The Carboxyl-terminal Domain of the Protein Kinase Fused Can Function As a Dominant Inhibitor of Hedgehog Signaling

Molecular and Cellular Biology. Mar, 2002  |  Pubmed ID: 11839821

The secreted protein hedgehog (Hh) plays a critical role in the developmental patterning of multiple tissues. In Drosophila melanogaster, a cytosolic multiprotein signaling complex appears necessary for Hh signaling. Genes that encode components of this Hh signaling complex (HSC) were originally identified and characterized based on their genetic interactions with hh, as well as with each other. It is only in recent years that the mechanistic functions of these components have begun to be unraveled. Here, we have investigated the relationship between two components of the HSC, the serine/threonine protein kinase Fused (Fu) and the kinesin-related protein Costal2 (Cos2). We have reconstituted a Fu/Cos2 complex in vitro and shown that Fu is able to directly associate with Cos2, forming a complex whose molecular size is similar to a previously described complex found in Drosophila cell extracts. We have also determined that the carboxyl-terminal domain of Fu is necessary and sufficient for the direct binding of Fu to Cos2. To validate the physiological relevance of this interaction, we overexpressed the carboxyl-terminal domain of Fu in wild-type flies. These flies exhibit a phenotype similar to that seen in fu mutants and consistent with an hh loss-of-function phenotype. We conclude that the carboxyl-terminal domain of Fu can function in a dominant negative manner, by preventing endogenous Fu from binding to Cos2. Thus, we provide the first evidence that Hh signaling can be compromised by targeting the HSC for disruption.

Identification of a Functional Interaction Between the Transmembrane Protein Smoothened and the Kinesin-related Protein Costal2

Current Biology : CB. Nov, 2003  |  Pubmed ID: 14614827

The hedgehog (Hh) family of morphogens plays important instructional roles in the development of numerous metazoan structures. Consistent with the role Hh homologs play in cell fate determination, aberrant Hh signaling results in numerous human pathologies. Hh signal transduction is initiated when Hh binds to its receptor Patched (Ptc), activating the transmembrane protein Smoothened (Smo). Smo transmits its activation signal to a microtubule-associated Hedgehog signaling complex (HSC). At a minimum, the HSC consists of the Kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the transcription factor Cubitus interruptus (Ci). In response to HSC activation, the ratio between repressor and activator forms of Ci is altered, determining the expression levels of various Hh target genes. The steps between Smo activation and signaling to the HSC have not been described. Here, we describe a functional interaction between Smo and Cos2, which is necessary for Hh signaling. We propose that this interaction is direct and allows for activation of Ci in response to Hh. This work fills in the last major gap in our understanding of the Hh signal transduction pathway by suggesting that no intermediate signal is required to connect Smo to the HSC.

The Kinesin-related Protein Costal2 Associates with Membranes in a Hedgehog-sensitive, Smoothened-independent Manner

The Journal of Biological Chemistry. Feb, 2004  |  Pubmed ID: 14645371

In Drosophila, Hedgehog (Hh) signal transduction has been shown to require a multiprotein complex (Hedgehog signaling complex (HSC)), which includes the Kinesin-related protein Costal2 (Cos2), the serine/threonine protein kinase Fused (Fu), and the transcription factor Cubitus interruptus (Ci). We present evidence that a biologically relevant fraction of the HSC is found in association with cellular membranes. We demonstrate that Cos2 is capable of tethering an exogenous protein to vesicular membranes and that Cos2 association with membranes is Hh-sensitive. In addition, we demonstrate that Cos2 associates with membranes in cells that lack the transmembrane protein Smoothened (Smo) through a domain of Cos2 distinct from its recently characterized Smo binding domain. We suggest that an Hh-regulated membrane binding activity of Cos2 is part of the mechanism by which Cos2 contributes to Hh signaling. We propose a model in which there are two distinct HSCs with discrete subcellular localizations and activities: one is endosome-associated and facilitates production of a repressor form of Ci (HSC-R), and one is Smo-associated and promotes Ci activation (HSC-A). In response to Hh and through interaction with Cos2, Smo mediates both inhibition of the endosome-associated HSC-R and activation of HSC-A at the plasma membrane.

Regulation of Hedgehog Signaling: a Complex Story

Biochemical Pharmacology. Mar, 2004  |  Pubmed ID: 15104233

The Hedgehog (Hh) signal transduction pathway plays critical instructional roles during development. Activating mutations in human Hh signaling components predispose to a variety of tumor types, and have been observed in sporadic tumors occurring in a wide range of organs. Multiple insights into the regulation of Hh signaling have been achieved through studies using Drosophila melanogaster as a model organism. In Drosophila, regulation of the transcription factor Cubitus interruptus (Ci) is the ultimate target of the Hh pathway. Ci is regulated through communication of the membrane proteins Patched (Ptc) and Smoothened (Smo) to the intracellular Hedgehog Signaling Complex (HSC) in response to a graded concentration of Hh ligand. The HSC consists of the Kinesin Related Protein, Costal2 (Cos2), the serine-threonine protein kinase. Fused (Fu) and Ci. In the absence of Hh stimulation, the HSC is involved in processing of Ci to a truncated repressor protein. In response to Hh binding to Ptc, processing of Ci is blocked to allow for accumulation of full-length Ci activator protein(s). Differential concentrations of Hh ligand stimulate production of Ci transcriptional activators of varying strength, which facilitate activation of distinct subsets of target genes. The mechanism(s) by which Ptc and Smo communicate with the HSC in response to differential ligand concentrations to regulate Ci function are not yet fully elucidated. Here, we review what is known about regulation of individual Hh signaling components, concentrating on the mechanisms by which the Hh signal is propagated through Smo to the HSC.

An Intramolecular Association Between Two Domains of the Protein Kinase Fused is Necessary for Hedgehog Signaling

Molecular and Cellular Biology. Dec, 2004  |  Pubmed ID: 15542847

The protein kinase Fused (Fu) is an integral member of the Hedgehog (Hh) signaling pathway. Although genetic studies demonstrate that Fu is required for the regulation of the Hh pathway, the mechanistic role that it plays remains largely unknown. Given our difficulty in developing an in vitro kinase assay for Fu, we reasoned that the catalytic activity of Fu might be highly regulated. Several mechanisms are known to regulate protein kinases, including self-association in either an intra- or an intermolecular fashion. Here, we provide evidence that Hh regulates Fu through intramolecular association between its kinase domain (DeltaFu) and its carboxyl-terminal domain (Fu-tail). We show that DeltaFu and Fu-tail can interact in trans, with or without the kinesin-related protein Costal 2 (Cos2). However, since the majority of Fu is found associated with Cos2 in vivo, we hypothesized that Fu-tail, which binds Cos2 directly, would be able to tether DeltaFu to Cos2. We demonstrate that DeltaFu colocalizes with Cos2 in the presence of Fu-tail and that this colocalization occurs on a subset of membrane vesicles previously characterized to be important for Hh signal transduction. Additionally, expression of Fu-tail in fu mutant flies that normally express only the kinase domain rescues the fu wing phenotype. Therefore, reestablishing the association between these two domains of Fu in trans is sufficient to restore Hh signal transduction in vivo. In such a manner we validate our hypothesis, demonstrating that Fu self-associates and is functional in an Hh-dependent manner. Our results here enhance our understanding of one of the least characterized, yet critical, components of Hh signal transduction.

Smoothened Regulates Activator and Repressor Functions of Hedgehog Signaling Via Two Distinct Mechanisms

The Journal of Biological Chemistry. Mar, 2006  |  Pubmed ID: 16423832

The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through the activation of the seven-transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. Here, we provide evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the Drosophila Gli homolog cubitus interruptus (Ci), in two distinct manners. We show that many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci.

Costal2 Functions As a Kinesin-like Protein in the Hedgehog Signal Transduction Pathway

Current Biology : CB. Aug, 2008  |  Pubmed ID: 18691888

The Hedgehog (Hh) signaling pathway initiates an evolutionarily conserved developmental program required for the proper patterning of many tissues [1]. Although Costal2 (Cos2) is a requisite component of the Hh pathway, its mechanistic role is not well understood. Because of its primary sequence, Cos2 was initially predicted to function as a kinesin-like protein [2]. However, evidence showing that Cos2 function might require kinesin-like properties has been lacking [2-6]. Thus, the prevailing dogma in the field is that Cos2 functions solely as a scaffolding protein [7, 8]. Here, we show that Cos2 motility is required for its biological function and that this motility may be Hh regulated. We show that Cos2 motility requires an active motor domain, ATP, and microtubules. Additionally, Cos2 recruits and transports other components of the Hh signaling pathway, including the transcription factor Cubitus interruptus (Ci). Drosophila expressing cos2 mutations that encode proteins that lack motility are attenuated in their ability to regulate Ci activity and exhibit phenotypes consistent with attenuated Cos2 function [9]. Combined, these results demonstrate that Cos2 motility plays an important role in its function, regulating the amounts and activity of Ci that ultimately interpret the level of Hh to which cells are exposed.

A Quantification of Pathway Components Supports a Novel Model of Hedgehog Signal Transduction

The Journal of Biological Chemistry. Oct, 2009  |  Pubmed ID: 19717563

The secreted protein Hedgehog (Hh) plays a critical instructional role during metazoan development. In Drosophila, Hh signaling is interpreted by a set of conserved, downstream effectors that differentially localize and interact to regulate the stability and activity of the transcription factor Cubitus interruptus. Two essential models that integrate genetic, cell biological, and biochemical information have been proposed to explain how these signaling components relate to one another within the cellular context. As the molar ratios of the signaling effectors required in each of these models are quite different, quantitating the cellular ratio of pathway components could distinguish these two models. Here, we address this important question using a set of purified protein standards to perform a quantitative analysis of Drosophila cell lysates for each downstream pathway component. We determine each component's steady-state concentration within a given cell, demonstrate the molar ratio of Hh signaling effectors differs more than two orders of magnitude and that this ratio is conserved in vivo. We find that the G-protein-coupled transmembrane protein Smoothened, an activating component, is present in limiting amounts, while a negative pathway regulator, Suppressor of Fused, is present in vast molar excess. Interestingly, despite large differences in the steady-state ratio, all downstream signaling components exist in an equimolar membrane-associated complex. We use these quantitative results to re-evaluate the current models of Hh signaling and now propose a novel model of signaling that accounts for the stoichiometric differences observed between various Hh pathway components.

The Full-length Unprocessed Hedgehog Protein is an Active Signaling Molecule

The Journal of Biological Chemistry. Jan, 2010  |  Pubmed ID: 19920144

The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as approximately 45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved.

Transcriptome-wide Identification of RNA-binding Protein and MicroRNA Target Sites by PAR-CLIP

Cell. Apr, 2010  |  Pubmed ID: 20371350

RNA transcripts are subject to posttranscriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases.

Multimeric Assembly and Biochemical Characterization of the Trax-translin Endonuclease Complex

Nature Structural & Molecular Biology. Jun, 2011  |  Pubmed ID: 21552261

Trax-translin heteromers, also known as C3PO, have been proposed to activate the RNA-induced silencing complex (RISC) by facilitating endonucleolytic cleavage of the siRNA passenger strand. We report on the crystal structure of hexameric Drosophila C3PO formed by truncated translin and Trax, along with electron microscopic and mass spectrometric studies on octameric C3PO formed by full-length translin and Trax. Our studies establish that Trax adopts the translin fold, possesses catalytic centers essential for C3PO's endoRNase activity and interacts extensively with translin to form an octameric assembly. The catalytic pockets of Trax subunits are located within the interior chamber of the octameric scaffold. Truncated C3PO, like full-length C3PO, shows endoRNase activity that leaves 3'-hydroxyl-cleaved ends. We have measured the catalytic activity of C3PO and shown it to cleave almost stoichiometric amounts of substrate per second.

Integrative Regulatory Mapping Indicates That the RNA-binding Protein HuR Couples Pre-mRNA Processing and MRNA Stability

Molecular Cell. Aug, 2011  |  Pubmed ID: 21723170

RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in polypyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.

New Insights in the Mechanism of MicroRNA-mediated Target Repression

Nature Structural & Molecular Biology. Nov, 2011  |  Pubmed ID: 22056803

Identification of RNA-protein Interaction Networks Using PAR-CLIP

Wiley Interdisciplinary Reviews. RNA. Dec, 2011  |  Pubmed ID: 22213601

All mRNA molecules are subject to some degree of post-transcriptional gene regulation (PTGR) involving sequence-dependent modulation of splicing, cleavage and polyadenylation, editing, transport, stability, and translation. The recent introduction of deep-sequencing technologies enabled the development of new methods for broadly mapping interaction sites between RNA-binding proteins (RBPs) and their RNA target sites. In this article, we review crosslinking and immunoprecipitation (CLIP) methods adapted for large-scale identification of target RNA-binding sites and the respective RNA recognition elements. CLIP methods have the potential to detect hundreds of thousands of binding sites in single experiments although the separation of signal from noise can be challenging. As a consequence, each CLIP method has developed different strategies to distinguish true targets from background. We focus on photoactivatable ribonucleoside-enhanced CLIP, which relies on the intracellular incorporation of photoactivatable ribonucleoside analogs into nascent transcripts, and yields characteristic sequence changes upon crosslinking that facilitate the separation of signal from noise. The precise knowledge of the position and distribution of binding sites across mature and primary mRNA transcripts allows critical insights into cellular localization and regulatory function of the examined RBP. When coupled with other systems-wide approaches measuring transcript and protein abundance, the generation of high-resolution RBP-binding site maps across the transcriptome will broaden our understanding of PTGR and thereby lead to new strategies for therapeutic treatment of genetic diseases perturbing these processes. WIREs RNA 2011. doi: 10.1002/wrna.1103 For further resources related to this article, please visit the WIREs website.