Transcriptional Regulation

The Drosophila larval hematopoietic system has been used as an in vivo model for the genetic and functional genomic analysis of oncogenic cell overproliferation. Ras regulates cell proliferation and differentiation in multicellular eukaryotes. To further elucidate the role of activated Ras in cell overproliferation, a collagen promoter-Gal4 strain was generated to overexpress RasV12 (Ras-act) in Drosophila hemocytes. Activated Ras causes a dramatic increase in the number of circulating larval hemocytes (blood cells); this increase is caused by cellular overproliferation. This phenotype is mediated by the Raf/MAPK pathway. The mutant hemocytes retain the ability to phagocytose bacteria as well as to differentiate into lamellocytes. Microarray analysis of hemocytes overexpressing RasV12 vs. Ras+ identified 279 transcripts that are differentially expressed threefold or more in hemocytes expressing activated Ras. This work demonstrates that it will be feasible to combine genetic and functional genomic approaches in the Drosophila hematopoietic system to systematically identify oncogene-specific downstream targets (Asha, 2003).

One overall finding is that many of the genes that are upregulated in Ras-act cells include genes that function in cell cycle regulation and DNA replication. These genes include both positive and negative regulators of cell proliferation. The cyclin-dependent kinase inhibitor dacapo (which antagonizes the function of cyclin E/cdk2 complexes), as well as the wee1 kinase (which inactivates cdc2), are both induced. There is currently no known function for either gene in promoting cell cycle progression. Thus the induction of these genes may represent a negative feedback mechanism that attempts to reduce cell proliferation under conditions of excessive cell proliferation. Another possibility is that these two genes have currently unknown roles in promoting cell cycle progression. The microarray data also show that regulators that promote all stages of cell cycle progression are induced, not only those that promote the G1/S transition. These data therefore suggest that both the G1/S and G2/M cell cycle transitions may be influenced by an increase in Ras activity (Asha, 2003).

Characterization of Wee enzymatic activity

The sequence conservation of Drosophila Wee strongly suggests that this protein acts as a Cdc2 inhibitory kinase, and this premise was examined directly in vitro. Assays were developed for Cdc2 phosphorylation, using haemmaglutinin (HA) epitope-tagged Drosophila Cdc2 produced by reticulocyte lysate translation, and GST-tagged Drosophila Cyclin B expressed in E. coli. Nuclear and cytoplasmic extracts were prepared from 0-12 hr Drosophila embryos, and tested for enzymatic modification of HA-Cdc2/Cyclin B complexes. Nuclear extracts contain an activity that tyrosine phosphorylated HA-DmCdc2, as detected by probing Western blots with an antibody to phosphotryosine. This phosphorylation is strongly stimulated by Drosophila Cyclin B. Phosphorylation of Drosophila Cdc2 alters its electrophoretic mobility. S35 labelling of the in vitro translated DmCdc2 allows detection of total Cdc2, and assessment of the efficiency with which input Cdc2 is modified. The shift in the mobility of the protein indicates that the majority of the protein is modified. While a number of new forms are seen, only two bands containing phospho-tyrosine were detected by the antibody (Campbell, 1995).

Like the nuclear extract, in vitro translated Drosophila Wee stimulates tryosine phosphorylation of Cdc2, but the latter reaction is more complete. Only the lower mobility phospho-tyrosine containing band is produced, and strong phosphorylation is seen in the absence of cyclin B. The more rapidly migrating phosphotryosine containing band is seen if cytoplasmic extract is included during the reaction, suggesting that it is produced by secondary modification by a separate activity. Indeed, even though the cytoplasmic extract on its own does not cause tryosine phosphorylation, it does modify Cdc2 to produce a more rapidly migrating form. Based on previous characterization this more rapidly migrating form is phosphorylated on Thr161. The Thr161 modifying activity appears to present in both cytoplasmic and nuclear extracts, but not in reticulocyte-translated protein extracts. Mutation of Tyr15 of Drosophila Cdc2 to Phe15 (Y15 to F15) blocks tryosine phosphorylation, suggesting that this is the residue that is modified by Dwee1. Furthermore, the effect of this mutation on the migration of the higher mobility form of Cdc2 suggests that efficient phosphorylation of both Tyr15 and Thr161 occurs when both in vitro translated Wee and cytoplasmic extract are present (Campbell, 1995).

It is concluded that Wee encodes a tyosine kinase that adds inhibitory phosphates to Cdc2 and that this activity is nuclear in embryos. Thus, the catalytic activity of Drosophila Wee is consistent with that expected for a Cdc2 inhibitory kinase and with its ability to compensate for loss of wee1 and mik1 functions in fission yeast (Campbell, 1995).

Protein Interactions

Drosophila Wee1 interacts with members of the γTURC and is required for proper mitotic-spindle morphogenesis and positioning

Wee1 kinases delay entry into mitosis by phosphorylating and inactivating cyclin-dependent kinase 1 (Cdk1). Loss of this activity in many systems, including Drosophila, leads to premature mitotic entry. Drosophila Wee1 (dwee1) mutant embryos show mitotic-spindle defects that include ectopic foci of microtubule organization, formation of multipolar spindles from adjacent centrosome pairs, and promiscuous interactions between neighboring spindles. Furthermore, centrosomes are displaced from the embryo cortex in mutants. These defects are not observed to the same extent in embryos in which nuclei also enter mitosis prematurely as a result of a lack of checkpoint control or in embryos with elevated Cdk1 activity. dWee1 physically interacts with members of the γ-tubulin ring complex (γTuRC), and γ-tubulin is phosphorylated in a dwee1-dependent manner in embryo extracts. Some of the abnormalities in dwee1 mutant embryos cannot be explained by premature entry into mitosis or bulk elevation of Cdk1 activity. Instead, dWee1 is also required for phosphorylation of gamma-tubulin, centrosome positioning, and mitotic-spindle integrity. A model is proposed to account for these requirements (Stempff, 2005).

dwee1 mutant embryos enter mitosis prematurely and form abnormal mitotic spindles. To determine whether premature mitotic entry and consequent centrosome inactivation account for the spindle abnormalities seen, dwee1 mutants, grp mutants, and irradiated wild-type embryos were compared. In all three cases, evidence of centrosome inactivation was seen: diminished astral microtubules and dispersal of both γ-tubulin and Dgrip84 from the centrosome in fixed embryos. Similar results were obtained from analyses of live dwee1 mutant embryos carrying the 17238-GFP transgene, in which GFP is inserted into a gene of unknown function and localizes to microtubules and centrosomes. dwee1 and grp mutant embryos and irradiated wild-type embryos show the loss of GFP signal on astral microtubules and at spindle poles during the syncytial blastoderm division cycles M12 and M13. In addition, all three groups display monopolar spindles, the inability to form a central spindle in M12 and M13, and the failure to fully separate centrosomes during interphase (Stempff, 2005).

Live analysis revealed three additional, phenotypes in cycles 12 and 13 of dwee1 mutants: (1) spindles with one to two ectopic microtubule-organizing centers (MTOC) within the single microtubule network (the ectopic sites change location dramatically within the confines of the spindle); (2) promiscuous interactions between adjacent spindles (in these situations, microtubules from one spindle appear to pull on a neighboring spindle), and (3) multipolar spindles that result from centrosome pairs of neighboring nuclei that interact. The last two phenotypes are also seen in fixed embryos. The first, however, was not seen -- ectopic MTOC may be too dynamic to be preserved during fixation. Importantly, these three phenotypes are absent in live irradiated wild-type embryos and present only at low frequencies in live grp mutants. These phenotypes are referred to as 'dwee1-specific' spindle defects (Stempff, 2005).

Centrosome inactivation in irradiated or grp mutant embryos is dependent on Chk2 function. Therefore, dwee1, chk2 double mutants were analyzed to further assess whether dwee1 spindle defects are caused by induction of the checkpoint. chk2 mutations are known to rescue cellularization in dwee1 mutants. As expected, mutation of chk2 also rescues the phenotypes, such as anastral spindles and the dispersal of γTURC in dwee1 mutants, that are characteristic of centrosome inactivation. However, dwee1, chk2 double mutants still display spindle interactions and form multipolar spindles (11 of 12 embryos in M12 or M13): the two dwee1-specific spindle defects are discernable in fixed embryos. On the basis of these data and the finding that grp mutants or irradiated embryos do not display dwee1-specific spindle defects to the same extent, it is concluded that spindle interactions and multipolar spindles in dwee1 mutants are not due to premature mitotic entry or to induction of chk2-dependent centrosome inactivation (Stempff, 2005).

Cdk1 activity is elevated during cortical syncytial cycles in dwee1 mutant embryos, and elevation of Cdk1 activity has been shown previously to affect spindle morphogenesis in precortical syncytial cycles. Therefore, the possibility that elevated Cdk1 activity is the cause of dwee1-specific spindle defects was addressed. To this end, fixed embryos from a fly stock with six copies of cyclin B, six cycB, were analyzed. Increasing cyclin B levels in embryos is known to increase Cdk1 activity. Consistently, it was found that (six cycB) embryos harbor higher CycB-Cdk1 activity than do wild-type embryos. More Cdk1 coprecipitates with cyclin B from six cycB embryos than from wild-type or dwee1 mutant embryos, suggesting that this increase in activity is due to the presence of more-active complexes, an idea that is consistent with previous observations that Cdk1 levels are not limiting in embryos. six cycB embryos display defects such as asynchronous divisions, but dwee1-specific spindle defects were not detected. It is concluded that a bulk elevation of Cdk1 activity cannot account for dwee1-specific spindle defects (Stempff, 2005).

CycB levels are reproducibly lower in dwee1 embryos than in wild-type embryos. The reason for this finding is not known, but nonetheless the possibility that reduction of CycB levels is the cause of dwee1-specific phenotypes was ruled out. This was done by analyzing embryos from mothers that are hemizygous for cycB and therefore have lower CycB levels. No evidence was found of spindle interactions or centrosome-positioning changes that resemble those of dwee1 mutant embryos (Stempff, 2005).

The interactions between neighboring centrosomes and spindles in dwee1 mutants are limited to cortical syncytial divisions. These are a subset of syncytial divisions that follow the migration of nuclei to the embryo cortex. During cortical divisions, nuclei and centrosomes closely abut the cortex, and their position is maintained by microtubule-filament- and actin-filament-dependent mechanisms. Specifically, astral microtubules nucleated by the centrosomes are proposed to interact with cortical actin to mediate the attachment of centrosomes, along with their associated nuclei, to the cortex. Physical separation of mitotic spindles in a syncytium is thought to occur by reorganization of F-actin caps into pseudocleavage furrows that surround each dividing nucleus. Pseudocleavage furrows form during prophase and metaphase, retract in anaphase, and are mostly absent in late anaphase and telophase (Stempff, 2005).

Pseudocleavage furrows in dwee1 mutants form with normal timing and reach similar depths as in wild-type embryos. Nuclei in dwee1 mutants, however, are positioned beyond the deepest part of the furrows in metaphase. Quantification of centrosome-cortex distance in wild-type embryos and dwee1 mutants in cycle 12 illustrates this phenotype. This cycle was chosen because it occurs after completion of cortical nuclear migration but before the onset of the centrosome-inactivation checkpoint, as evidenced by anastral spindles. Displacement of centrosomes from the cortex is observed in both interphase and mitosis of cycle 12 in dwee1 mutants and is only partially rescued by the chk2 mutation. It is suggested that centrosome and nuclear displacement in dwee1 mutants has two underlying components: one is a consequence of the Chk2-mediated checkpoint, and the other is a more direct result of loss of dwee1. That is, dwee1 is required to promote centrosome-cortex interaction, which is thought to be dependent on centrosomal microtubules and cortical actin. The displacement of centrosomes from the cortex in dwee1 mutants could distance mitotic spindles from the protection of pseudocleavage furrows in prophase and metaphase, thereby allowing interactions between adjacent spindles (Stempff, 2005).

six cycB embryos show normal localization of nuclei and centrosomes, suggesting that bulk elevation of Cdk1 activity in dwee1 mutants cannot account for the cortical detachment of centrosomes. Additionally, spindle interactions in dwee1 mutants also initiate when pseudocleavage furrows are normally absent (anaphase and telophase). Therefore, a furrow-independent mechanism may operate to keep spindles apart in anaphase and telophase in a dwee1-dependent fashion (Stempff, 2005).

To further address the requirement for dWee1 in centrosome and spindle function, dWee1-containing protein complexes were purified and dWee1-interacting proteins were identified by mass spectrometry. The heat-inducible HA-dWee1 transgene used as a source of dWee1 has been shown to partially rescue dwee1 mutant embryos when expressed in the mothers, indicating that the product is functional. HA-dWee1 was induced in embryos, purified on an anti-HA antibody column, and eluted with a HA-dipeptide. Analysis of eluates by SDS-PAGE and mass spectrometry identified peptides that matched dWee1 and Drosophila γ-tubulin ring proteins (Dgrips) 163, 128, 91, 84, and 71. The identities of Dgrip84, Dgrip91, and HA-dWee1 were confirmed by Western blotting. Note that γ-tubulin was not detected in the HA-dWee1 eluates because a strong background band in the 50 kDa range (the MW of γ-tubulin), likely the IgG heavy chain, prevented analysis of that region of the gel (Stempff, 2005).

Because the above experiments were performed with overexpressed, tagged dWee1, it was necessary to ensure that endogenous dWee1 interacts with γTuRC. Dgrip91 and γ-tubulin was readily detected in immunoprecipitates by using an antibody against dWee1. It was not possible, however, to detect dWee1 in immunoprecipitates by using an antibody against γ-tubulin or Dgrip91. This may be because it is possible, at best, to precipitate approximately 5% of total protein present with each antibody. The presence of γ-tubulin, a structural component of the cytoskeleton, at a higher concentration than dWee1, a regulatory kinase, is a likely scenario, and it could explain why no dWee1 is seen in immunoprecipitates of γ-tubulin (Stempff, 2005).

To determine whether the kinase dWee1 influences the phosphorylation status of proteins it binds to, γ-tubulin (which is known to be phosphorylated in budding yeast) was examined. Two-dimensional (2D) gel electrophoresis followed by Western blotting revealed that Drosophila γ-tubulin separates as a series of five spots in the first dimension. Two of the more acidic isoforms are phosphatase sensitive, suggesting that Drosophila γ-tubulin is a phosphoprotein. The phosphatase-sensitive acidic forms are absent or severely diminished in extracts from dwee1 mutant embryos (Stempff, 2005).

Given that interphase is truncated in dwee1 mutants, the possibility was addressed that loss of γ-tubulin phosphorylation is a consequence of changes in cell cycle profile. However, extracts from grp mutant embryos that exhibit truncated interphases retain the phosphatase-sensitive γ-tubulin isoforms. It is concluded that interphase shortening does not lead to loss of γ-tubulin phosphorylation and that dwee1 is required for γ-tubulin phosphorylation in vivo. Despite testing a range of conditions, it has not been possible to phosphorylate γ-tubulin with recombinant dWee1 in vitro, although GST-dWee1 readily autophosphorylates and phosphorylates Cdk1 in these assays. Either dWee1 regulates γ-tubulin phosphorylation indirectly or γ-tubulin phosphorylation by dWee1 requires a cofactor (Stempff, 2005).

The known role of Wee1 homologs in cell cycle regulation is accomplished through a single substrate, Cdk1. Elevation of bulk Cdk1 activity in an otherwise wild-type background, however, does not produce dwee1-specific phenotypes. Therefore, if dWee1 influences spindle organization or positioning via Cdk1, it would have to regulate Cdk1 locally, at the embryo cortex for example. The attachment of centrosomes to the cortex is mediated by microtubules. In the absence of dWee1, Cdk1 activity would be higher locally, i.e., between the centrosome and the cortex, and could inhibit microtubule growth in this region, leading to the displacement of centrosomes. This idea is consistent with observations that increased Cdk1 activity destabilizes microtubules during nuclear migration in Drosophila embryos. In six cycB embryos, dWee1 could still inhibit Cdk1 locally to allow normal nuclear and spindle positioning. This possibility can be addressed with a Cdk1 mutant that cannot be phosphorylated by dwee1 and should mimic the loss of dwee1. Attempts were made to introduce into syncytial embryos (which are prezygotic transcription) such a mutant in which Y14 and T15 have been altered, Cdk1AF, by expressing it in females. Unfortunately, females fail to lay eggs after induction of Cdk1AF, suggesting disruption of oogenesis and precluding further analysis (Stempff, 2005).

An alternate approach to introduction of Cdk1AF is to induce dWee1-antagonizing phosphatases, Cdc25string and Cdc25twine, in embryos. Indeed, such experiments have been described before. Increasing the maternal Cdc25 gene dose by up to 4-fold in various combinations of the two Drosophila Cdc25 homologs produces increased mRNA and protein in embryos and leads to an extra syncytial nuclear division before cellularization. This division and preceding syncytial divisions, however, are normal in fixed and live embryos. No mitotic abnormalities, which are readily apparent throughout dwee1 mutant embryos, were seen in embryos with elevated Cdc25. This is consistent with the observation that bulk elevation of Cdk1 activity does not produce dwee1-specific phenotypes. Instead, localized regulation of Cdk1 by dWee1, which would still be present in embryos harboring extra Cdc25, could explain the apparently normal divisions in these embryos (Stempff, 2005).

Another explanation for spindle phenotypes in dwee1 mutants is suggested by the finding that dWee1 shows physical interaction with components of the γTuRC and that dwee1 influences the phosphorylation status of γ-tubulin in vivo. In this model, dWee1 promotes the phosphorylation of γ-tubulin, either directly or indirectly. In dwee1 mutants, loss of γ-tubulin phosphorylation could compromise microtubule-dependent attachments between centrosomes and the cortex. A test of this model will require identification and mutation of dwee1-dependent phosphoacceptor residues in γ-tubulin. Interestingly, the budding-yeast γ-tubulin homolog, Tub4p, is phosphorylated on a tyrosine residue during G1, but the responsible kinase has yet to be identified. A phosphomimetic mutation of Tub4p affects the number and organization of microtubules and causes transient nuclear-positioning abnormalities. Thus, it is possible that in both yeast and fly, the phosphorylation status of γ-tubulin plays a role in centrosome and nuclear positioning via interactions with the cortex (Stempff, 2005).

At present, it is not possible to distinguish between the two above explanations for dwee1 phenotypes; the explanations are not mutually exclusive. However, neither is predicted by previous models that describe how Wee1 homologs act to regulate entry into mitosis. Human Wee1, for example, resides in the nucleus during interphase and is proposed to prevent nuclear accumulation of Cdk1 activity and nuclear-envelope breakdown, an initiating event in mitosis. Such models can explain Wee1's role in regulating when mitosis occurs, but the current results indicate that Wee1 can also regulate where (relative to the cortex) mitosis occurs (Stempff, 2005).

The first 11 nuclear divisions proceed normally in dwee1 mutants. Therefore, dwee1-dependent regulation of spindle organization or positioning is not essential for mitosis per se. In cycle 12, nuclei are at the cortex and at twice the density (i.e., closer together) compared to those in cycle 11. It is reasoned that manifestation of dwee1-specific spindle interactions in later cortical cycles is a consequence of increasing nuclear density with each cycle that brings neighboring spindles closer together. In such a situation, protection offered by actin furrows may be essential to keep spindles apart. Detachment of centrosomes from the cortex would distance the spindles from furrows, allowing neighbors to interact (Stempff, 2005).

It is not known whether dwee1 also plays a role in spindle morphogenesis and centrosome positioning in cell cycles beyond cortical syncytial cycles. It is known, however, that dwee1 is needed to ensure fidelity of cell division in larvae; larval neuroblasts in dwee1 mutants show elevated mitotic index and ploidy. It would be interesting to determine the basis for this requirement and whether dwee1 has a role in the positioning of the spindle in cell divisions where a specific cortical attachment of the spindle is required, such as in the asymmetric cell divisions of neuroblast lineages (Stempff, 2005).

This study presents several lines of data that collectively suggest a requirement for dWee1 in centrosome function and spindle morphogenesis. Importantly, these roles translate into a requirement for dWee1 in not only temporal but also spatial regulation of mitosis. Two mechanistic models, which are not mutually exclusive, have been offered to account for these results: localized regulation of Cdk1 by dWee1 and phosphoregulation of γTuRC. Further analysis will be needed to test these models, but it is clear that the requirement for dWee1 cannot be explained by simple regulation of bulk Cdk1 activity. In this regard, Wee1 homologs may be likened to other kinases, such as Plk and Aurora B, that have multiple roles in mitosis through multiple substrates. Localized activity of master regulatory kinases such as these is likely to coordinate many distinct cell-division events (such as spindle movements, chromosome segregation, and cytokinesis) to allow faithful segregation of genetic information into daughter cells (Stempff, 2005).

Dual phosphorylation of Cdk1 coordinates cell proliferation with key developmental processes in Drosophila

Eukaryotic organisms use conserved checkpoint mechanisms that regulate Cdk1 by inhibitory phosphorylation to prevent mitosis from interfering with DNA replication or repair. In metazoans, this checkpoint mechanism is also used for coordinating mitosis with dynamic developmental processes. Inhibitory phosphorylation of Cdk1 is catalyzed by Wee1 kinases that phosphorylate tyrosine 15 (Y15) and dual-specificity Myt1 kinases found only in metazoans that phosphorylate Y15 and the adjacent threonine (T14) residue. Despite partially redundant roles in Cdk1 inhibitory phosphorylation, Wee1 and Myt1 serve specialized developmental functions that are not well understood. Wild type and phospho-acceptor mutant Cdk1 proteins were expresses in order to investigate how biochemical differences in Cdk1 inhibitory phosphorylation influence Drosophila imaginal development. Phosphorylation of Cdk1 on Y15 appeared to be crucial for developmental and DNA damage-induced G2 phase checkpoint arrest, consistent with other evidence that Myt1 is the major Y15-directed Cdk1 inhibitory kinase at this stage of development. Expression of non-inhibitable Cdk1 also caused chromosome defects in larval neuroblasts that were not observed with Cdk1(Y15F) mutant proteins that were phosphorylated on T14, implicating Myt1 in a novel mechanism promoting genome stability. Collectively, these results suggest that dual inhibitory phosphorylation of Cdk1 by Myt1 serves at least two functions during development. Phosphorylation of Y15 is essential for the pre-mitotic checkpoint mechanism, whereas T14 phosphorylation facilitates accumulation of dually inhibited Cdk1-Cyclin B complexes that can be rapidly activated once checkpoint-arrested G2 phase cells are ready for mitosis (Ayeni, 2013).

Combining genetic perturbations and proteomics to examine kinase-phosphatase networks in Drosophila embryos

Connecting phosphorylation events to kinases and phosphatases is key to understanding the molecular organization and signaling dynamics of networks. This study has generated a validated set of transgenic RNA-interference reagents for knockdown and characterization of all protein kinases and phosphatases present during early Drosophila melanogaster development. These genetic tools enable collection of sufficient quantities of embryos depleted of single gene products for proteomics. As a demonstration of an application of the collection, multiplexed isobaric labeling was used for quantitative proteomics to derive global phosphorylation signatures associated with kinase-depleted embryos to systematically link phosphosites with relevant kinases. This strategy uncovers kinase consensus motifs and prioritizes phosphoproteins for kinase target validation. This approach was validated by providing auxiliary evidence for Wee kinase-directed regulation of the chromatin regulator Stonewall. Further, it was shown how correlative phosphorylation at the site level can indicate function, as exemplified by Sterile20-like kinase-dependent regulation of Stat92E (Sopko, 2014).

wee: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation and Overexpression | References

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