The heparan sulfate-fibroblast growth factor signaling system in liver growth and function

The heparan sulfate (HS)-fibroblast growth factor (FGF) signaling system is a ubiquitous regulator that senses local environmental changes and mediates cell-to-cell communication. This system consists of three mutually interactive components. These are regulatory polypeptides (FGF), FGF receptor (FGFR) and heparan sulfate proteoglycans (FGFRHS). All four FGFR genes are expressed in the adult liver. Expression of the FGFR1–3 genes is generally associated with non-parenchymal cells while expression of the FGFR4 gene is associated with parenchymal hepatocytes. We showed that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (−/−) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7α-hydroxylase (Cyp7a), the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. These results indicated that FGFR4 was not directly involved in liver growth but exerted negative control on liver bile acid synthesis. This was confirmed in transgenic mice overexpressing the constitutively active human FGFR4 in livers. The transgenic mice exhibited decreased fecal bile acid excretion, bile acid pool size, and expression of Cyp7a. Introduction of this constitutively active human FGFR4 into FGFR4 (−/−) mice restored the inhibition of bile acid synthesis. Activation of the c-Jun N-terminal Kinase (JNK) pathway by FGFR4 correlated with the repressive effect on bile acid synthesis. ^ To determine whether FGFR4 played a broader role in liver-specific metabolic function, we examined the impact of both acute and chronic exposure to CCl 4 in FGFR4 (−/−) mice. Following acute CCl4 exposure, the FGFR4 (−/−) mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair, with no apparent effect on liver cell proliferation and restoration of cellularity. Chronic CCl4 exposure resulted in severe fibrosis in livers of FGFR4 (−/−) mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8 hr delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl 4-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. ^ Of the 23 FGF polypeptides, FGF1 and FGF2 are present at significant levels in the liver. To determine whether FGF1 and FGF2 played a role in CCl 4-induced liver injury and fibrosis, we examined the impact of both acute and chronic exposure to CCl4 in both wild-type and FGF1-FGF2 double-knockout mice. Following acute CCl4 exposure, FGF1(−/−)FGF2(−/−) mice exhibited accelerated liver injury, overall normal liver growth and repair, and decreased liver collagen α1(I) induction. Liver fibrosis resulting from chronic CCl4 exposure was markedly decreased in livers of FGF1(−/−)FGF2(−/−) mice compared to wild-type mice. This study suggests a role for FGF1 and FGF2 in hepatic fibrogenesis. ^ In summary, our three part study shows that specific components of the ubiquitous HS-FGF signaling family in the liver context interfaces with metabolite- and xenobiotic-controlled networks to regulate liver function, but has no apparent direct effect on liver cell growth. ^

The role of the sonic hedgehog signaling pathway in epidermal homeostasis

Non-melanoma skin cancer is the most frequently diagnosed malignancy in the United States of which basal cell carcinoma (BCC) accounts for 65%. It has recently been determined that deregulation of the sonic hedgehog (shh) pathway leads to the development of BCC. Shh, gli-1, gli-2 gli-3, ptc and smo are overexpressed in BCC and overexpression of these genes in the epidermis results in formation of BCC-like tumors. Despite these observations, the mechanisms by which the pathway controls epidermal homeostasis and the development of the malignant phentotype are unknown. This study assessed the role of the shh pathway in epidermal homeostasis through regulation of apoptosis and differentiation. ^ The anti-apoptotic protein, bcl-2 is overexpressed in BCC, however transcriptional regulators of bcl-2 in the epidermis are unknown. Transient transfection of primary keratinocytes with gli-1 resulted in an increase of bcl-2 expression. Database analysis revealed seven candidate gli binding sites on the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. An N-terminal mutant of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. The region −428 to −420 was found to be important for gli-1 regulation through gel shift, luciferase assays and site-directed mutagenesis. ^ In order to assess the ability of the shh pathway to regulate keratinocyte differentiation, HaCaT keratinocytes overexpressing sonic hedgehog, were grown in organotypic raft culture. Overexpression of shh induced a basal cell phenotype compared to vector control, as evidenced by transmural staining of cytokeratin 14 and altered Ki67 staining. Shh also induced keratinocyte invasion into the underlying collagen. This was associated with increased phosphorylation of EGFR, jnk and raf and increased expression of c-jun, mmp-9 and Ki67. Interestingly, shh overexpression in HaCaTs did not induce the typical downstream effects of shh signaling, suggesting a gli-independent mechanism. Sonic hedgehog's ability to induce an invasive phenotype was found to be dependent on activation of the EGF pathway as inhibition of EGFR activity with AG1478 and c-225 was able to reduce the invasiveness of HaCaT shh keratinocytes, whereas treatment with EGF augmented the invasiveness of the HaCaT shh clones. ^ These studies reveal the importance of the sonic hedgehog pathway in epidermal homeostasis by regulation of apoptosis through bcl-2, and control of keratinocyte differentiation and invasion through activation of the EGF pathway. They further suggest potential mechanisms by which deregulation of the shh pathway may lead to the development of the malignant phenotype. ^

Receptor tyrosine kinase HER2-mediated signaling pathways in metastasis and angiogenesis

Metastasis, the major cause of morbidity and mortality in most cancers, is a highly organized and organ-selective process. The receptor tyrosine kinase HER2 enhances tumor metastasis, however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the malignant cancer cells to specific organs. Here we show that HER2 enhances the expression of CXCR4 by increasing CXCR4 protein synthesis and inhibiting its degradation. We also observed significant correlation between HER2 and CXCR4 expression in human breast tumor tissues, and an association between CXCR4 expression and a poor overall survival rate in patients with breast cancer. Furthermore, we found that CXCR4 is required for HER2-induced invasion, migration, and adhesion activities in vitro . Finally we established stable transfectants using retroviral RNA interference to inhibit CXCR4 expression and showed that the CXCR4 is required for HER2-mediated lung metastasis in vivo. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and homing to metastatic organs, and establish a functional link between the receptor tyrosine kinase HER2 and the chemokine receptor CXCR4 signaling pathways. ^ The HER2 overexpression activates PI-3K/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia inducible factors (HIF) are the key regulator for angiogenesis and energy metabolism, and thereby enhance tumor growth and metastasis. HIF activation occurs in the majority of human cancers, including the HER2 overexpressing cancer cells. Previous reports suggested that increased PI-3K/Akt may activate HIF pathway in various tumors, but the detail mechanism is still not completely understood. Here we found that HER2/PI-3K/Akt pathway induces HIF-1α activation, which is independent of hypoxia, but relatively weaker than hypoxic stimulation. This phenomenon was further observed in Akt knock out mouse embryonic fibroblast cells. The PI-3K/Akt pathway does not affect HIF-1α binding with its E3 ligase VHL, but enhances the binding affinity between HIF-1α and β unit. Furthermore, we found Akt phosphorylates HIF-1β at serine 271 and further regulated HIF transcriptional activity. Our findings provided one mechanism that HER2 induce HIF activation via Akt to promote angiogenesis, and this process is independent on hypoxia, which may have implications in the oncogenic activity of HER2 and PI-3K/Akt pathway. ^

A novel mechanism for regulation of calmodulin signaling by RC3

RC3, also known as neurogranin, is a small neuronal IQ domain protein whose only known function is to bind calmodulin (CaM). The hypothesis tested in this work was that RC3 alters the dynamics of the interaction of Ca 2+-CaM with CaM-kinase II, so that there is less CaM-kinase II activation for a given Ca2+ stimulus. To evaluate this hypothesis, we investigated the affinity and kinetics of the interactions of CaM with Ca 2+, RC3 and CaM-kinase II. We quantitated the interaction of the four CaM-kinase II isoforms with CaM and found that the KD for binding of CaM to CaM-kinase II ranged from 7 nM to 60 nM. Using stopped-flow fluorimetry, we determined the kinetics of the interaction of Ca2+-CaM with αCaM-kinase II, and found that the association rate constant is 2.1 × 10 M −1s−1 and the dissociation rate constant is 1.6 s−1. We investigated the effects of RC3 and αCaM-kinase II on the affinity of CaM for Ca2+ and found that both proteins alter the rate of dissociation of Ca2+ from CaM. RC3 increases the rate of dissociation of Ca2+ from the C-terminal binding sites of CaM from 9 s−1 to ∼500 s−1 , while αCaM-kinase II causes a decrease in the rate of dissociation from all four Ca2+ binding sites. Measurement of the rate of dissociation of Ca2+ from CaM in the presence of both RC3 and αCaM-kinase II revealed a role for RC3 in accelerating the dissociation of the Ca 2+-CaM-αCaM-kinase II complex at the end of a Ca2+ signal. We characterized the interaction of RC3 with apo-CaM and Ca 2+-CaM and found that the KD for both of these interactions is about 1 μM. We also directly tested whether RC3 slowed the dynamics of the binding of CaM to αCaM-kinase II and found that RC3 had no effect for large changes in Ca2+, and a modest effect for small changes in Ca2+ levels. Our overall conclusion is that the ability of RC3 to alter the interaction of Ca2+ with CaM allows RC3 to alter the dynamics of interaction of CaM with Ca2+-dependent targets such as CaM-kinase II. ^

The dynamics of calmodulin signaling revealed using optical methods

The Ca2+-binding protein calmodulin (CaM) is a key transducer of Ca2+ oscillations by virtue of its ability to bind Ca 2+ selectively and then interact specifically with a large number of downstream enzymes and proteins. It remains unclear whether Ca2+ -dependent signaling alone can activate the full range of Ca 2+/CaM regulated processes or whether other regulatory schemes in the cell exist that allow specific targeting of CaM to subsets of Ca 2+/CaM binding sites or regions of the cell. Here we investigate the possibility that alterations of the availability of CaM may serve as a potential cellular mechanism for regulating the activation of CaM-dependent targets. By utilizing sensitive optical techniques with high spatial and temporal resolution, we examine the intracellular dynamics of CaM signaling at a resolution previously unattainable. After optimizing and characterizing both the optical methods and fluorescently labeled probes for intracellular measurements, the diffusion of CaM in the cytoplasm of HEK293 cells was analyzed. It was discovered that the diffusion characteristics of CaM are similar to that of a comparably sized inert molecule. Independent manipulation of experimental parameters, including increases in total concentrations of CaM and intracellular Ca2+ levels, did not change the diffusion of CaM in the cytoplasm. However, changes in diffusion were seen when the concentration of Ca2+/CaM-binding targets was increased in conjunction with elevated Ca2+. This indicates that CaM is not normally limiting for the activation of Ca 2+/CaM-dependent enzymes in HEK293 cells but reveals that the ratio of CaM to CaM-dependent targets is a potential mechanism for changing CaM availability. Next we considered whether cellular compartmentalization may act to regulate concentrations of available Ca2+/CaM in hippocampal neurons. We discovered changes in diffusion parameters of CaM under elevated Ca2+ conditions in the soma, neurite and nucleus which suggest that either the composition of cytoplasm is different in these compartments and/or they are composed of unique families of CaM-binding proteins. Finally, we return to the HEK293 cell and for the first time directly show the intracellular binding of CaM and CaMKII, an important target for CaM critical for neuronal function and plasticity. Furthermore, we analyzed the complex binding stoichiometry of this molecular interaction in the basal, activated and autophosphorylated states of CaMKII and determined the impact of this binding on CaM availability in the cell. Overall these results demonstrate that regulation of CaM availability is a viable cellular mechanism for regulating the output of CaM-dependent processes and that this process is tuned to the specific functional needs of a particular cell type and subcellular compartment. ^

Potential Role of Adenosine Signaling in Acetic Acid Activation of Murine Sensory Neurons

Chronic respiratory illnesses are a significant cause of morbidity and mortality, and acute changes in respiratory function often lead to hospitalization. Air pollution is known to exacerbate asthma, but the molecular mechanisms of this are poorly understood. The current studies were aimed at clarifying the roles of nerve subtypes and purinergic receptors in respiratory reflex responses following exposure to irritants. In C57Bl/6J female mice, inspired adenosine produced sensory irritation, shown to be mediated mostly by A-delta fibers. Secondly, the response to inhaled acetic acid was discovered to be dually influenced by C and A-delta fibers, as indicated by the observed effects of capsaicin pretreatment, which selectively destroys TRPV1-expressing fibers (mostly C fibers) and pretreatment with theophylline, a nonselective adenosine receptor antagonist. The responses to both adenosine and acetic acid were enhanced in the ovalbumin-allergic airway disease model, although the particular pathway altered is still unknown.

Molecular and genetic analyses of Fgf signaling during cardiac outflow tract development

Epithelial-mesenchymal tissue interactions regulate the development of derivatives of the caudal pharyngeal arches (PAs) to govern the ultimate morphogenesis of the aortic arch and outflow tract (OFT) of the heart. Disruption of these signaling pathways is thought to contribute to the pathology of a significant proportion of congenital cardiovascular defects in humans. In this study, I tested whether Fibroblast Growth Factor 15 (Fgf15), a secreted signaling molecule expressed within the PAs, is an extracellular mediator of tissue interactions during PA and OFT development. Analyses of Fgf15−/− mouse embryonic hearts revealed abnormalities primarily localized to the OFT, correlating with aberrant cardiac neural crest cell behavior. The T-box-containing transcription factor Tbx1 has been implicated in the cardiovascular defects associated with the human 22q11 Deletion Syndromes, and regulates the expression of other Fgf family members within the mouse PAs. However, expression and genetic interaction studies incorporating mice deficient for Tbx1, its upstream regulator, Sonic Hedgehog (Shh), or its putative downstream effector, Fgf8, indicated that Fgf15 functions during OFT development in a manner independent of these factors. Rather, analyses of compound mutant mice indicated that Fgf15 and Fgf9, an additional Fgf family member expressed within the PAs, genetically interact, providing insight into the factors acting in conjunction with Fgf15 during OFT development. Finally, in an effort to further characterize this Fgf15-mediated developmental pathway, promoter deletion analyses were employed to isolate a 415bp sequence 7.1Kb 5′ to the Fgf15 transcription start site both necessary and sufficient to drive reporter gene expression within the epithelium of the PAs. Sequence comparisons among multiple mammalian species facilitated the identification of evolutionarily conserved potential trans-acting factor binding sites within this fragment. Subsequent studies will investigate the molecular pathway(s) through which Fgf15 functions via identification of factors that bind to this element to govern Fgf15 gene expression. Furthermore, targeted deletion of this element will establish the developmental requirement for pharyngeal epithelium-derived Fgf15 signaling function. Taken as a whole, these data demonstrate that Fgf15 is a component of a novel, Tbx1-independent molecular pathway, functioning within the PAs in a manner cooperative with Fgf9, required for proper development of the cardiac OFT. ^

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

Studies of MEKK2 activation mechanisms and its biological role in LPS/TLR4 signaling

MEKK2 is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that controls the MAPK and IKK-NF-κB pathways. The MAPK and IKK pathways are intracellular signaling networks that are crucial for the Toll-like receptor (TLR) mediated innate immunity, cellular stress and many other physiological responses. Members of the MAP3K family are central to the activation of these processes. However, the molecular mechanisms underlying stimuli-mediated MAP3K activation remain largely unknown. In this study, we identified a key phosphoserine residue, Ser-519 in MEKK2, and its equivalent site Ser-526 in MEKK3 within their activation loop whose phosphorylation are essential for their optimal activation. Mutation of this regulatory serine to an alanine severely impaired MEKK2 activation and MEKK2 signaling to its downstream targets. To demonstrate that physiological stimuli induce this serine phosphorylation, we generated an antibody that specifically recognizes the phosphorylated serine residue. We found that many, but not all, of the MAPK agonists, including the TLR ligands, growth factors, cytokines and cellular stresses, induced this regulatory serine phosphorylation in MEKK2, suggesting an involvement of MEKK2 in the activation of the MAPK cascade leading to different cellular responses. We further investigated the specific role of MEKK2 in LPS/TLR4 signaling by using MEKK2−/− mice. We found that MEKK2 was selectively required for LPS-induced ERK1/2 activation, but not JNK, p38 or NF-κB activation. We also found that MEKK2 was involved in TLR4 dependent induction of proinflammatory cytokines and LPS-induced septic shock. In conclusion, we identified a key regulatory serine residue in the activation loop of MEKK2 whose phosphorylation is a key sensor of receptor- and cellular stress-mediated signals. We also demonstrated that MEKK2 is crucial for TLR4-mediated innate immunity. ^

The biologic and signaling effects of Tiam1 overexpression in colorectal carcinoma

Cellular migration is essential to many normal cellular processes. In tumor cells, aberrant activation of the normal pathways regulating migration is one of the critical steps in the development of metastasis. Previously, I demonstrated for the first time that overexpression of Tiam1, a guanine nucleotide exchange factor (GNEF) for small G proteins in the Rho family, could alter migration in colorectal tumor cells. ^ This dissertation focuses on the roles of Tiam1 in promoting cell migration, survival, and metastasis of colorectal carcinoma cells, utilizing the model system I developed. To determine the in vivo phenotype of the migratory cell lines, athymic nude mice were injected with cells into the orthotopic site. Several of the mice injected with cells of increased migratory potential had metastases. Thus, the in vitro selection for increased migration resulted in increased metastatic potential in vivo, and therefore, the Tiam1-overexpressing cells provide a model to examine signal transduction pathways important to this process. ^ To examine effects of Tiam1 signaling on small G proteins critical to cellular functions associated with migration, I examined the activation status of the small G proteins Rac, Rho, and Cdc42. The cells of increased migratory potential have increased GTP-bound Rac and Rho, compared to control SW480 cells. Cells that overexpress Tiam1 are more migratory and are resistant to detachment-induced death, or anoikis. To determine which effects and phenotypes were Tiam1-specific, we utilized siRNA to downregulate Tiam1 expression. These results demonstrate that Tiam1 is sufficient but not required for the migration of colorectal carcinoma cells in our model system, and that the biologically selected cells have additional changes that promote migration besides the increase in Tiam1. I also show that Tiam1 protects colorectal carcinoma cells from detachment-induced death, but is not required for anoikis resistance in the biologically selected migratory cells. ^ In summary, my studies demonstrate a heretofore-unknown regulator of phenotypes critical to the development of colorectal carcinoma metastases, overexpression of Tiam1. Understanding the mechanism by which Tiam1 contributes to cellular migration and metastasis is crucial to developing desperately needed new therapies for colorectal carcinoma. ^

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

Molecular mechanisms of prostacyclin receptor signaling: The intracellular domains in G protein coupling

To better understand the mechanisms of how the human prostacyclin receptor (1P) mediates vasodilation and platelet anti-aggregation through Gs protein coupling, a strategy integrating multiple approaches including high resolution NMR experiments, synthetic peptide, fluorescence spectroscopy, molecular modeling, and recombinant protein was developed and used to characterize the structure/function relationship of important segments and residues of the IP receptor and the α-subunit of the Gs protein (Gαs). The first (iLP1) and third (iLP3) intracellular loops of the IP receptor, as well as the Gαs C-terminal domain, relevant to the Gs-mediated IP receptor signaling, were first identified by observation of the effects of the mini gene-expressed corresponding protein segments in HEK293 cells which co-expressed the receptor and Gαs. Evidence of the IP iLP1 domain interacted with the Gαs C-terminal domain was observed by fluorescence and NMR spectroscopic studies using a constrained synthetic peptide, which mimicked the IP iLP1 domain, and the synthetic peptide, which mimicked Gαs C-terminal domain. The solution structural models and the peptide-peptide interaction of the two synthetic protein segments were determined by high resolution NMR spectroscopy. The important residues in the corresponding domains of the IP receptor and the Gαs predicted by NMR chemical shift mapping were used to guide the identification of their protein-protein interaction in cells. A profile of the residues Arg42 - Ala48 of the IP iLP1 domain and the three residues Glu392 ∼ Leu394 of the Gαs C-terminal domain involved in the IP/Gs protein coupling were confirmed by recombinant proteins. The data revealed an intriguing speculation on the mechanisms of how the signal of the ligand-activated IP receptor is transmitted to the Gs protein in regulating vascular functions and homeostasis, and also provided substantial insights into other prostanoid receptor signaling. ^

p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

The p120-catenin/Kaiso signaling pathway

The canonical and non-canonical Wnt signaling pathways appear to interact with one another as a network in development, or when hyper-activated, in the progression of disease. A much studied key mediator of the canonical Wnt pathway, β-catenin, is characterized by a central armadillo-repeat domain that engages in multiple protein-protein interactions, such as those with cadherins functioning at cell-cell contact regions. In the nucleus, β-catenin forms a complex with the repressor TCF/LEF, promoting the activation of genes participating in processes such as proliferation, differentiation and stem cell survival. Somewhat similarly, the p120-catenin binds the distinct transcriptional repressor Kaiso, relieving Kaiso-mediated repression to promote gene activation. Here, employing Xenopus laevis, I report upon both downstream and upstream aspects of the p120-catenin/Kaiso pathway which was previously poorly understood. I first show that Kaiso, a BTB/POZ zinc-finger family member, directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1 and c-Myc) in conjunction with TCF. Depletion or dominant-negative inhibition of xKaiso results in Siamois de-repression, while xKaiso over-expression induces additional Siamois repression through recruitment of N-CoR co-repressor and chromatin modifications. Functional interdependencies are further corroborated by the capacity of Kaiso to suppress β-catenin-induced axis duplication. Thus, my work inter-relates the p120-catenin/Kaiso and β-catenin/TCF pathways at the level of specific gene promoters important in development and cancer progression. Regarding upstream aspects of the p120-catenin/Kaiso pathway, I collaboratively identified p120 in association with Frodo, a protein previously identified as a component of the canonical (β-catenin dependent) Wnt pathway. I determined that canonical Wnt signals result in Frodo-mediated stabilization of p120-catenin, resulting in the sequestration of Kaiso to the cytoplasm and thereby the activation (relief of repression) of gene targets. Developmental evidence supporting this view included findings that Frodo has the capacity to partially rescue Kaiso over-expression phenotypes in early Xenopus embryos. Taken together, my studies point to the convergence of p120-catenin/Kaiso and β-catenin/TCF signaling pathways at the level of gene transcription as well as at more upstream points during vertebrate development. ^