RESUMO
Mature tropical urban trees are susceptible to root and trunk rot caused by pathogenic fungi. A metagenomic survey of such fungi was carried out on 210 soil and tissue samples collected from 134 trees of 14 common species in Singapore. Furthermore, 121 fruiting bodies were collected and barcoded. Out of the 22,067 OTUs (operational taxonomic units) identified, 10,646 OTUs had annotation information, and most were either ascomycetes (63.4%) or basidiomycetes (22.5%). Based on their detection in the diseased tissues and surrounding soils and/or the presence of fruiting bodies, fourteen basidiomycetes (nine Polyporales, four Hymenochaetales, one Boletales) and three ascomycetes (three species of Scytalidium) were strongly associated with the diseased trees. Fulvifomes siamensis affected the largest number of tree species surveyed. The association of three fungi was further supported by in vitro wood decay studies. Genetic heterogeneity was common in the diseased tissues and fruiting bodies (Ganoderma species especially). This survey identified the common pathogenic fungi of tropical urban trees and laid the foundation for early diagnosis and targeted mitigation efforts. It also illustrated the complexity of fungal ecology and pathogenicity.
RESUMO
The p21-activated kinases (PAKs) are one of the first direct kinase targets of Ras-related small GTPases to be discovered and have emerged as central players in growth factor signaling networks that regulate morphogenetic processes. In some situations, PAKs control cell proliferation, but their wider role involves establishing cell polarity and promoting cellular plasticity via changes in the actin cytoskeleton. PAKs have been shown to impact on three important areas of human health, namely, cancer, brain function, and virus infection. We review the mechanisms and targets of PAKs in these contexts and provide an overview of the ways in which inhibitors might act to arrest tumor growth, combat virus infection, and promote cell apoptosis. Although PAKs are most abundant in the brain, there are few details of how they might be operating in this context. The advent of new and more selective PAK inhibitors promises new avenues of treatment and allows us to probe in greater detail the importance of PAK biology.
Assuntos
Encefalopatias/enzimologia , Proteínas de Neoplasias/fisiologia , Neoplasias/enzimologia , Proteínas do Tecido Nervoso/fisiologia , Processamento de Proteína Pós-Traducional , Viroses/enzimologia , Quinases Ativadas por p21/fisiologia , Apoptose/fisiologia , Ciclo Celular/fisiologia , Citoesqueleto/metabolismo , Escherichia coli Êntero-Hemorrágica/fisiologia , Ativação Enzimática , Infecções por Escherichia coli/enzimologia , Humanos , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/patologia , Proteínas Oncogênicas/fisiologia , Fosfoproteínas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
Collapsin response mediator proteins are ubiquitously expressed from multiple genes (CRMPs 1-5) and play important roles in dividing cells and during semaphorin 3A (Sema3A) signaling. Nonetheless, their mode of action remains opaque. Here we carried out in vivo and in vitro assays that demonstrate that CRMPs are a new class of microtubule-associated protein (MAP). In experiments with CRMP1 or CRMP2 and their derivatives, only the C-terminal region (residues 490-572) mediated microtubule binding. The in vivo microtubule association of CRMPs was abolished by taxol or epothilone B, which is highly unusual. CRMP2-depleted cells exhibited destabilized anaphase astral microtubules and altered spindle position. In a cell-based assay, all CRMPs stabilized interphase microtubules against nocodazole-mediated depolymerization, with CRMP1 being the most potent. Remarkably, a 82-residue C-terminal region of CRMP1 or CRMP2, unrelated to other microtubule binding motifs, is sufficient to stabilize microtubules. In cells, we demonstrate that glycogen synthase kinase-3ß (GSK3ß) inhibition potentiates this activity. Thus, CRMPs are a new class of MAP that binds through a unique motif, but in common with others such as Tau, is antagonized by GSK3ß. This regulation is consistent with such kinases being critical for the Sema3A (collapsin) pathway. These findings have implications for cancer and neurodegeneration.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Paclitaxel/farmacologia , Moduladores de Tubulina/farmacologia , Motivos de Aminoácidos , Anáfase/efeitos dos fármacos , Anáfase/fisiologia , Animais , Células COS , Chlorocebus aethiops , Epotilonas/farmacologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Estrutura Terciária de Proteína , Ratos , Semaforina-3A/genética , Semaforina-3A/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.
Assuntos
Coanoflagelados/citologia , Coanoflagelados/enzimologia , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Ligantes , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Receptor tyrosine kinases couple a wide variety of extracellular cues to cellular responses. The class III subfamily comprises the platelet-derived growth factor receptor, c-Kit, Flt3 and c-Fms, all of which relay cell proliferation signals upon ligand binding. Accordingly, mutations in these proteins that confer ligand-independent activation are found in a subset of cancers. These mutations cluster in the juxtamembrane (JM) and catalytic tyrosine kinase domain (TKD) regions. In the case of acute myeloid leukemia (AML), the juxtamembrane (named ITD for internal tandem duplication) and TKD Flt3 mutants differ in their spectra of clinical outcomes. Although the mechanism of aberrant activation has been largely elucidated by biochemical and structural analyses of mutant kinases, the differences in disease presentation cannot be attributed to a change in substrate specificity or signaling strength of the catalytic domain. This review discusses the latest literature and presents a working model of differential Flt3 signaling based on mis-localized juxtamembrane autophosphorylation, to account for the disease variation. This will have bearing on therapeutic approaches in a complex disease such as AML, for which no efficacious drug yet exists.
Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Membrana Celular/metabolismo , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/metabolismo , Modelos Biológicos , Estrutura Terciária de Proteína , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/químicaRESUMO
ACK1 (activated Cdc42-associated kinase 1), a cytoplsmic tyrosine kinase, is implicated in metastatic behavior, cell spreading and migration, and epidermal growth factor receptor (EGFR) signaling. The function of ACK1 in the regulation of receptor tyrosine kinases requires a C-terminal region that demonstrates a significant homology to the EGFR binding domain of MIG6. In this study, we have identified additional receptor tyrosine kinases, including Axl, leukocyte tyrosine kinase, and anaplastic lymphoma kinase, that can bind to the ACK1/MIG6 homology region. Unlike the interaction between MIG6 and EGFR, our data suggest that these receptor tyrosine kinases require the adaptor protein Grb2 for efficient binding, which interacts with highly conserved proline-rich regions that are conserved between ACK1 and MIG6. We have focused on Axl and compared how ACK1/Axl differs from the ACK1/EGFR axis by investigating effects of knockdown of endogenous ACK1. Although EGFR activation promotes ACK1 turnover, Axl activation by GAS6 does not; interestingly, the reciprocal down-regulation of GAS6-stimulated Axl is blocked by removing ACK1. Thus, ACK1 functions in part to control Axl receptor levels. Silencing of ACK1 also leads to diminished ruffling and migration in DU145 and COS7 cells upon GAS6-Axl signaling. The ability of ACK1 to modulate Axl and perhaps anaplastic lymphoma kinase (altered in anaplastic large cell lymphomas) might explain why ACK1 can promote metastatic and transformed behavior in a number of cancers.
Assuntos
Proteína Adaptadora GRB2/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Movimento Celular , Chlorocebus aethiops , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2/classificação , Proteína Adaptadora GRB2/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Dados de Sequência Molecular , Proteínas Oncogênicas/classificação , Proteínas Oncogênicas/genética , Filogenia , Proteínas Tirosina Quinases/classificação , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas , Interferência de RNA , Receptores Proteína Tirosina Quinases/classificação , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Receptor Tirosina Quinase AxlRESUMO
Fractionation of brain extracts and functional biochemical assays identified PP2Calpha, a serine/threonine phosphatase, as the major biochemical activity inhibiting PAK1. PP2Calpha dephosphorylated PAK1 and p38, both of which were activated upon hyperosmotic shock with the same kinetics. In comparison to growth factors, hyperosmolality was a more potent activator of PAK1. Therefore we characterize the PAK signaling pathway in the hyperosmotic shock response. Endogenous PAKs were recruited to the p38 kinase complex in a phosphorylation-dependent manner. Overexpression of a PAK inhibitory peptide or dominant negative Cdc42 revealed that p38 activation was dependent on PAK and Cdc42 activities. PAK mutants deficient in binding to Cdc42 or PAK-interacting exchange factor were not activated. Using a panel of kinase inhibitors, we identified PI3K acting upstream of PAK, which correlated with PAK repression by pTEN overexpression. RNA interference knockdown of PAK expression reduced stress-induced p38 activation and conversely, PP2Calpha knockdown increased its activation. Hyperosmotic stress-induced PAK translocation away from focal adhesions to the perinuclear compartment and resulted in disassembly of focal adhesions, which are hallmarks of PAK activation. Inhibition of PAK by overexpression of PP2Calpha or the kinase inhibitory domain prevented sorbitol-induced focal adhesion dissolution. Inhibition of MAPK pathways showed that MEK-ERK signaling but not p38 is required for full PAK activation and focal adhesion turnover. We conclude that 1) PAK plays a required role in hyperosmotic signaling through the PI3K/pTEN/Cdc42/PP2Calpha/p38 pathway, and 2) PAK and PP2Calpha modulate the effects of this pathway on focal adhesion dynamics.
Assuntos
Adesões Focais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Fosfatase 2/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática/fisiologia , Adesões Focais/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Pressão Osmótica , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Proteína Fosfatase 2/genética , Estrutura Terciária de Proteína/fisiologia , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Genetic studies have implicated the cytosolic juxtamembrane region of the Kit receptor tyrosine kinase as an autoinhibitory regulatory domain. Mutations in the juxtamembrane domain are associated with cancers, such as gastrointestinal stromal tumors and mastocytosis, and result in constitutive activation of Kit. Here we elucidate the biochemical mechanism of this regulation. A synthetic peptide encompassing the juxtamembrane region demonstrates cooperative thermal denaturation, suggesting that it folds as an autonomous domain. The juxtamembrane peptide directly interacted with the N-terminal ATP-binding lobe of the kinase domain. A mutation in the juxtamembrane region corresponding to an oncogenic form of Kit or a tyrosine-phosphorylated form of the juxtamembrane peptide disrupted the stability of this domain and its interaction with the N-terminal kinase lobe. Kinetic analysis of the Kit kinase harboring oncogenic mutations in the juxtamembrane region displayed faster activation times than the wild-type kinase. Addition of exogenous wild-type juxtamembrane peptide to active forms of Kit inhibited its kinase activity in trans, whereas the mutant peptide and a phosphorylated form of the wild-type peptide were less effective inhibitors. Lastly, expression of the Kit juxtamembrane peptide in cells which harbor an oncogenic form of Kit inhibited cell growth in a Kit-specific manner. Together, these results show the Kit kinase is autoinhibited through an intramolecular interaction with the juxtamembrane domain, and tyrosine phosphorylation and oncogenic mutations relieved the regulatory function of the juxtamembrane domain.