Protein tyrosine kinase Abl promotes hepatitis C virus particle assembly via interaction with viral substrate activator NS5A.

Previously, we reported that knockdown of Abl protein tyrosine kinase by shRNA or pharmacological inhibition suppresses particle assembly of J6/JFH1 strain-derived hepatitis C virus (HCV) in Huh-7.5 cells. However, the detailed mechanism by which Abl regulates HCV replication remained unclear. In this study, we established Abl-deficient (Abl-) cells through genome editing and compared HCV production between Abl- cells expressing WT or kinase-dead Abl and parental Huh-7.5 cells. Our findings revealed that Abl expression was not required from the stages of virus attachment and entry to viral gene expression; however, the kinase activity of Abl was necessary for the assembly of HCV particles. Reconstitution experiments using human embryonic kidney 293T cells revealed that phosphorylation of Tyr412 in the activation loop of Abl was enhanced by coexpression with the viral nonstructural protein 5A (NS5A) and was abrogated by the substitution of NS5A Tyr330 with Phe (Y330F), suggesting that NS5A functions as a substrate activator of Abl. Abl-NS5A association was also attenuated by the Y330F mutation of NS5A or the kinase-dead Abl, and Abl Tyr412 phosphorylation was not enhanced by NS5A bearing a mutation disabling homodimerization, although the association of Abl with NS5A was still observed. Taken together, these results demonstrate that Abl forms a phosphorylation-dependent complex with dimeric NS5A necessary for viral particle assembly, but that Abl is capable of complex formation with monomeric NS5A regardless of tyrosine phosphorylation. Our findings provide the foundation of a molecular basis for a new hepatitis C treatment strategy using Abl inhibitors.

Cumulative mechanism of several major imatinib-resistant mutations in Abl kinase.

Despite the outstanding success of the cancer drug imatinib, one obstacle in prolonged treatment is the emergence of resistance mutations within the kinase domain of its target, Abl. We noticed that many patient-resistance mutations occur in the dynamic hot spots recently identified to be responsible for imatinib's high selectivity toward Abl. In this study, we provide an experimental analysis of the mechanism underlying drug resistance for three major resistance mutations (G250E, Y253F, and F317L). Our data settle controversies, revealing unexpected resistance mechanisms. The mutations alter the energy landscape of Abl in complex ways: increased kinase activity, altered affinity, and cooperativity for the substrates, and, surprisingly, only a modestly decreased imatinib affinity. Only under cellular adenosine triphosphate (ATP) concentrations, these changes cumulate in an order of magnitude increase in imatinib's half-maximal inhibitory concentration (IC50). These results highlight the importance of characterizing energy landscapes of targets and its changes by drug binding and by resistance mutations developed by patients.

c-Abl phosphorylation of Yin Yang 1's conserved tyrosine 254 in the spacer region modulates its transcriptional activity.

Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate for c-Abl kinase phosphorylation at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA, and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies reveal that c-Abl mediated phosphorylation of YY1 regulates YY1's transcriptional ability in vivo. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1's transcriptional activity, linking YY1 regulation with c-Abl tyrosine kinase signaling pathways.

Cyclophilin A promotes cell migration via the Abl-Crk signaling pathway.

Cyclophilin A (CypA) is overexpressed in a number of human cancer types, but the mechanisms by which the protein promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds the CrkII adaptor protein and prevents it from switching to the inhibited state. CrkII influences cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binding site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly upregulated.

Dynamic multi-site phosphorylation by Fyn and Abl drives the interaction between CRKL and the novel scaffolding receptors DCBLD1 and DCBLD2.

Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL-SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL-SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL-SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.

Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding.

RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.

Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency.

BACKGROUND: The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID). OBJECTIVE: We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency. METHODS: We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of γH2AX accumulation was studied after ionizing radiation. RESULTS: Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect. CONCLUSIONS: Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.

Abl2/Arg controls dendritic spine and dendrite arbor stability via distinct cytoskeletal control pathways.

Rho family GTPases coordinate cytoskeletal rearrangements in neurons, and mutations in their regulators are associated with mental retardation and other neurodevelopmental disorders (Billuart et al., 1998; Kutsche et al., 2000; Newey et al., 2005; Benarroch, 2007). Chromosomal microdeletions encompassing p190RhoGAP or its upstream regulator, the Abl2/Arg tyrosine kinase, have been observed in cases of mental retardation associated with developmental defects (Scarbrough et al., 1988; James et al., 1996; Takano et al., 1997; Chaabouni et al., 2006; Leal et al., 2009). Genetic knock-out of Arg in mice leads to synapse, dendritic spine, and dendrite arbor loss accompanied by behavioral deficits (Moresco et al., 2005; Sfakianos et al., 2007). To elucidate the cell-autonomous mechanisms by which Arg regulates neuronal stability, we knocked down Arg in mouse hippocampal neuronal cultures. We find that Arg knockdown significantly destabilizes dendrite arbors and reduces dendritic spine density by compromising dendritic spine stability. Inhibiting RhoA prevents dendrite arbor loss following Arg knockdown in neurons, but does not block spine loss. Interestingly, Arg-deficient neurons exhibit increased miniature EPSC amplitudes, and their remaining spines exhibit larger heads deficient in the actin stabilizing protein cortactin. Spine destabilization in Arg knockdown neurons is prevented by blocking NMDA receptor-dependent relocalization of cortactin from spines, or by forcing cortactin into spines via fusion to an actin-binding region of Arg. Thus, Arg employs distinct mechanisms to selectively regulate spine and dendrite stability: Arg dampens activity-dependent disruption of cortactin localization to stabilize spines and attenuates Rho activity to stabilize dendrite arbors.

Indispensable functions of ABL and PDGF receptor kinases in epithelial adherence of attaching/effacing pathogens under physiological conditions.

Enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium are attaching-and-effacing (A/E) pathogens that cause intestinal inflammation and diarrhea. The bacteria adhere to the intestinal epithelium, destroy microvilli, and induce actin-filled membranous pedestals but do not invade the mucosa. Adherence leads to activation of several host cell kinases, including FYN, n-SRC, YES, ABL, and ARG, phosphorylation of the bacterial translocated intimin receptor, and actin polymerization and pedestal formation in cultured cells. However, marked functional redundancy appears to exist between kinases, and their physiological importance in A/E pathogen infections has remained unclear. To address this question, we employed a novel dynamic in vitro infection model that mimics transient and short-term interactions in the intestinal tract. Screening of a kinase inhibitor library and RNA interference experiments in vitro revealed that ABL and platelet-derived growth factor (PDGF) receptor (PDGFR) kinases, as well as p38 MAP kinase, have unique, indispensable roles in early attachment of EPEC to epithelial cells under dynamic infection conditions. Studies with mutant EPEC showed that the attachment functions of ABL and PDGFR were independent of the intimin receptor but required bacterial bundle-forming pili. Furthermore, inhibition of ABL and PDGFR with imatinib protected against infection of mice with modest loads of C. rodentium, whereas the kinases were dispensable for high inocula or late after infection. These results indicate that ABL and PDGFR have indispensable roles in early A/E pathogen attachment to intestinal epithelial cells and for in vivo infection with limiting inocula but are not required for late intimate bacterial attachment or high inoculum infections.

STAT1 acts as a tumor promoter for leukemia development.

The tumor suppressor STAT1 is considered a key regulator of the surveillance of developing tumors. Here, we describe an unexpected tumor-promoting role for STAT1 in leukemia. STAT1(-/-) mice are partially protected from leukemia development, and STAT1(-/-) tumor cells induce leukemia in RAG2(-/-) and immunocompetent mice with increased latency. The low MHC class I protein levels of STAT1(-/-) tumor cells enable efficient NK cell lysis and account for the enhanced tumor clearance. Strikingly, STAT1(-/-) tumor cells acquire increased MHC class I expression upon leukemia progression. These findings define STAT1 as a tumor promoter in leukemia development. Furthermore, we describe the upregulation of MHC class I expression as a general mechanism that allows for the escape of hematopoietic malignancies from immune surveillance.

Importance of PKCδ signaling in fractionated-radiation-induced expansion of glioma-initiating cells and resistance to cancer treatment.

Brain tumors frequently recur or progress as focal masses after treatment with ionizing radiation. However, the mechanisms underlying the repopulation of tumor cells after radiation have remained unclear. In this study, we show that cellular signaling from Abelson murine leukemia viral oncogene homolog (Abl) to protein kinase Cδ (PKCδ) is crucial for fractionated-radiation-induced expansion of glioma-initiating cell populations and acquisition of resistance to anticancer treatments. Treatment of human glioma cells with fractionated radiation increased Abl and PKCδ activity, expanded the CD133-positive (CD133(+)) cell population that possesses tumor-initiating potential and induced expression of glioma stem cell markers and self-renewal-related proteins. Moreover, cells treated with fractionated radiation were resistant to anticancer treatments. Small interfering RNA (siRNA)-mediated knockdown of PKCδ expression blocked fractionated-radiation-induced CD133(+) cell expansion and suppressed expression of glioma stem cell markers and self-renewal-related proteins. It also suppressed resistance of glioma cells to anticancer treatments. Similarly, knockdown of Abl led to a decrease in CD133(+) cell populations and restored chemotherapeutic sensitivity. It also attenuated fractionated-radiation-induced PKCδ activation, suggesting that Abl acts upstream of PKCδ. Collectively, these data indicate that fractionated radiation induces an increase in the glioma-initiating cell population, decreases cellular sensitivity to cancer treatment and implicates activation of Abl-PKCδ signaling in both events. These findings provide insights that might prove pivotal in the context of ionising-radiation-based therapeutic interventions for brain tumors.

Enhanced expression of the PDGFR/Abl signaling pathway in aromatase inhibitor-resistant breast cancer.

BACKGROUND: We have found that the platelet-derived growth factor receptor (PDGFR)/Abl signaling pathway is up-regulated as a determinant of the acquisition of resistance to estrogen deprivation in vitro. We aimed to determine its clinical relevance in aromatase inhibitor (AI)-resistant breast cancer. PATIENTS AND METHODS: We identified a cohort of 45 patients with estrogen receptor-positive breast cancer who had been treated with an AI, subsequently relapsed and had biopsy material available from both the presentation and post-AI recurrent lesion. PDGFRα, PDGFRß and Abl expression was assessed in formalin-fixed paraffin-embedded sections. RESULTS: Tumor protein expression of PDGFRα (1.39-fold, P=0.0065), PDGFRß (4.32-fold, P=0.006) and Abl (1.8-fold, P=0.001) was increased at the point of relapse. Tumor and stromal expression of PDGFRα as well as PDGFRß was significantly correlated in pre-treatment and relapse samples. High post-treatment tumor and stromal PDGFRß levels were associated with a short time to treatment failure (TTF). Expression of PDGFRα in relapsing tumor specimens was correlated with Abl expression and Ki67 levels. Furthermore, changes in Abl correlated significantly with changes in ER expression. CONCLUSIONS: These clinical data support a role for enhanced PDGF/Abl signaling in AI-resistant disease and provide a rationale for targeting the pathway in endocrine-resistant breast cancer.

The tyrosine kinase Abl is a component of macrophage podosomes and is required for podosome formation and function.

Myeloid leukocytes form actin-based plasma membrane protrusions, called podosomes, that are implicated in myeloid cell recruitment into tissues and cell migration within the interstitium. In this study, we show that tyrosine kinases of the Abl family are present in podosomes formed by murine and human macrophages. Silencing of Abl expression in bone marrow-derived macrophages and monocyte-derived macrophages by siRNA or Abl enzymatic inhibition with imatinib resulted in the disassembly of macrophage podosomes and the reduction of their capacity to degrade an extracellular matrix and migrate through matrigel matrices and endothelial cell monolayers. Additionally, macrophages deficient in Src-family kinases, which cross-talk with Abl in regulating macrophage migration, also demonstrated podosome disassembly. These findings suggest that podosome disassembly induced by Abl targeting may inhibit podosome-dependent functions such as leukocyte recruitment into inflammatory sites and osteoclast-dependent bone resorption.

Abl regulates smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 activation.

Abl is a nonreceptor tyrosine kinase that has a role in regulating migration and adhesion of nonmuscle cells as well as smooth muscle contraction. The role of Abl in smooth muscle cell proliferation has not been investigated. In this study, treatment with endothelin-1 (ET-1) and platelet-derived growth factor (PDGF) increased Abl phosphorylation at Tyr(412) (an indication of Abl activation) in vascular smooth muscle cells. To assess the role of Abl in smooth muscle cell proliferation, we generated stable Abl knockdown cells by using lentivirus-mediated RNA interference. ET-1- and PDGF-induced cell proliferation was attenuated in Abl knockdown cells compared with cells expressing control shRNA and uninfected cells. Abl silencing also arrested cell cycle progression from G(0)/G(1) to S phase. Furthermore, activation of smooth muscle cells with ET-1 and PDGF induced phosphorylation of ERK1/2 and Akt. Abl knockdown attenuated ERK1/2 phosphorylation in smooth muscle cells stimulated with ET-1 and PDGF. However, Akt phosphorylation upon stimulation with ET-1 and PDGF was not reduced. Because Abl is known to regulate actin polymerization in smooth muscle, we also evaluated the effects of inhibition of actin polymerization on phosphorylation of ERK1/2. Pretreatment with the actin polymerization inhibitor latrunculin-A also blocked ERK1/2 phosphorylation during activation with ET-1 and PDGF. The results suggest that Abl may regulate smooth muscle cell proliferation by modulating actin dynamics and ERK1/2 phosphorylation during mitogenic activation.

SH2-containing inositol 5'-phosphatase inhibits transformation of Abelson murine leukemia virus.

v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) transforms pre-B cells. Transformation requires the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is antagonized by SH2-containing inositol 5'-phosphatase (SHIP), raising the possibility that v-Abl modulates PI3K signaling through SHIP. Consistent with this, we show that v-Abl expression reduces levels of full-length p145 SHIP in a v-Abl kinase activity-dependent fashion. This event requires signals from the Abl SH2 domain but not the carboxyl terminus. Forced expression of full-length SHIP significantly reduces Ab-MLV pre-B-cell transformation. Therefore, reduction of SHIP protein by v-Abl is a critical component in Ab-MLV transformation.

Tyrosine kinase inhibitors: why does the current process of clinical development not apply to them?

The robust clinical activity of imatinib and trastuzumab for treatment of chronic myeloid leukemia, gastrointestinal stromal tumors, and breast cancer has demonstrated that blocking pathogenic tyrosine kinases can alter the natural history of human tumors. On the other hand, EGF receptor inhibitors have shown overall modest activity. The contrast in the development of these agents implies that both molecular target dependence and patient selection are essential for the successful outcome of this process. We will contrast lessons derived from the development of inhibitors of Abl, c-Kit, HER2/neu (erbB2), and EGFR, highlight successes and limitations in the field, and propose new approaches for clinical development of tyrosine kinase inhibitor therapy.

Vitexin isolated from Prosopis cineraria leaves induce apoptosis in K-562 leukemia cells via inhibition of the BCR-ABL-Ras-Raf pathway.

OBJECTIVES: Leukemia is one of the severe cancer types all around the globe. Even though some chemotherapeutic drugs are available for treating leukemia, they have various side effects. As an alternative approach, herbal drugs are focused on current research to overcome leukemia. The present work was conducted to investigate the antileukemic mechanism of active phytochemical vitexin, which was isolated from ethno-medicine (Prosopis cineraria leaf) used by traditional healers of West Bengal, India. METHODS: Antiproliferative mechanisms of selected phyto-compound against K-562 cells were evaluated using cellular uptake, morphological changes, DNA fragmentation, mitochondrial membrane potential and signaling pathways analysis. KEY FINDINGS: Vitexin exhibited cytotoxicity by reducing mitochondrial membrane potential (32.40%) and causing DNA fragmentation (84.15%). The western blotting study indicated inhibition of cell survival proteins (BCR, ABL, H-RAS, N-RAS, K-RAS and RAF) and expression of apoptotic proteins (p38, BAX and caspase-9) in leukemia cells upon treatment with vitexin. CONCLUSIONS: Based on the results, presently investigated phyto-compound vitexin could be considered for developing safe and natural drugs to treat leukemia after conducting suitable preclinical and clinical trials.

Tip60-mediated acetylation activates transcription independent apoptotic activity of Abl.

BACKGROUND: The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR), is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown. RESULTS: We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (KXXK--X3-5--SGS) is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase. CONCLUSION: Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function.

Abl family tyrosine kinases regulate sialylated ganglioside receptors for polyomavirus.

Sialylated lipids serve as cellular receptors for polyomaviruses. Using pharmacological inhibitors and cell lines derived from knockout mice, we demonstrate that Abl family tyrosine kinases are required for replication of mouse polyomavirus and BK virus, a human polyomavirus associated with allograft failure following kidney transplantation. We show that decreasing Abl family kinase activity results in low levels of cell surface ganglioside receptors for mouse polyomavirus and that inhibition of sialidase activity promotes virion binding in the absence of Abl family kinase activity. These data provide evidence that Abl family kinases reduce ganglioside turnover in the plasma membrane by inhibiting host cell sialidase activity. Thus, Abl family kinases regulate the susceptibility of cells to polyomavirus infection by modulating gangliosides required for viral attachment.

Guidance of myocardial patterning in cardiac development by Sema6D reverse signalling.

Cardiac chamber formation involves dynamic changes in myocardial organization, including trabeculation and expansion of the compact layer. The positional cues that regulate myocardial patterning, however, remain unclear. Through ligation of the Plexin-A1 receptor, the transmembrane-type semaphorin Sema6D regulates endocardial cell migration. Here, we demonstrate that knockdown of either Sema6D or Plexin-A1 leads to the generation of a small, thin ventricular compact layer and to defective trabeculation. In the heart, expression of the Plexin-A1 extracellular domain alone can rescue the defective trabeculation induced by suppression of Plexin-A1, but not that resulting from defective Sema6D expression. This indicates that reverse signalling by Sema6D occurs within the myocardium. In a ligand-dependent manner, Abl kinase is recruited to the cytoplasmic tail of Sema6D and activated, resulting in phosphorylation of Enabled and dissociation from Sema6D. Constitutive activation of Sema6D signalling enhances the migration of myocardial cells into the trabeculae, whereas inhibition arrests cells within the compact layer. Thus, Sema6D coordinates both compact-layer expansion and trabeculation, functioning as both a ligand and a receptor for Plexin-A1.