Basic HTML Version
Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
187
The extracellular loops of Smoothened play a regulatory role in Hedgehog signaling.
Candace E. Carroll, Marada Suresh, Daniel Stewart, J. Xiaoxi
Oyang, Stacey K. Ogden. Biochemistry, St Jude Children's Research Hospital, Memphis, TN.
The Hedgehog (Hh) signaling pathway plays a critical role during metazoan development and is frequently inappropriately activated in cancer.
Smoothened (Smo), a transmembrane protein that is a member of the G-protein coupled receptor (GPCR) superfamily, is absolutely required for the
transduction of ligand-induced pathway activation. The extracellular loops (EC) of canonical GPCRs harbor cysteine residues that form disulfide bonds that
affect both the active and inactive state by regulating receptor confirmation, dimerization and/or ligand binding. In the present study, we mutated the
conserved cysteine residues of Smo and tested for effects on Hh signal transduction. While mutating EC3 cysteines did not reveal altered signaling compared
to wild type Smo, investigations of EC1 and EC2 mutants reveal ligand-independent activation of the Hh pathway in both mammalian and Drosophila in
vitro systems. Indirect immunofluorescence reveals altered sub-cellular localization of both the EC1 and EC2 mutants, suggesting that these mutants are
signaling in a manner inconsistent with canonical Hh signaling. Further biochemical studies reveal a possible disulfide bond between an EC1 and the EC2
cysteine, which is seen in canonical GPCRs. Our studies reveal previously uncharacterized functional roles for Smo EC1 and EC2.
202A
A sensitized suppressor/enhancer screen for Hedgehog pathway components identifies the kinase
Darkener of apricot
as a direct regulator of the
transcription factor Ci.
Ryan R Hurtado
1
, Cheng Du
2
, Leonard Rabinow
3
, Robert Holmgren
1
. 1) Northwestern University, Evanston, IL; 2) Univ. Nebraska
Medical Center, Omaha, Nebraska; 3) Université de Paris XI, Paris, France.
As a key mediator of embryonic development, the Hedgehog (Hh) morphogen acts in a signaling gradient to specify the fates of nearby cells. In adults,
aberrant Hedgehog signaling is involved in a variety of carcinomas. While much is known about Hh signaling, many gaps of understanding in the pathway
still remain.
Using the
Drosophila
wing as a model for Hh signaling we performed an RNAi based suppressor/enhancer screen in a
fu
1
sensitized genetic background
looking for novel proteins involved in the Hh pathway. UAS-RNAi lines for all available kinases, phosphatases, acetylases and deacetylases as well as 1590
randomly selected genes were screened. Using the wing specific MS1096-Gal4 driver individual genes were knocked down and the distance between the 3rd
and 4th wing vein, which is directly regulated by Hh, was assessed. Our screen was able to successfully identify previously known pathway members as well
as 21 novel suppressors and 59 novel enhancers of
fu
1
. Many hits have shown interesting effects on Hh target gene expression and follow-up experiments are
being performed.
Knockdown of the kinase Darkener of Apricot (doa) enhanced
fu
1
in adult wings and reduced expression of the Hh target gene
dpp
in wing discs. Genetic
mutations in
doa
rescue the wing expansion phenotype as well as reduce ectopic target gene expression in wing discs of a Hh overexpression mutant Hh
mrt
.
A highly conserved
doa
consensus site at S199 of the Hh transcription factor Ci, known to be phosphorylated
in vivo
exists. In a S2R+ cell luciferase assay,
glutamate substitution at S199 results in higher Ci activity. Current experiments are investigating whether this increased activity can be recapitulated by
overexpression of
doa
both by luciferase experiments
in vitro
as well as with transgenics
in vivo
.
203B
Altered localization of activating Smoothened mutants.
Suresh Marada, Candace Carroll, Daniel Stewart, Jessica Ouyang, Stacey Ogden. Biochemistry,
St. Jude Children's Research Hospital, Memphis, TN.
Hedgehog (Hh) signal transduction plays an essential role in patterning and organizing tissues during metazoan development. Smoothened (Smo), the
obligate signal transducing molecule of the Hh pathway, is predicted to function as a G-protein coupled receptor. In the absence of Hh, Smo localizes to
intracellular vesicles, and upon Hh stimulation, translocates to the plasma membrane and undergoes a conformational shift of its intracellular domains to
facilitate signaling to downstream effectors. Numerous studies have examined the contribution of the Smo intracellular tail to signaling. However, few
studies have examined the contribution of Smo extracellular domains to its regulation and signaling. In this study we generated a set of novel Smo mutants
by replacing highly conserved extracellular loop cysteines with alanines. These Smo mutants, when expressed in Drosophila wings and embryos, induce Hh
gain-of-function phenotypes. Flip-out clonal analysis revealed that these mutants trigger ectopic stabilization of the Hh pathway transcriptional effector
cubitus interruptus (Ci), and ectopic expression of Hh target genes
patched (ptc)
and
decapentaplegic (dpp)
, suggesting that these mutants are constitutively
active. Based on these phenotypes we predicted that these activating Smo mutants would localize to the plasma membrane in a Hh-independent manner. On
the contrary, subcellular localization studies in Clone 8 and S2 cells revealed that these activating Smo mutants are not localized to plasma membrane, either
in the absence or presence of Hh, but are instead retained in the endoplasmic reticulum (ER). These results argue that the activating Smo mutants can induce
high-level signaling from the ER.
204C
The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates Fused by promoting its dimerization and
phosphorylation.
Qing Shi
1
, Shuang Li
1
, Jianhang Jia
2
, Jin Jiang
1
. 1) Department of Developmental Biology, UT Southwestern Medical Center at Dallas,
Dallas, TX 75390, USA; 2) Markey Cancer Center and Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536,
USA.
Hedgehog (Hh) transduces signal by regulating the subcellular localization and conformational state of the GPCR-like protein Smoothened (Smo) but how
Smo relays the signal to cytoplasmic signaling components remains poorly understood. Here, by colocalization study and fluorescence resonance energy
transfer (FRET) analysis, we find that Hh-induced Smo conformational change recruits Costal2 (Cos2)/Fused (Fu) and promotes Fu kinase domain
dimerization. By performing mutagenesis analysis and using phospho-specific antibodies, we show that induced dimerization through the Fu kinase domain
activates Fu by inducing multi-site phosphorylation of its activation loop (AL) and that phospho-mimetic mutations of AL activate the Hh pathway.
Interestingly, we observe that graded Hh signals progressively increase Fu kinase domain dimerization and AL phosphorylation, suggesting that Hh activates
Fu in a dose-dependent manner. Moreover, we find that activated Fu regulates Cubitus interruptus (Ci) by both promoting its transcriptional activator
activity and inhibiting its proteolysis into a repressor form. We provide evidence that activated Fu exerts these regulations by interfering with the formation
of Ci-Sufu and Ci-Cos2-kinase complexes that normally inhibit Ci activity and promote its processing. Taken together, our results suggest that Hh-induced
Smo conformational change facilitates the assembly of active Smo-Cos2-Fu signaling complexes that promote Fu kinase domain dimerization,