The protein kinase Pelle mediates feedback regulation in the Drosophila Toll signaling pathway.

Dorsoventral polarity in the Drosophila embryo is established through a signal transduction cascade triggered in ventral and ventrolateral regions. Activation of a transmembrane receptor, Toll, leads to localized recruitment of the adaptor protein Tube and protein kinase Pelle. Signaling through these components directs degradation of the IkappaB-like inhibitor Cactus and nuclear translocation of the Rel protein Dorsal. Here we show through confocal immunofluorescence microscopy that Pelle functions to downregulate the signal-dependent relocalization of Tube. Inactivation of the Pelle kinase domain, or elimination of the Tube-Pelle interaction, dramatically increases Tube recruitment to the ventral plasma membrane in regions of active signaling. We also characterize a large collection of pelle alleles, identifying the molecular lesions in these alleles and their effects on Pelle autophosphorylation, Tube phosphorylation and Tube relocalization. Our results point to a mechanism operating to modulate the domain or duration of signaling downstream from Tube and Pelle.     

TAK1 mitogen-activated protein kinase kinase kinase is activated by autophosphorylation within its activation loop

TAK1, a member of the mitogen-activated kinase kinase kinase family, is activated in vivo by various cytokines, including interleukin-1 (IL-1), or when ectopically expressed together with the TAK1-binding protein TAB1. However, this molecular mechanism of activation is not yet understood. We show here that endogenous TAK1 is constitutively associated with TAB1 and phosphorylated following IL-1 stimulation. Furthermore, TAK1 is constitutively phosphorylated when ectopically overexpressed with TAB1. In both cases, dephosphorylation of TAK1 renders it inactive, but it can be reactivated by preincubation with ATP. A mutant of TAK1 that lacks kinase activity is not phosphorylated either following IL-1 treatment or when coexpressed with TAB1, indicating that TAK1 phosphorylation is due to autophosphorylation. Furthermore, mutation to alanine of a conserved serine residue (Ser-192) in the activation loop between kinase domains VII and VIII abolishes both phosphorylation and activation of TAK1. These results suggest that IL-1 and ectopic expression of TAB1 both activate TAK1 via autophosphorylation of Ser-192.     

The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation.

Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.     

The Drosophila Toll signaling pathway

The identification of the Drosophila melanogaster Toll pathway cascade and the subsequent characterization of TLRs have reshaped our understanding of the immune system. Ever since, Drosophila NF-κB signaling has been actively studied. In flies, the Toll receptors are essential for embryonic development and immunity. In total, nine Toll receptors are encoded in the Drosophila genome, including the Toll pathway receptor Toll. The induction of the Toll pathway by gram-positive bacteria or fungi leads to the activation of cellular immunity as well as the systemic production of certain antimicrobial peptides. The Toll receptor is activated when the proteolytically cleaved ligand Spatzle binds to the receptor, eventually leading to the activation of the NF-κB factors Dorsal-related immunity factor or Dorsal. In this study, we review the current literature on the Toll pathway and compare the Drosophila and mammalian NF-κB pathways.     

Inhibition of ERK-MAP kinase signaling by RSK during Drosophila development

Although p90 ribosomal S6 kinase (RSK) is known as an important downstream effector of the ribosomal protein S6 kinase/extracellular signal-regulated kinase (Ras/ERK) pathway, its endogenous role, and precise molecular function remain unclear. Using gain-of-function and null mutants of RSK, its physiological role was successfully characterized in Drosophila. Surprisingly, RSK-null mutants were viable, but exhibited developmental abnormalities related to an enhanced ERK-dependent cellular differentiation such as ectopic photoreceptor- and vein-cell formation. Conversely, overexpression of RSK dramatically suppressed the ERK-dependent differentiation, which was further augmented by mutations in the Ras/ERK pathway. Consistent with these physiological phenotypes, RSK negatively regulated ERK-mediated developmental processes and gene expressions by blocking the nuclear localization of ERK in a kinase activity-independent manner. In addition, we further demonstrated that the RSK-dependent inhibition of ERK nuclear migration is mediated by the physical association between ERK and RSK. Collectively, our study reveals a novel regulatory mechanism of the Ras/ERK pathway by RSK, which negatively regulates ERK activity by acting as a cytoplasmic anchor in Drosophila.     

Mammalian Raf-1 is activated by mutations that restore Raf signaling in Drosophila.

An interaction with the Ras proto-oncogene product is a requirement for Raf-1 activation in many signaling cascades. The significance of this interaction is demonstrated by the fact that a mutation preventing the Ras-Raf interaction severely impairs the function of both mammalian (Raf-1) and Drosophila (D-Raf) Raf proteins. In D-Raf, however, dominant intragenic mutations have been identified that suppress the effect of the Ras-binding site (RBS) mutation. To address the mechanism by which these mutations restore Raf signaling, we have introduced the suppressor mutations into the analogous residues of mammalian Raf-1. Here, we show that rather than compensating for the RBS mutation by restoring the Ras-Raf-1 interaction, the suppressor mutations increase the enzymatic and biological activity of Raf-1, allowing Raf-1 to signal in the absence of Ras binding. Surprisingly, we find that while one of the suppressor mutations (P181L) increases the basal kinase activity of Raf-1, it also abolishes the ability of wild-type Raf-1 to become activated by Ras. This mutation occurs in the cysteine-rich domain (CRD) of Raf-1 and demonstrates the importance of this region for a productive Ras-Raf interaction. Finally, we present evidence that the most activating suppressor mutation (G498S) increases Raf-1 activity by introducing a novel phosphorylation site into the L12 activation loop of the Raf-1 kinase domain.     

Oocyte aging is controlled by mitogen‐activated protein kinase signaling

Oogenesis is one of the first processes to fail during aging. In women, most oocytes cannot successfully complete meiotic divisions already during the fourth decade of life. Studies of the nematode Caenorhabditis elegans have uncovered conserved genetic pathways that control lifespan, but our knowledge regarding reproductive aging in worms and humans is limited. Specifically, little is known about germline internal signals that dictate the oogonial biological clock. Here, we report a thorough characterization of the changes in the worm germline during aging. We found that shortly after ovulation halts, germline proliferation declines, while apoptosis continues, leading to a gradual reduction in germ cell numbers. In late aging stages, we observed that meiotic progression is disturbed and crossover designation and DNA double‐strand break repair decrease. In addition, we detected a decline in the quality of mature oocytes during aging, as reflected by decreasing size and elongation of interhomolog distance, a phenotype also observed in human oocytes. Many of these altered processes were previously attributed to MAPK signaling variations in young worms. In support of this, we observed changes in activation dynamics of MPK‐1 during aging. We therefore tested the hypothesis that MAPK controls oocyte quality in aged worms using both genetic and pharmacological tools. We found that in mutants with high levels of activated MPK‐1, oocyte quality deteriorates more rapidly than in wild‐type worms, whereas reduction of MPK‐1 levels enhances quality. Thus, our data suggest that MAPK signaling controls germline aging and could be used to attenuate the rate of oogenesis quality decline.     

Mob as tumor suppressor is activated by Hippo kinase for growth inhibition in Drosophila

Tissue growth and organ size are determined by coordinated cell proliferation and apoptosis in development. Recent studies have demonstrated that Hippo (Hpo) signaling plays a crucial role in coordinating these processes by restricting cell proliferation and promoting apoptosis. Here we provide evidence that the Mob as tumor suppressor protein, Mats, functions as a key component of the Hpo signaling pathway. We found that Mats associates with Hpo in a protein complex and is a target of the Hpo serine/threonine protein kinase. Mats phosphorylation by Hpo increases its affinity with Warts (Wts)/large tumor suppressor (Lats) serine/threonine protein kinase and ability to upregulate Wts catalytic activity to target downstream molecules such as Yorkie (Yki). Consistently, our epistatic analysis suggests that mats acts downstream of hpo. Coexpression analysis indicated that Mats can indeed potentiate Hpo-mediated growth inhibition in vivo. Our results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals.     

果蝇Toll和IMD信号通路中的功能结构域

功能结构域在蛋白相互关系中起着重要的作用,在细胞信号通路中,上、下游信号分子结构域间的相互作用,传递着信号.赖氨酸型肽聚糖( Lys-typePGN)(来自革兰氏阳性菌)和β-1,3葡聚糖(β-1,3 glucan)(来自真菌)激活果蝇Toll信号通路;二氨基庚二酸型肽聚糖(DAP-type PGN)和脂多糖粗多糖(L...     

TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development

Transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) is a member of the MAPKKK superfamily and has been characterized as a component of the TGF-beta/bone morphogenetic protein signaling pathway. TAK1 function has been extensively studied in cultured cells, but its in vivo function is not fully understood. In this study, we isolated a Drosophila homolog of TAK1 (dTAK1) which contains an extensively conserved NH(2)-terminal kinase domain and a partially conserved COOH-terminal domain. To learn about possible endogenous roles of TAK1 during animal development, we generated transgenic flies which express dTAK1 or the mouse TAK1 (mTAK1) gene in the fly visual system. Ectopic activation of TAK1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by TAK1 signaling. Expression of a dominant negative form of dTAK during embryonic development resulted in various embryonic cuticle defects including dorsal open phenotypes. Our results strongly suggest that in Drosophila melanogaster, TAK1 functions as a MAPKKK in the JNK signaling pathway and participates in such diverse roles as control of cell shape and regulation of apoptosis.     

Dictyostelium myosin II heavy-chain kinase A is activated by autophosphorylation: studies with Dictyostelium myosin II and synthetic peptides

One of the major sites phosphorylated on the Dictyostelium myosin II heavy chain by the Dictyostelium myosin II heavy-chain kinase A (MHCK A) is Thr-2029. Two synthetic peptides based on the sequence of the Dictyostelium myosin II heavy chain around Thr-2029 have been synthesized: MH-1 (residues 2020-2035; RKKFGESEKTKTKEFL-amide) and MH-2 (residues 2024-2035). Both peptides are substrates for MHCK A and are phosphorylated to a level of 1 mol of phosphate/mol. Tryptic digests indicate that the peptides are phosphorylated on the threonine corresponding to Thr-2029. When assays are initiated by the addition of MHCK A, the rate of phosphate incorporation into the peptides increases progressively for 4-6 min. The increasing activity of MHCK A over this time period is a result of autophosphorylation. Although each 130-kDa subunit of MHCK A can incorporate up to 10 phosphate molecules, 3 molecules of phosphate per subunit are sufficient to completely activate the kinase. Autophosphorylated MHCK A displays Vmax values of 2.2 and 0.6 mumol.min-1.mg-1 and Km values of 100 and 1200 microM with peptides MH-1 and MH-2, respectively. Unphosphorylated MHCK A displays a 50-fold lower Vmax with MH-1 but only a 2-fold greater Km. In the presence of Dictyostelium myosin II, the rate of autophosphorylation of MHCK A is increased 4-fold. If assays are performed at 4 degrees C (to slow the rate of MHCK A autophosphorylation), autophosphorylation can be shown to increase the activity of MHCK A with myosin II.     

Alcohol resistance in Drosophila is modulated by the Toll innate immune pathway

A growing body of evidence has shown that alcohol alters the activity of the innate immune system and that changes in innate immune system activity can influence alcohol‐related behaviors. Here, we show that the Toll innate immune signaling pathway modulates the level of alcohol resistance in Drosophila. In humans, a low level of response to alcohol is correlated with increased risk of developing an alcohol use disorder. The Toll signaling pathway was originally discovered in, and has been extensively studied in Drosophila. The Toll pathway is a major regulator of innate immunity in Drosophila, and mammalian Toll‐like receptor signaling has been implicated in alcohol responses. Here, we use Drosophila‐specific genetic tools to test eight genes in the Toll signaling pathway for effects on the level of response to ethanol. We show that increasing the activity of the pathway increases ethanol resistance whereas decreasing the pathway activity reduces ethanol resistance. Furthermore, we show that gene products known to be outputs of innate immune signaling are rapidly induced following ethanol exposure. The interaction between the Toll signaling pathway and ethanol is rooted in the natural history of Drosophila melanogaster.     

A dimeric kinase assembly underlying autophosphorylation in the p21 activated kinases

The microtubule-associated protein tau is impacted in neurodegeneration and dementia through its deposition in the form of paired helical filaments in Alzheimer's disease neurofibrillary tangles and through mutations linking it to the autosomal dominant disorder frontotemporal dementia with Parkinsonism. When isolated in solution tau is intrinsically unstructured and does not fold, while the conformation of the protein in the microtubule-bound state remains uncharacterized. Here we show that the repeat region of tau, which has been reported both to mediate tau microtubule interactions and to constitute the proteolysis-resistant core of disease-associated tau aggregates, associates with lipid micelles and vesicles and folds into an ordered structure upon doing so. In addition to providing the first structural insights into a folded state of tau, our results support a role for lipid membranes in mediating tau function and tau pathology.     

A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase.

Antibodies to the human Shc adaptor protein were used to isolate a cDNA encoding a Drosophila Shc protein (dShc) by screening an expression library. The dshc gene, which maps to position 67B-C on the third chromosome, encodes a 45-kDa protein that is widely expressed throughout the Drosophila life cycle. In flies, the dShc protein physically associates with the activated Drosophila epidermal growth factor receptor homolog (DER) and is inducibly phosphorylated on tyrosine by DER. The 45-kDa dShc protein is closely related both in overall organization and in amino acid sequence (46% identity) to the 52-kDa mammalian Shc isoform. In addition to a C-terminal Src homology 2 (SH2) domain, dShc contains an N-terminal phosphotyrosine-binding (PTB) domain, which associates in vitro with the autophosphorylated DER receptor tyrosine kinase and with phosphopeptides containing an Asn-Pro-X-pTyr motif, where pTyr stands for phosphotyrosine. A potential binding site for the dShc PTB domain is located at Tyr-1228 of DER. These results indicate that the shc gene has been conserved in evolution, as have the binding properties of the Shc PTB and SH2 domains. Despite the close relationship between the Drosophila and mammalian Shc proteins, dShc lacks the high-affinity Grb2-binding site found in mammalian Shc, suggesting that Shc proteins may have functions in addition to regulation of the Ras pathway.     

Inhibition of homologous recombination by DNA-dependent protein kinase requires kinase activity, is titratable, and is modulated by autophosphorylation

How a cell chooses between nonhomologous end joining (NHEJ) and homologous recombination (HR) to repair a double-strand break (DSB) is a central and largely unanswered question. Although there is evidence of competition between HR and NHEJ, because of the DNA-dependent protein kinase (DNA-PK)'s cellular abundance, it seems that there must be more to the repair pathway choice than direct competition. Both a mutational approach and chemical inhibition were utilized to address how DNA-PK affects HR. We find that DNA-PK's ability to repress HR is both titratable and entirely dependent on its enzymatic activity. Still, although requisite, robust enzymatic activity is not sufficient to inhibit HR. Emerging data (including the data presented here) document the functional complexities of DNA-PK's extensive phosphorylations that likely occur on more than 40 sites. Even more, we show here that certain phosphorylations of the DNA-PK large catalytic subunit (DNA-PKcs) clearly promote HR while inhibiting NHEJ, and we conclude that the phosphorylation status of DNA-PK impacts how a cell chooses to repair a DSB.     

Nuclear translocation of activated MAP kinase is developmentally regulated in the developing Drosophila eye

In proneural groups of cells in the morphogenetic furrow of the developing Drosophila eye phosphorylated mitogen activated protein kinase (MAPK) antigen is held in the cytoplasm for hours. We have developed a reagent to detect nuclear MAPK non-antigenically and report our use of this reagent to confirm that MAPK nuclear translocation is regulated by a second mechanism in addition to phosphorylation. This "cytoplasmic hold" of activated MAPK has not been observed in cell culture systems. We also show that MAPK cytoplasmic hold has an essential function in vivo: if it is overcome, developmental patterning in the furrow is disrupted.