The challenging task of mitotic cell divisions is to generate two genetically identical daughter cells from a single precursor cell. To accomplish this task, a complex regulatory network evolved, which ensures that all events critical for the duplication of cellular contents and their subsequent segregation occur in the correct order, at specific intervals and with the highest possible fidelity. Transitions between cell cycle stages are triggered by changes in the phosphorylation state and levels of components of the cell cycle machinery. Entry into S-phase and M-phase are mediated by cyclin-dependent kinases (Cdks), serine-threonine kinases that require a regulatory cyclin subunit for their activity. Resetting the system to the interphase state is mediated by protein phosphatases (PPs) that counteract Cdks by dephosphorylating their substrates. To avoid futile cycles of phosphorylation and dephosphorylation, Cdks and PPs must be regulated in a manner such that their activities are mutually exclusive.
Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.
Animals detect and respond to temperature changes in their environment. Responses can be choice based to achieve an optimum environment for physiological function, or escape reflexes as temperature reaches noxious levels. Both types of response can be observed in Drosophila.1,2 Recent descriptions of temperature responses in Drosophila have uncovered mechanisms and pathways involved in optimal temperature detection in flies 3 that appear to be shared in nociceptive perception in mammals. 4 Mutations in a number of Transient Receptor Potential (TRP) receptors have been identified. Behavioral analysis of TRP receptor mutants and the expression patterns of the TRP proteins have helped to identify components of the underlying neurocircuitry. 1,3,[5][6][7] We describe here a robust behavioral escape response from larvae to rising temperature. The behavior closely resembles previously described nocifensive responses elicited by touch with a heated probe set at 39-41°C producing a corkscrewing avoidance of the stimulus.1 A later modification of this stimulus application employed a temperature regulated thermal probe. 8 For both studies, duration and force of contact between probe and larvae is a function of the testers' judgement and dexterity. In an attempt to remove this potential operator variability, we applied a thermal stimulus to larvae by immersion in a water droplet and subsequent heating of the water to noxious levels. In practical terms, we placed a larva in 30 μl of water at room temperature on a petri dish lid. The lid was then transferred to a hotplate surface set to 70°C. Care was taken to ensure that the petri dish lid consistently came into close contact with the hotplate. As our baseline, we used a fine temperature probe (a thermocouple) to determine the rate of temperature increase in the water droplet so that we have an exact determination of the temperature of the droplet at any time-point ( fig. 1B) Keywords: nociception, nocifensive, TRP receptor, Drosophila larvae dish holding the droplet and larva to the hot plate, we recorded the time at which the larva initiated the characteristic nocifensive corkscrew roll. We use our time/temperature relationship curve to infer the temperature in the droplet of water when the escape response is activated. In our assay, the escape response in wildtype larvae was evoked at 28-29°C ( fig. 1c). We determined the escape response temperature for larvae of our lab stock of w 1118 , and two wild-type stocks, Canton-S and Oregon-R. The three wild-type stocks all responded at a temperature just above 29°C (29.3°C, 29.8°C and 29.4°C respectively). In larvae mutant for the TRP receptor painless (pain 1 ), or functionally blocked by the expression of tetanus toxin light chain 10 in the class IV da sensory neurons, the response occurred at 33-34°C ( fig. 1c). Control animals expressing inactive tetanus toxin light chain respond at 28.1°C. This suggests that the painless TRP receptor may be involved in responses to temperature around 29°C in this behavioral test.In additi...
Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by promoting RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 loading to sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.
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