corepressor proteins. Moreover, the full repression activity of CtIP requires a PLDLS domain that is also necessary for the interaction with CtBP. We propose that E2F-mediated repression involves at least two events, either the recruitment of a histone deacetylase or the recruitment of the CtIP/CtBP corepressor complex.

The gene defective in Huntington's disease encodes a protein, huntingtin, with unknown function. Antisera generated against three separate regions of huntingtin identified a single high molecular weight protein of approximately 320 kDa on immunoblots of human neuroblastoma extracts. The same protein species was detected in human and rat cortex synaptosomes and in sucrose density gradients of vesicle-enriched fractions, where huntingtin immunoreactivity overlapped with the distribution of vesicle membrane proteins (SV2, transferrin receptor, and synaptophysin). Immunohistochemistry in human and rat brain revealed widespread cytoplasmic labeling of huntingtin within neurons, particularly cell bodies and dendrites, rather than the more selective pattern of axon terminal labeling characteristic of many vesicle-associated proteins. At the ultrastructural level, immunoreactivity in cortical neurons was detected in the matrix of the cytoplasm and around the membranes of the vesicles. The ubiquitous cytoplasmic distribution of huntingtin in neurons and its association with vesicles suggest that huntingtin may have a role in vesicle trafficking.