Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

Syndecan-2 Regulates the Migratory Potential of Melanoma Cells

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is a critical mediator in the tumorigenesis of colon carcinoma cells. We explored the function of syndecan-2 in melanoma, one of the most invasive types of cancers, and found that the expression of this protein was elevated in tissue samples from both nevus and malignant human melanomas but not in melanocytes of the normal human skin tissues. Similarly, elevated syndecan-2 expression was observed in various melanoma cell lines. Overexpression of syndecan-2 enhanced migration and invasion of melanoma cells, whereas the opposite was observed when syndecan-2 levels were knocked down using small inhibitory RNAs. Syndecan-2 expression was enhanced by fibroblast growth factor-2, which is known to stimulate melanoma cell migration; however, α-melanocyte-stimulating hormone decreased syndecan-2 expression and melanoma cell migration and invasion in a melanin synthesis-independent manner. Furthermore, syndecan-2 overexpression rescued the migration defects induced by α-melanocyte-stimulating hormone treatment. Together, these data strongly suggest that syndecan-2 plays a crucial role in the migratory potential of melanoma cells.The syndecans, a family of four transmembrane cell surface heparan sulfate proteoglycans, mainly serving as a co-receptor, regulate the adhesion-dependent signal transduction of a variety of cell types, including cancer cells (1, 2). Cell adhesion receptors or co-receptors play a critical role in the neoplastic transformation of normal cells by regulating the induction of cancer-specific cellular behavior and morphology. Thus, cancer cells probably express and utilize a distinct set of syndecans in the regulation of cancer cell growth.Several reports have linked altered syndecan expression to various elements of cancer cell growth. Loss of syndecan-1 correlates with shorter survival times in patients with squamous cell carcinoma of the head, neck, and lung (3) as well as multiple myeloma (4); loss of syndecan-1 is also associated with an elevated potential for metastasis in patients with hepatocellular and colorectal carcinomas (5, 6). Previous studies have shown that syndecan-1 regulates tumor activity in pancreatic (7), gastric (8), and breast carcinomas (9). Syndecan-1 may thus play multiple roles in tumorigenic activity and perform various tissue- and/or tumor stage-specific functions (10). Syndecan-4 expression is reduced in colon carcinoma cells (11, 12) and appears to correlate with increased tumorigenic activity (e.g. cell migration and invasion (13)), implying that syndecan-4 functions as a tumor suppressor.Syndecan-2 is also known to play a crucial role in the regulation of cancer activity. Increased levels of syndecan-2 confer an invasive phenotype in lung (14) and colon cancer cells (15). Reduction in syndecan-2 expression induces cells to switch from the transformed phenotype to flattened monolayers (8) and reduces tumorigenic activity in colon adenocarcinoma and fibrosarcoma cells (8, 16). In addition, syndecan-2 is highly expressed in the microvasculature of mouse gliomas and has been shown to regulate angiogenesis in microvascular endothelial cells (17). On the other hand, an inverse correlation between syndecan-2 expression and metastatic potential has been found in Lewis lung carcinoma cell lines (6). Therefore, changes in syndecan-2 expression may directly or indirectly regulate cancer growth.Melanoma is the most aggressive malignant tumor of melanocytes. Although found predominantly in the skin, primary melanomas are also known to occur in the bowel and eye (18). Malignant melanoma is notoriously one of the most difficult cancers to treat (19). Therefore, identifying and understanding molecules that regulate the aggressive melanoma phenotype is essential for predicting the likelihood of metastasis. Interestingly, previous studies have shown that melanoma cells acquire the ability to recognize components of the extracellular matrix (ECM)² via the ectopic expression of different ECM receptors during invasion of the basement membrane (20). Indeed, invadopodia, the dynamic organelle-like structures that form actin-rich protrusions with ECM proteolytic activity, adhere to and digest collagens, laminins, and fibronectin (21). The adhesive properties of invadopodia are primarily attributed to integrins, a large family of heterodimeric transmembrane receptors composed of α and β subunits (22). For example, β₁ integrins localize within the invadopodia of melanoma cells (23), and the α₅β₁ integrins are enriched peripherally in invadopodia, where they stabilize invadopodia protrusion (24). Ectopic stimulation of α₆β₁ integrin with laminin peptides or with β₁ or α₆ integrin stimulatory antibodies increases invadopodia activity and melanoma invasiveness (23). The invasive behavior of melanoma cells can be attributed to increased cell motility caused by changes in cytoskeletal organization and altered contacts with the ECM. Thus, cell adhesion receptors may play a crucial role in the acquisition of highly migratory behavior.Syndecan-2 acts as a key regulator of cancer cells, suggesting that syndecan-2 may contribute to the aggressive phenotype and metastatic potential of melanoma. Here, we report that syndecan-2 plays a pivotal role in the migratory activity of melanoma cells.     

Heparan Sulfate Proteoglycan Modulation of Wnt5A Signal Transduction in Metastatic Melanoma Cells

Heparan sulfate proteoglycans (HSPGs) are important modulators for optimizing signal transduction of many pathways, including the Wnt pathways. We demonstrate that HSPG glycosaminoglycan levels increased with increasing metastatic potential of melanoma cells. Previous studies have demonstrated that Wnt5A increases the invasiveness of melanoma cells. We further demonstrate that HSPGs potentiate Wnt5A signaling, since enzymatic removal of the HSPG backbone resulted in a decrease in cellular Wnt5A levels, an increase in secreted Wnt5A in cell media, a decrease in downstream signaling, and ultimately, a decrease in invasiveness. Specifically, syndecan 1 and syndecan 4 expression correlated to Wnt5A expression and melanoma malignancy. Knockdown of syndecan 1 or 4 caused decreases in cell invasion, which could be restored by treating the cells with recombinant Wnt5A. These data indicate that syndecan 1 and 4 correlate to increased metastatic potential in melanoma patients and are an important component of the Wnt5A autocrine signaling loop, the activation of which leads to increased metastasis of melanoma.The American Cancer Society estimates that in 2009 there will be 68,720 new cases of melanoma in this country with ∼8,650 deaths. Recent studies have demonstrated that the non-canonical Wnt pathway, also known as the Wnt/Ca²⁺ pathway, plays an important role in increasing the metastatic potential of melanoma cells (1–5). Studies from our laboratory demonstrated that increasing Wnt5A, which mediates the non-canonical Wnt/Ca²⁺ signaling pathway, increased melanoma metastasis (1–3), and silencing Wnt5A levels via siRNA³ decreased invasion (2, 3). In addition, we have shown that Wnt5A acts via protein kinase C (PKC) to mediate the motility of melanoma cells via the inhibition of metastasis suppressors and an initiation of the epithelial to mesenchymal transition, characterized by the loss of E-cadherin and the up-regulation of Snail (2).Wnt signaling can be mediated by heparan sulfate proteoglycans (HSPGs) which are important signal transduction modulators. They mediate fibroblast growth factor, Hedgehog, epidermal growth factor, transforming growth factor-β, and WNT signaling pathways (6–11). HSPGs consist of two types, cell surface and basement membrane-associated HSPGs (12). Cell surface HSPGs are glycoproteins with covalently attached unbranched and modified sugar chains known as glycosaminoglycans (GAGs). There are two types of cell surface HSPGs, known as glypicans and syndecans (11, 13). Glypicans are attached to the cell surface via a glycosylphosphatidylinositol anchor, whereas syndecans are type 1 transmembrane proteins. HSPG GAG side chains are unbranched chains of modified repeating disaccharide units of N-acetylglucosamine and glucuronic acid. They are joined to the core protein via a tetrasaccharide linker attached to a serine residue. Following synthesis, these chains undergo modification with the addition of sulfates by N- and O-sulfation (14). The sulfation status determines to which specific portion of the GAG chains ligands, such as Wnt, will attach. The heparan sulfate endosulfatases Sulf1 and Sulf2 are cell surface enzymes that control growth factor signaling. The regulation of the 6-O-sulfation states by these endosulfatases changes the affinity of the GAG chains for ligand binding (15–17). Following sulfation modification, HSPGs can regulate signaling by dimerization (with other HSPGs or canonical signaling receptors), stabilization, or transport of the ligand to or away from the high affinity receptors (18–20). In addition, studies have suggested that the core proteins themselves may also play an important role in cell phenotype and function (21).HSPGs have been implicated in a number of pathological conditions, such as Simpson-Golabi-Behmel overgrowth syndrome (22), fibrodysplasia ossificans progressiva (23), and Alzheimer disease (24). In addition, HSPGs are overexpressed in many forms of cancer, including prostate cancer and melanoma (25, 26). Importantly, in cancer, proteoglycans can have both tumor-promoting and tumor-suppressing activities. This depends on the type of protein core, the GAGs attached, and the localization of the proteoglycan and the molecules they associate with. In addition, the tumor subtype, stages, and degree of tumor differentiation also affect the function of HSPGs (27). HSPGs are cleaved by heparanases or heparin lyases (heparinases), which have been shown to have differing effects on tumor cell activity. For example, treating cancer cells with heparanase-1, which cleaves heparin-like regions (specifically HLGAG sites with O-sulfated l-iduronic acid residues), results in an increase in both tumor growth and metastatic dissemination (28). However, treating tumor cells with heparinase III, which more specifically cleaves HSPGs (i.e. unsulfated d-glucorinic acid, heparan-sulfate-like regions) results in an inhibition of their metastatic capacity (29). Importantly, heparanase I cleaves only certain side chains, where heparinase III treatment cleaves the entire backbone of the HSPG. It is likely that cleavage of specific side chains facilitates cell motility by releasing cells from adhesion to neighboring cells, whereas cleavage of the entire molecule decreases the availability of secreted ligands to their receptors, especially those involved in autocrine signaling, such as Wnt5A.Historically, Wnt5A has been quite difficult to purify from cell culture media, despite the fact that it is a secreted protein. Further, in melanoma cells, Wnt5A appears to be signaling in an autocrine fashion (1, 2). These two observations, together with the fact that Wnt5A undergoes glycosylation (30), led us to hypothesize that HSPGs might be involved in increasing the availability of Wnt5A to its receptor, resulting in an increase in autocrine signaling and ultimately an increase in cellular invasion. In this study, we explore this hypothesis and investigate the role of HSPGs in the Wnt5A signaling cascade in metastatic melanoma cells.     

Evaluating Melanoma Drug Response and Therapeutic Escape with Quantitative Proteomics

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.Despite excitement about the development of targeted therapy strategies for cancer, few cures have been achieved. In patients with BRAF mutant melanoma, treatment with small molecule BRAF inhibitors typically follows a course of response and tumor shrinkage followed by eventual relapse and resistance (mean progression-free survival is ∼5.3 months) (1). Resistance to BRAF inhibitors is typically accompanied by reactivation of the MAPK signaling pathway, an effect mediated through activating mutations in NRAS and MEK1/2, genomic amplification of BRAF, increased expression of CRAF and Cot, and the acquisition of BRAF splice-form mutants (2–5). There is also evidence that increased PI3K/AKT signaling, resulting from the genetic inactivation of PTEN and NF1 and increased receptor tyrosine kinase (RTK)¹ signaling, may be involved in acquired BRAF inhibitor resistance (5–7). Many of the signaling proteins implicated in the escape from BRAF inhibitor therapy are clients of heat shock protein (HSP)-90 (8). Preclinical evidence now indicates that HSP90 inhibitors can overcome acquired and intrinsic BRAF inhibitor resistance, and clinical trials have been initiated to evaluate the BRAF/HSP90 combination in newly diagnosed patients (8, 9).Although targeted therapy strategies have been promising in BRAF mutant melanoma, few options currently exist for the 15–20% of melanoma patients whose tumors harbor activating NRAS mutations (10). Although there is some evidence that MEK inhibitors have activity in NRAS mutant melanoma patients, responses tend to be short-lived (mean progression-free survival ∼3 months) and resistance is nearly inevitable (11). Our emerging experience suggests that oncogene-driven signaling networks are highly robust with the capacity to rapidly adapt (12, 13). The future success of targeted therapy for melanoma and other cancers will depend upon the development of strategies that identify and overcome these adaptive escape mechanisms.The evaluation of targeted therapy responses in patients has proved to be challenging. The clinical development of HSP90 inhibitors has been hampered in part by the lack of a good pharmacodynamic assay for measuring HSP90 inhibition within tumor specimens (14). Additionally, very little is known about the adaptive changes that occur following the inhibition of MEK/ERK signaling in NRAS mutant melanoma. To address these issues, the optimal technique is liquid chromatography-multiple reaction monitoring mass spectrometry, which been shown to be highly reproducible and portable across laboratories (15–18).In addition to these technical developments, LC-MRM has also been shown to have excellent application to the study of biological pathways, including phosphotyrosine signaling, β-catenin signaling in colon cancer, and the evasion of apoptosis following BRAF inhibition in PTEN null melanoma (19–21). This technique can also be readily translated from cell line models to patient specimens. Here, we have developed a novel multiplexed LC-MRM assay to quantify the expression of >80 key signaling proteins in cell line models and fine needle aspirates from accessible melanoma lesions (22). In this study, we present the proof-of-principle for monitoring multiple signaling proteins in melanomas treated with either HSP90 or MEK inhibitors. Through this method, we identify the degradation of key HSP90 client proteins in vivo and elucidate a novel mechanism of adaptation to MEK inhibition through increased RTK signaling.     

Helicobacter pylori CagA Causes Mitotic Impairment and Induces Chromosomal Instability

Infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric carcinoma. The cagA gene product CagA, which is delivered into gastric epithelial cells, specifically binds to and aberrantly activates SHP-2 oncoprotein. CagA also interacts with and inhibits partitioning-defective 1 (PAR1)/MARK kinase, which phosphorylates microtubule-associated proteins to destabilize microtubules and thereby causes epithelial polarity defects. In light of the notion that microtubules are not only required for polarity regulation but also essential for the formation of mitotic spindles, we hypothesized that CagA-mediated PAR1 inhibition also influences mitosis. Here, we investigated the effect of CagA on the progression of mitosis. In the presence of CagA, cells displayed a delay in the transition from prophase to metaphase. Furthermore, a fraction of the CagA-expressing cells showed spindle misorientation at the onset of anaphase, followed by chromosomal segregation with abnormal division axis. The effect of CagA on mitosis was abolished by elevated PAR1 expression. Conversely, inhibition of PAR1 kinase elicited mitotic delay similar to that induced by CagA. Thus, CagA-mediated inhibition of PAR1, which perturbs microtubule stability and thereby causes microtubule-based spindle dysfunction, is involved in the prophase/metaphase delay and subsequent spindle misorientation. Consequently, chronic exposure of cells to CagA induces chromosomal instability. Our findings reveal a bifunctional role of CagA as an oncoprotein: CagA elicits uncontrolled cell proliferation by aberrantly activating SHP-2 and at the same time induces chromosomal instability by perturbing the microtubule-based mitotic spindle. The dual function of CagA may cooperatively contribute to the progression of multistep gastric carcinogenesis.Helicobacter pylori is a spiral-shaped bacterium first described in 1984 by Marshall and Warren (1). H. pylori inhabits at least half of the world''s human population. Clinically isolated H. pylori strains can be divided into two major subtypes based on their ability to produce a 120- to 145-kDa protein called cytotoxin-associated gene A antigen (CagA)² (2–5). More than 90–95% of H. pylori strains isolated in East Asian countries such as Japan, Korea, and China are cagA-positive, whereas 40–50% of those isolated in Western countries are cagA-negative. Infection with a cagA-positive H. pylori strain is associated with severe atrophic gastritis, peptic ulcerations, and gastric adenocarcinoma (6–12).H. pylori cagA-positive strains deliver the CagA protein into host cells via the cag pathogenicity island-encoded type IV secretion system (4, 5, 13, 14). Translocated CagA then localizes to the inner surface of the plasma membrane, where it undergoes tyrosine phosphorylation by Src family kinases or Abl kinase at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs present in the C-terminal region of CagA (15–17). Tyrosine-phosphorylated CagA then binds specifically to SHP-2 tyrosine phosphatase and deregulates its phosphatase activity (18–21). Recent studies have revealed that gain-of-function mutations of SHP-2 are associated with a variety of human malignancies, indicating that SHP-2 is a bona fide human oncoprotein. Furthermore, transgenic expression of CagA in mice induces gastrointestinal and hematological malignancies in a manner that is dependent on CagA tyrosine phosphorylation (22). These findings suggest a critical role of CagA-SHP-2 interaction in the oncogenic potential of CagA.A polarized epithelial monolayer is characterized by the presence of well developed cell-cell interaction apparatuses such as tight junctions and adherens junctions. The tight junctions act as a paracellular barrier in polarized epithelial cells and play an essential role in the establishment and maintenance of epithelial cell polarity by delimiting the apical and basolateral membrane domains. CagA disrupts the tight junctions and causes loss of epithelial apical-basal polarity (23, 24). The disruption of tight junctions by CagA is mediated by the specific interaction of CagA with partitioning-defective 1 (PAR1) (25, 26). PAR1 is a serine/threonine kinase originally isolated in Caenorhabditis elegans and highly conserved from yeast to humans (27, 28). In mammals, there are four PAR1 isoforms, which may have redundant roles in polarity regulation. PAR1 acts as a master regulator for the regulation of cell polarity in various cell systems. During epithelial polarization, PAR1 specifically localizes to the basolateral membrane, whereas atypical PKC complexed with PAR3 and PAR6 (aPKC complex) specifically localizes to the apical membrane as well as the tight junctions (29–31). This asymmetric distribution of the two kinases, PAR1 and aPKC complex, ensures formation and maintenance of epithelial apical-basal polarity. Notably, mammalian PAR1 kinases were originally identified as microtubule affinity-regulating kinases (MARKs), which phosphorylate microtubule-associated proteins (MAPs) such as Tau, MAP2, and MAP4 on their tubulin-binding repeats. The PAR1/MARK-dependent phosphorylation causes MAPs to detach from and thereby destabilize microtubules (32, 33). Importantly, microtubules form a mitotic spindle, which plays an indispensable role in chromosomal alignment and separation during mitosis, raising the possibility that PAR1 regulates mitosis through controlling stability of the mitotic spindle. Indeed, during mitosis, MAPs undergo a severalfold higher level of phosphorylation (34, 35), and microtubule dynamics increase ∼20-fold (36). This in turn raises the intriguing possibility that CagA influences chromosomal stability by subverting MAP phosphorylation through systemic inhibition of PAR1.In this study, the effects of CagA on microtubule-dependent cellular events, especially dynamics of the mitotic spindle and chromosomal segregation during mitosis, were examined. The results of this work provide evidence that CagA perturbs mitotic spindle checkpoint and thereby causes chromosomal instability. Given the role of chromosomal instability in cell transformation, the newly identified CagA activity may play a crucial role in the development of gastric carcinoma.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Analysis of Non-canonical Fibroblast Growth Factor Receptor 1 (FGFR1) Interaction Reveals Regulatory and Activating Domains of Neurofascin

Fibroblast growth factor receptors (FGFRs) are important for many different mechanisms, including cell migration, proliferation, differentiation, and survival. Here, we show a new link between FGFR1 and the cell adhesion molecule neurofascin, which is important for neurite outgrowth. After overexpression in HEK293 cells, embryonal neurofascin isoform NF166 was able to associate with FGFR1, whereas the adult isoform NF186, differing from NF166 in additional extracellular sequences, was deficient. Pharmacological inhibitors and overexpression of dominant negative components of the FGFR signaling pathway pointed to the activation of FGFR1 after association with neurofascin in neurite outgrowth assays in chick tectal neurons and rat PC12-E2 cells. Both extra- and intracellular domains of embryonal neurofascin isoform NF166 were able to form complexes with FGFR1 independently. However, the cytosolic domain was both necessary and sufficient for the activation of FGFR1. Cytosolic serine residues 56 and 100 were shown to be essential for the neurite outgrowth-promoting activity of neurofascin, whereas both amino acid residues were dispensable for FGFR1 association. In conclusion, the data suggest a neurofascin intracellular domain, which activates FGFR1 for neurite outgrowth, whereas the extracellular domain functions as an additional, regulatory FGFR1 interaction domain in the course of development.The four known fibroblast growth factor receptors (FGFRs),² which are targeted by a large family of 22 fibroblast growth factor ligands, represent a highly diverse signaling system important for migration, proliferation, differentiation, and survival of many different cell types (1, 2). fibroblast growth factor activation of FGFR leads to the activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and phospholipase Cγ (PLCγ), depending on the cellular system under study. Non-canonical FGFR interactions with NCAM, cadherins, and syndecan via extracellular domains were also described (1). However, the contribution of intracellular interactions of FGFR1 with further membrane co-receptors is poorly understood. Only cytosolic interaction between FGFRs and EphA4 have been described that are involved in mutual transphosphorylation (3).The cell adhesion molecule neurofascin is important for cell-cell communication in the nervous system (4, 5). Neurofascin regulates many different functions in the brain, suggesting that it functions as a key regulator for both developing and differentiated neural cells. Different alternatively spliced neurofascin isoforms are expressed in different cells and at different times of development (6). Embryonal neurofascin NF166 is important for neurite outgrowth and guidance (7, 8). Recently, a role for neurofascin NF166 for early processes of inhibitory synaptogenesis at the axon hillock and for the positioning of inhibitory synapses at the axon initial segment has been proven (9, 10).In the more developed nervous system, NF166 is replaced by NF186, which is inhibitory for neurite outgrowth (11). NF186 is linked to the cortical actin cytoskeleton via ankyrin_G (12). Clustering of voltage-gated sodium channels both at axon initial segments and at the nodes of Ranvier is conferred by neurofascin NF186 (13, 14). A further cytosolic interaction partner is the PDZ molecule syntenin-1 (15).Despite the well known functional importance of neurofascin in the nervous system, corresponding signaling pathways have not been investigated. In contrast, signaling by the related molecules NCAM and L1 have been studied with regard to the induction of neurite outgrowth in greater detail (for a review, see Refs. 16–18). Both NCAM and L1 induce neurite outgrowth through activation of FGFR1 (19–23). NCAM may further undergo lateral interactions with PrP (prion precursor protein) or GFRα, which is part of the glia-derived neurotrophic factor receptor (24, 25). In addition to FGFR1 interaction, both L1 and NCAM are connected to non-receptor tyrosine kinases. However, whereas NCAM employs the non-receptor kinase c-Fyn as an upstream component, L1 is linked to c-Src (26, 27). L1 converges with NCAM signaling upstream of the MAPK pathway at the level of Raf (18, 21, 28, 29). NCAM may induce alternative signaling pathways, including protein kinase A-dependent signaling or G-proteins (18, 30). NCAM signaling to the nucleus may include activation of CREB and c-Fos or NF-κB (29, 31, 32).Here, we elucidate the molecular mechanisms of neurofascin-FGFR1 interaction for neurite outgrowth. We show that both cytosolic and the extracellular domains are important for the association of FGFR1 with neurofascin. Although the cytosolic domain represents a critical determinant for FGFR1 activation, the extracellular sequences of neurofascin act as a regulator for FGFR1-dependent signal transduction in the course of development.     

Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 5 Desensitization and Internalization by G Protein-coupled Receptor Kinase 2 in Neurons

The uncoupling of metabotropic glutamate receptors (mGluRs) from heterotrimeric G proteins represents an essential feedback mechanism that protects neurons against receptor overstimulation that may ultimately result in damage. The desensitization of mGluR signaling is mediated by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). Unlike mGluR1, the attenuation of mGluR5 signaling in HEK 293 cells is reported to be mediated by a phosphorylation-dependent mechanism. However, the mechanisms regulating mGluR5 signaling and endocytosis in neurons have not been investigated. Here we show that a 2-fold overexpression of GRK2 leads to the attenuation of endogenous mGluR5-mediated inositol phosphate (InsP) formation in striatal neurons and siRNA knockdown of GRK2 expression leads to enhanced mGluR5-mediated InsP formation. Expression of a catalytically inactive GRK2-K220R mutant also effectively attenuates mGluR5 signaling, but the expression of a GRK2-D110A mutant devoid in Gα_q/11 binding increases mGluR5 signaling in response to agonist stimulation. Taken together, these results indicate that the attenuation of mGluR5 responses in striatal neurons is phosphorylation-independent. In addition, we find that mGluR5 does not internalize in response to agonist treatment in striatal neuron, but is efficiently internalized in cortical neurons that have higher levels of endogenous GRK2 protein expression. When overexpressed in striatal neurons, GRK2 promotes agonist-stimulated mGluR5 internalization. Moreover, GRK2-mediated promotion of mGluR5 endocytosis does not require GRK2 catalytic activity. Thus, we provide evidence that GRK2 mediates phosphorylation-independent mGluR5 desensitization and internalization in neurons.Glutamate is the major excitatory neurotransmitter in the mammalian brain and functions to activate two distinct classes of receptors (ionotropic and metabotropic) to regulate a variety of physiological functions (1–3). Ionotropic glutamate receptors, such as NMDA, AMPA, and kainate receptors, are ligand-gated ion channels, whereas metabotropic glutamate receptors (mGluRs)⁵ are members of the G protein-coupled receptor (GPCR) superfamily (4–7). mGluRs modulate synaptic activity via the activation of heterotrimeric G proteins that are coupled to a variety of second messenger cascades. Group I mGluRs (mGluR1 and mGluR5) are coupled to the activation of Gα_q/11 proteins, which stimulate the activation of phospholipase Cβ1 resulting in diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃) formation, release of Ca²⁺ from intracellular stores and subsequent activation of protein kinase C.The attenuation of GPCR signaling is mediated in part by G protein-coupled receptor kinases (GRKs), which phosphorylate GPCRs to promote the binding of β-arrestin proteins that uncouple GPCRs from heterotrimeric G proteins (8–10). GRK2 has been demonstrated to contribute to the phosphorylation and desensitization of both mGluR1 and mGluR5 in human embryonic kidney (HEK 293) cells (11–17). GRK4 is also implicated in mediating the desensitization of mGluR1 signaling in cerebellar Purkinje cells, but does not contribute to the desensitization of mGluR5 (14, 15). In addition, GRK4 plays a major role in mGluR1 internalization (13, 14). A role for GRK2 in promoting mGluR1 internalization is less clear as different laboratories have obtained discordant results (11, 14, 15, 16). However, the only study examining the role of GRK2 in regulating mGluR1 endocytosis in a native system reported that GRK2 knockdown had no effect upon mGluR1 internalization in cerebellar Purkinje cells (14).GRK2 is composed of three functional domains: an N-terminal regulator of G protein signaling (RGS) homology (RH) domain, a central catalytic domain, and a C-terminal G_βγ binding pleckstrin homology domain (18). In HEK 293 cells, mGluR1 desensitization is not dependent on GRK2 catalytic activity. Rather the GRK2 RH domain interacts with both the second intracellular loop domain of mGluR1 and the α-subunit of Gα_q/11 and attenuates second messenger responses by disrupting the mGluR1/Gα_q/11 signaling complexes (12, 19–21). Although the molecular mechanism underlying GRK2-mediated attenuation of mGluR1 signaling is relatively well established in HEK 293 cells, the role of GRK2 in regulating the desensitization of mGluRs in neurons remains to be determined. Moreover, it is not known whether GRK2-dependent attenuation of mGluR5 signaling is mediated by the same phosphorylation-independent mechanism that has been described for mGluR1. In a previous study, GRK2-mediated mGluR5 desensitization was reported to be phosphorylation-dependent, based on the observation that the overexpression of a catalytically inactive GRK2 (K220R) did not attenuate mGluR5 signaling (15). In the present study, we examined whether a 2-fold overexpression of GRK2 in primary mouse striatal neurons to match GRK2 expression levels found in the cortex results in increased agonist-stimulated desensitization and internalization of endogenous mGluR5. We report here that GRK2 mediates phosphorylation-independent mGluR5 desensitization and internalization. Furthermore, GRK2 knockdown causes an increase in mGluR5 signaling, demonstrating that endogenous GRK2 plays a role in mGluR5 desensitization.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.