SGC-GAK-1: A Chemical Probe for Cyclin G Associated Kinase (GAK).

We describe SGC-GAK-1 (11), a potent, selective, and cell-active inhibitor of cyclin G-associated kinase (GAK), together with a structurally related negative control SGC-GAK-1N (14). 11 was highly selective in an in vitro kinome-wide screen, but cellular engagement assays defined RIPK2 as a collateral target. We identified 18 as a potent RIPK2 inhibitor lacking GAK activity. Together, this chemical probe set can be used to interrogate GAK cellular biology.

Cyclin G and the Polycomb Repressive complexes PRC1 and PR-DUB cooperate for developmental stability.

In Drosophila, ubiquitous expression of a short Cyclin G isoform generates extreme developmental noise estimated by fluctuating asymmetry (FA), providing a model to tackle developmental stability. This transcriptional cyclin interacts with chromatin regulators of the Enhancer of Trithorax and Polycomb (ETP) and Polycomb families. This led us to investigate the importance of these interactions in developmental stability. Deregulation of Cyclin G highlights an organ intrinsic control of developmental noise, linked to the ETP-interacting domain, and enhanced by mutations in genes encoding members of the Polycomb Repressive complexes PRC1 and PR-DUB. Deep-sequencing of wing imaginal discs deregulating CycG reveals that high developmental noise correlates with up-regulation of genes involved in translation and down-regulation of genes involved in energy production. Most Cyclin G direct transcriptional targets are also direct targets of PRC1 and RNAPolII in the developing wing. Altogether, our results suggest that Cyclin G, PRC1 and PR-DUB cooperate for developmental stability.

Drosophila Cyclin G Is a Regulator of the Notch Signalling Pathway during Wing Development.

Notch signalling regulates a multitude of differentiation processes during Drosophila development. For example, Notch activity is required for proper wing vein differentiation which is hampered in mutants of either the receptor Notch, the ligand Delta or the antagonist Hairless. Moreover, the Notch pathway is involved in several aspects of Drosophila oogenesis as well. We have identified Drosophila Cyclin G (CycG) as a molecular interaction partner of Hairless, the major antagonist in the Notch signalling pathway, in vitro and in vivo. Loss of CycG was shown before to cause female sterility and to disturb the architecture of the egg shell. Nevertheless, Notch dependent processes during oogenesis appeared largely unaffected in cycG mutant egg chambers. Loss of CycG modified the dominant wing phenotypes of Notch, Delta and Hairless mutants. Whereas the Notch loss of function phenotype was ameliorated by a loss of CycG, the phenotypes of either Notch gain of function or of Delta or Hairless loss of function were enhanced. In contrast, loss of CycG had only a minor effect on the wing vein phenotype of mutants affecting the EGFR signalling pathway emphasizing the specificity of the interaction of CycG and Notch pathway members.

A triangular connection between Cyclin G, PP2A and Akt1 in the regulation of growth and metabolism in Drosophila.

Size and weight control is a tightly regulated process, involving the highly conserved Insulin receptor/target of rapamycin (InR/TOR) signaling cascade. We recently identified Cyclin G (CycG) as an important modulator of InR/TOR signaling activity in Drosophila. cycG mutant flies are underweight and show a disturbed fat metabolism resembling TOR mutants. In fact, InR/TOR signaling activity is disturbed in cycG mutants at the level of Akt1, the central kinase linking InR and TORC1. Akt1 is negatively regulated by protein phosphatase PP2A. Notably the binding of the PP2A B'-regulatory subunit Widerborst (Wdb) to Akt1 is differentially regulated in cycG mutants, presumably by a direct interaction of CycG and Wdb. Since the metabolic defects of cycG mutant animals are abrogated by a concomitant loss of Wdb, CycG presumably influences Akt1 activity at the PP2A nexus. Here we show that Well rounded (Wrd), another B' subunit of PP2A in Drosophila, binds CycG similar to Wdb, and that its loss ameliorates some, but not all, of the metabolic defects of cycG mutants. We propose a model, whereby the binding of CycG to a particular B'-regulatory subunit influences the tissue specific activity of PP2A, required for the fine tuning of the InR/TOR signaling cascade in Drosophila.

Asymmetric flies: the control of developmental noise in Drosophila.

What are the sources of phenotypic variation and which factors shape this variation are fundamental questions of developmental and evolutionary biology. Despite this simple formulation and intense research, controversy remains. Three points are particularly discussed: (1) whether adaptive developmental mechanisms buffering variation exist at all; (2) if yes, do they involve specific genes and processes, i.e., different from those involved in the development of the traits that are buffered?; and (3) whether different mechanisms specifically buffer the various sources of variation, i.e., genetic, environmental and stochastic, or whether a generalist process buffers them all at once. We advocate that experimental work integrating different levels of analysis will improve our understanding of the origin of phenotypic variation and thus help answering these contentious questions. In this paper, we first survey the current views on these issues, highlighting potential sources of controversy. We then focus on the stochastic part of phenotypic variation, as measured by fluctuating asymmetry, and on current knowledge about the genetic basis of developmental stability. We report our recent discovery that an individual gene, Cyclin G, plays a central role-adaptive or not-in developmental stability in Drosophila. ( 1) We discuss the implications of this discovery on the regulation of organ size and shape, and finally point out open questions.

Dorso-ventral axis formation of the Drosophila oocyte requires Cyclin G.

In general, cyclins control the cell cycle. Not so the atypical cyclins, which are required for diverse cellular functions such as for genome stability or for the regulation of transcription and translation. The atypical Cyclin G (CycG) gene of Drosophila has been involved in the epigenetic regulation of abdominal segmentation, cell proliferation and growth, based on overexpression and RNAi studies, but detailed analyses were hampered by the lack of a cycG mutant. For further investigations, we subjected the cycG locus to a detailed molecular analysis. Moreover, we studied a cycG null mutant that we recently established. The mutant flies are homozygous viable, however, the mutant females are sterile and produce ventralized eggs. Here we show that this egg phenotype is primarily a consequence of a defective Epidermal Growth Factor Receptor (EGFR) signalling pathway. By using different read outs, we demonstrate that cycG loss is tantamount to lowered EGFR signalling. Inferred from epistasis experiments, we conclude that CycG promotes the Grk signal in the oocyte. Abnormal accumulation but regular secretion of the Grk protein suggests defects of Grk translation in cycG mutants rather than transcriptional regulation. Accordingly, protein accumulation of Vasa, which acts as an oocyte specific translational regulator of Grk in the oocyte is abnormal. We propose a role of cycG in processes that regulate translation of Grk and hence, influence EGFR-mediated patterning processes during oogenesis.

Cyclin G is involved in meiotic recombination repair in Drosophila melanogaster.

Cyclin G (CycG) belongs to the atypical cyclins, which have diverse cellular functions. The two mammalian CycG genes, CycG1 and CycG2, regulate the cell cycle in response to cell stress. Detailed analyses of the role of the single Drosophila cycG gene have been hampered by the lack of a mutant. We generated a null mutant in the Drosophila cycG gene that is female sterile and produces ventralised eggs. This phenotype is typical of the downregulation of epidermal growth factor receptor (EGFR) signalling during oogenesis. Ventralised eggs are also observed in mutants (for example, mutants of the spindle class) that are defective in meiotic DNA double-strand break repair. Double-strand breaks (DSBs) induce a meiotic checkpoint by activating Mei-41 kinase (the Drosophila ATR homologue), thereby indirectly causing dorsoventral patterning defects. We provide evidence for the role of CycG in meiotic checkpoint control. The increased incidence of DSBs in cycG mutant germaria may reflect inefficient DSB repair. Therefore, the downregulation of Mei-W68 (an endonuclease that induces meiotic DSBs), Mei-41, or Drosophila melanogaster Chk2 (a downstream kinase that initiates the meiotic checkpoint) rescues the cycG mutant eggshell phenotype. In vivo, CycG associates with Rad9 and BRCA2. These two proteins are components of the 9-1-1 complex, which is involved in sensing DSBs and in activating meiotic checkpoint control. Therefore, we propose that CycG has a role in an early step of meiotic recombination repair, thereby affecting EGFR-mediated patterning processes during oogenesis.

The effects of simultaneous combined exposure to CDMA and WCDMA electromagnetic fields on rat testicular function.

Wireless mobile phones and other telecommunication devices are used extensively in daily life. We therefore examined the effects of combined exposure to radiofrequency electromagnetic fields (RF-EMF) on rat testicular function, specifically with respect to sensitive processes such as spermatogenesis. Male rats were exposed to single code division multiple access (CDMA) and wideband code division multiple access (WCDMA) RF signals for 12 weeks. The RF exposure schedule comprised 45 min/day, 5 days/week for a total of 12 weeks. The whole-body average specific absorption rate (SAR) of CDMA and WCDMA was 2.0 W/kg each or 4.0 W/kg in total. We then investigated the correlates of testicular function such as sperm count in the cauda epididymis, testosterone concentration in the blood serum, malondialdehyde concentrations in the testes and epididymis, frequency of spermatogenesis stages, and appearance of apoptotic cells in the testes. We also immunoblotted for p53, bcl2, GADD45, cyclin G, and HSP70 in the testes of sham- and combined RF-exposed animals. Based on the results, we concluded that simultaneous exposure to CDMA and WCDMA RF-EMFs at 4.0 W/kg SAR did not have any observable adverse effects on rat spermatogenesis.

Drosophila melanogaster Cyclin G coordinates cell growth and cell proliferation.

Mammalian Cyclins G1 and G2 are unconventional cyclins whose role in regulating the cell cycle is ambiguous. Cyclin G1 promotes G2/M cell cycle arrest in response to DNA damage whereas ectopic expression of CCNG2, that encodes Cyclin G2, induces G1/S cell cycle arrest. The only Drosophila Cyclin G was previously shown to be a transcriptional regulator that interacts with the chromatin factor Corto and controls expression of the homeotic gene Abdominal B. It is very close to mammalian Cyclin G1 and G2 except in its N-terminal region, that interacts with Corto, and that seems to have been acquired in dipterans. Ubiquitous misregulation of Cyclin G (CycG) using transgenic lines lengthens development and induces phenotypes suggesting growth or proliferation defects. Using tissue-specific misregulation of CycG and FACS, we show that overproduction of Cyclin G produces small cells whereas shortage produces large cells, suggesting that Cyclin G negatively regulates cell growth. Furthermore, overexpression of CycG lengthens the cell cycle, with a prominent effect on G1 and S phases. Genetic interactions with Cyclin E suggest that Cyclin G prevents G1 to S transition and delays S phase progression. Control of cell growth and cell cycle by Cyclin G might be achieved via interaction with a network of partners, notably the cyclin-dependent kinases CDK4 and CDK2.