Péter Deák

DOS, a Novel Pleckstrin Homology Domain–Containing Protein Required for Signal Transduction between Sevenless and Ras1 in Drosophila

The specification of the R7 photoreceptor cell in the developing eye of Drosophila is dependent upon activation of the Sevenless (SEV) receptor tyrosine kinase. By screening for mutations that suppress signaling via a constitutively activated SEV protein, we have identified a novel gene, daughter of sevenless (dos). DOS is required not only for signal transduction via SEV but also in other receptor tyrosine kinase signaling pathways throughout development. The presence of an amino-terminally located pleckstrin homology domain and many potential tyrosine phosphorylation sites suggests that DOS functions as an adaptor protein able to interact with multiple signaling molecules. Our genetic analysis demonstrates that DOS functions upstream of Ras1 and defines a signaling pathway that is independent of direct binding of the DRK SH2/SH3 adaptor protein to the SEV receptor tyrosine kinase.

APC/CFzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. Here, we report that APC/CFzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. We demonstrate that compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, our data suggest that the activity of APC/CFzr/Cdh1 during the endocycle is not continuous but is cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, our data suggest a model in which the cyclic activity of APC/CFzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. We propose that, as is observed in mitotic cycles, during endocycles, APC/CFzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression.

Deletion of proteasomal subunit S5a/Rpn10/p54 causes lethality, multiple mitotic defects and overexpression of proteasomal genes in Drosophila melanogaster

The regulatory complex of the 26S proteasome is responsible for the selective recognition and binding of multiubiquitinated proteins. It was earlier shown that the subunit S5a/Rpn10/p54 of the regulatory complex is the only cellular protein capable of binding multiubiquitin chains in an in vitro overlay assay. The role of this subunit in substrate selection, however, is a subject of debate, following the observation that its deletion in Saccharomyces cerevisiae is not lethal and instead causes only a mild phenotype. To study the function of this subunit in higher eukaryotes, a mutant Drosophila strain was constructed by deleting the single copy gene encoding subunit S5a/Rpn10/p54. This deletion caused larval-pupal polyphasic lethality, multiple mitotic defects, the accumulation of higher multimers of ubiquitinated proteins and a huge accumulation of defective 26S proteasome particles. Deletion of the subunit S5a/Rpn10/p54 does not destabilise the regulatory complex and does not disturb the assembly of the regulatory complex and the catalytic core. The pupal lethality is a consequence of the depletion of the maternally provided 26S proteasome during the larval stages and a sudden increase in the proteasomal activity demands during the first few hours of pupal development. The huge accumulation of the fully assembled 26S proteasome in the deletion mutant and the lack of free subunits or partially assembled particles indicate that there is a highly coordinated accumulation of all the subunits of the 26S proteasome. This suggests that in higher eukaryotes, as with yeast, a feedback circuit coordinately regulates the expression of the proteasomal genes, and this adjusts the actual proteasome concentration in the cells according to the temporal and/or spatial proteolytic demands.

Mutations in mákos, a Drosophila gene encoding the Cdc27 subunit of the anaphase promoting complex, enhance centrosomal defects in polo and are suppressed by mutations in twins/aar, which encodes a regulatory subunit of PP2A

The gene mákos (mks) encodes the Drosophila counterpart of the Cdc27 subunit of the anaphase promoting complex (APC/C). Neuroblasts from third-larval-instar mks mutants arrest mitosis in a metaphase-like state but show some separation of sister chromatids. In contrast to metaphase-checkpoint-arrested cells, such mutant neuroblasts contain elevated levels not only of cyclin B but also of cyclin A. Mutations in mks enhance the reduced ability of hypomorphic polo mutant alleles to recruit and/or maintain the centrosomal antigens γ-tubulin and CP190 at the spindle poles. Absence of the MPM2 epitope from the spindle poles in such double mutants suggests Polo kinase is not fully activated at this location. Thus, it appears that spindle pole functions of Polo kinase require the degradation of early mitotic targets of the APC/C, such as cyclin A, or other specific proteins. The metaphase-like arrest of mks mutants cannot be overcome by mutations in the spindle integrity checkpoint gene bub1, confirming this surveillance pathway has to operate through the APC/C. However, mutations in the twins/aar gene, which encodes the 55kDa regulatory subunit of PP2A, do suppress the mks metaphase arrest and so permit an alternative means of initiating anaphase. Thus the APC/C might normally be required to inactivate wild-type twins/aar gene product.

Moulting hormone regulates its receptor level in Drosophila melanogaster

Abstract The moulting hormone (MH) receptor level has been measured in embryonic first and third larval stages of D. melanogaster . Fluctuating receptor levels were found in all stages investigated with prominent peaks in 16 h embryos, 18 h first instar and 13 and 33 h third instar larvae. The receptor level peaks occur subsequent to MH peaks. Experimental 20-hydroxyecdysone feeding resulted in a rapid increase of the receptor level, which was dependent upon protein synthesis. When the conversion of ecdysone to 20-hydroxyecdysone was effectively inhibited by the drug, methyrapone, ecdysone was not effective in this receptor level augmentation. The receptor selectively binds 20-hydroxyecdysone only among the several ecdysteroids present in the embryo, providing further evidence that 20-hydroxyecdysone is the active hormone. The different efficacy of these steroids parallels the extent of receptor binding, suggesting the involvement of the receptor in its augmentation. The role of the receptor level in mediating the action of MH is discussed.

Developmental-stage-specific regulation of the polyubiquitin receptors in Drosophila melanogaster

Recognition of polyubiquitylated substrates by the proteasome is a highly regulated process that requires polyubiquitin receptors. We show here that the concentrations of the proteasomal and extraproteasomal polyubiquitin receptors change in a developmentally regulated fashion. The stoichiometry of the proteasomal p54/Rpn10 polyubiquitin receptor subunit, relative to that of other regulatory particle (RP) subunits falls suddenly at the end of embryogenesis, remains low throughout the larval stages, starts to increase again in the late third instar larvae and remains high in the pupae, adults and embryos. A similar developmentally regulated fluctuation was observed in the concentrations of the Rad23 and Dsk2 extraproteasomal polyubiquitin receptors. Depletion of the polyubiquitin receptors at the end of embryogenesis is due to the emergence of a developmentally regulated selective proteolytic activity. To follow the fate of subunit p54/Rpn10 in vivo, transgenic Drosophila melanogaster lines encoding the N-terminal half (NTH), the C-terminal half (CTH) or the full-length p54/Rpn10 subunit were established in the inducible Gal4-UAS system. The daughterless-Gal4-driven whole-body expression of the full-length subunit or its NTH did not produce any detectable phenotypic changes, and the transgenic products were incorporated into the 26S proteasome. The transgene-encoded CTH was not incorporated into the 26S proteasome, caused third instar larval lethality and was found to be multi-ubiquitylated. This modification, however, did not appear to be a degradation signal because the half-life of the CTH was over 48 hours. Accumulation of the CTH disturbed the developmentally regulated changes in subunit composition of the RP and the emergence of the selective proteolytic activity responsible for the depletion of the polyubiquitin receptors. Build-up of subunit p54/Rpn10 in the RP had already started in 84-hour-old larvae and reached the full complement characteristic of the non-larval developmental stages at the middle of the third instar larval stage, just before these larvae perished. Similar shifts were observed in the concentrations of the Rad23 and Dsk2 polyubiquitin receptors. The postsynthetic modification of CTH might be essential for this developmental regulation, or it might regulate an essential extraproteasomal function(s) of subunit p54/Rpn10 that is disturbed by the expression of an excess of CTH.

The isolation and genetic analysis of aCaenorhabditis elegants translocation (szT1) strain bearing an X-chromosome balancer

A single X-chromosome balancer-bearingCelegans ♂+, as a founder of a strain (AF1), was isolated directly from Fl progeny of irradiated+dpy-8unc-3/lon-2++ hermaphrodites on the basis of the absence of recombinant F2 categories. The balancer chromosome (Bal-X-1) suppresses recombination over a two-thirds section of the X chromosome (between genesdpy-8 andlet-2) and is associated with a reciprocal translocation between linkage groups (LG) X and I. Animals homozygous for the translocation (szT1(X:1)) are nonviable. Hermaphrodites heterozygous for the translocation segregate male selfprogeny at a frequency of 0.08-0.12.Bal-X-l carries the marker mutationlon-2(e678) and can be detected cytologically. This balancer chromosome proved useful for rnaimaininga number of X-linked lethal mutations and deficiencies inC. elegans.

Structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex in Drosophila melanogaster

The anaphase-promoting complex/cyclosome or APC/C is a key regulator of chromosome segregation and mitotic exit in eukaryotes. It contains at least 11 subunits, most of which are evolutionarily conserved. The most abundant constituents of the vertebrate APC/C are the four structurally related tetratrico-peptide repeat (TPR) subunits, the functions of which are not yet precisely understood. Orthologues of three of the TPR subunits have been identified in Drosophila. We have shown previously that one of the TPR subunits of the Drosophila APC/C, Apc3 (also known as Cdc27 or Mákos), is essential for development, and perturbation of its function results in mitotic cyclin accumulation and metaphase-like arrest. In this study we demonstrate that the Drosophila APC/C associates with a new TPR protein, a genuine orthologue of the vertebrate Apc7 subunit that is not found in yeasts. In addition to this, transgenic flies knocked down for three of the TPR genes Apc6 (Cdc16), Apc7 and Apc8 (Cdc23), by RNA interference were established to investigate their function. Whole-body expression of subunit-specific dsRNA efficiently silences these genes resulting in only residual mRNA concentrations. Apc6/Cdc16 and Apc8/Cdc23 silencing induces developmental delay and causes different pupal lethality. Cytological examination showed that these animals had an elevated level of apoptosis, high mitotic index and delayed or blocked mitosis in a prometaphase-metaphase-like state with overcondensed chromosomes. The arrested neuroblasts contained elevated levels of cyclin B but, surprisingly, cyclin A appeared to be degraded normally. Contrary to the situation for the Apc6/Cdc16 and Apc8/Cdc23 genes, the apparent loss of Apc7 function does not lead to the above abnormalities. Instead, the Apc7 knocked down animals and null mutants are viable and fertile, although they display mild chromosome segregation defects and anaphase delay. Nevertheless, the Apc7 subunit shows synergistic genetic interaction with Apc8/Cdc23 that, together with the phenotypic data, assumes a limited functional role for Apc7. Taken together, these data suggest that the structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex.

Ubiquitylation of drosophila p54/Rpn10/S5a regulates its interaction with the UBA-UBL polyubiquitin receptors

Analysis of the in vivo ubiquitylation of the p54/Rpn10 polyubiquitin receptor subunit of the Drosophila 26S proteasome revealed that the site of ubiquitylation is the C-terminal cluster of lysines, which is conserved in higher eukaryotes. Extraproteasomal p54 was extensively multiubiquitylated, but only very modest modification was detected in the proteasome-assembled subunit. Ubiquitylation of p54 seriously jeopardizes one of its most important functions, i.e., the interaction of its ubiquitin-interacting motifs with the ubiquitin-like domain of Dsk2 and Rad23 extraproteasomal polyubiquitin receptors. This modification of p54 supports the previous notion that p54 is a shuttling subunit of the 26S proteasome with a specific extraproteasomal function. This assumption is supported by the observation that, while transgenic p54 can fully rescue the lethal phenotype of the Δp54 null mutation, its derivative from which the cluster of conserved lysines is deleted shifts the lethality from the early pupa to pharate adult stage but cannot rescue the Δp54 mutation, suggesting that ubiquitylated extraproteasomal p54 has an essential role in the pupa-adult transition.

Overexpression of Dsk2/dUbqln results in severe developmental defects and lethality in Drosophila melanogaster that can be rescued by overexpression of the p54/Rpn10/S5a proteasomal subunit

Polyubiquitin receptors execute the targeting of polyubiquitylated proteins to the 26S proteasome. In vitro studies indicate that disturbance of the physiological balance among different receptor proteins impairs the proteasomal degradation of polyubiquitylated proteins. To study the physiological consequences of shifting the in vivo equilibrium between the p54/Rpn10 proteasomal and the Dsk2/dUbqln extraproteasomal polyubiquitin receptors, transgenic Drosophila lines were constructed in which the overexpression or RNA interference‐mediated silencing of these receptors can be induced. Flies overexpressing Flag–p54 were viable and fertile, without any detectable morphological abnormalities, although detectable accumulation of polyubiquitylated proteins demonstrated a certain level of proteolytic disturbance. Flag–p54 was assembled into the 26S proteasome and could fully complement the lethal phenotype of a p54 null mutant Drosophila line. The overexpression of Dsk2 caused severe morphological abnormalities in the late pupal stages, leading to pharate adult lethality, accompanied by a huge accumulation of highly polyubiquitylated proteins. The lethal phenotype of Dsk2 overexpression could be rescued in a double transgenic line coexpressing Flag–Dsk2 and Flag–p54. Although the double transgenic line was viable and fertile, it did not restore the proteolytic defects; the accumulation of the highly polyubiquitylated proteins was even more severe in the double transgenic line. Significant differences were found in the Dsk2–26S proteasome interaction in Drosophila melanogaster as compared with Saccharomyces cerevisiae. In yeast, Dsk2 can interact only with ΔRpn10 proteasomes and not with the wild‐type one. In Drosophila, Dsk2 does not interact with Δp54 proteasomes, but the interaction can be fully restored by complementing the Δp54 deletion with Flag–p54.