The role of RPA2 phosphorylation in homologous recombination in response to replication arrest.

Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR.

The PDZ domain protein CAL interacts with mGluR5a and modulates receptor expression.

In this study, we investigated the association of metabotropic glutamate receptor subtype-5a (mGluR5a) with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL). Using glutathione-S-transferase pull-down techniques, we found that mGluR5a directly interacted with CAL, with the C-terminus of the receptor binding to the PSD95/Discslarge/ZO-1 homology domain of CAL. The last four amino acids (S-S-S-L) of the C-terminus of the receptor were essential determinants for the interaction. Co-immunoprecipitation experiments and immunofluorescence assays revealed that full-length mGluR5a also associated with intact CAL in vivo, an observation consistent with the results from studies on fragment interactions in vitro. Functionally, upon co-expression with mGluR5a, CAL profoundly inhibited the ubiquitination of mGluR5a and enhanced receptor expression at the protein level but not at the mRNA level. These findings reveal that mGluR5a protein expression is physiologically regulated via its interaction with CAL. These results also suggest a molecular mechanism by which mGluR5a protein expression may be regulated at the post-translational level by the CAL protein, possibly by blocking ubiquitination-dependent receptor degradation.

Beta-adrenoceptors, but not dopamine receptors, mediate dopamine-induced ion transport in late distal colon of rats.

Dopamine, an important modulator in the gastrointestinal system, induces concentration-dependent transepithelial ion transport in the distal colon of the rat, as shown by a decrease in the short-circuit current, and acts in a segmentally dependent manner. However, the receptor(s) that mediates dopamine-induced ion transport is unknown. We have investigated the receptor mechanisms underlying dopamine-induced colonic ion transport by means of short-circuit current recording, real-time polymerase chain reaction, and Western blotting analysis, plus gene transfection and enzyme-linked immunosorbance assay. mRNA transcripts of adrenoceptors (alpha, beta) and dopaminergic receptors (D(1) and D(2)) were detected in the rat late distal colonic mucosa, with beta(2) displaying the highest expression. A similar result was found in human colorectal mucosa (equivalent of late distal colon in rat). Pretreatment with a beta(1)-adrenoceptor antagonist (CGP-20712A) and a beta(2)-adrenoceptor antagonist (ICI 118,551) inhibited the dopamine-induced short-circuit current response by 52.59% and 92.51%, respectively. However, neither dopamine D(1) receptor antagonist SCH-23390 nor dopamine D(2) receptor antagonist sulpiride blocked the effect of dopamine. Protein expression of both beta(1)- and beta(2)-adrenoceptors was found in the mucosa of rat distal colon and human sigmoid colon and rectum. Dopamine significantly increased intracellular cAMP levels in COS-7 cells transfected with beta(1)- or beta(2)-adrenoceptors. Thus, beta-adrenoceptors (mainly beta(2)-adrenoceptors), but not dopamine receptors, mediate dopamine-induced ion transport in the late distal colon of the rat. This extends our knowledge of the late distal colon (rats) or colorectum (human) and provides further experimental evidence that might aid the prevention, diagnosis, and clinical therapy of human colorectal diseases.

The mTOR inhibitor rapamycin suppresses DNA double-strand break repair.

mTOR (mammalian target of rapamycin) signaling plays a key role in the development of many tumor types. Therefore, mTOR is an attractive target for cancer therapeutics. Although mTOR inhibitors are thought to have radiosensitization activity, the molecular bases remain largely unknown. Here we show that treating MCF7 breast cancer cells with rapamycin (an mTOR inhibitor) results in significant suppression of homologous recombination (HR) and nonhomologous end joining (NHEJ), two major mechanisms required for repairing ionizing radiation-induced DNA DSBs. We observed that rapamycin impaired recruitment of BRCA1 and Rad51 to DNA repair foci, both essential for HR. Moreover, consistent with the suppressive role of rapamycin on both HR and NHEJ, persistent radiation-induced DSBs were detected in cells pretreated with rapamycin. Furthermore, the frequency of chromosome and chromatid breaks was increased in cells treated with rapamycin before and after irradiation. Thus our results show that radiosensitization by mTOR inhibitors occurs via disruption of the major two DNA DSB repair pathways.

A novel association of mGluR1a with the PDZ scaffold protein CAL modulates receptor activity.

Metabotropic glutamate receptor subtype 1a (mGluR1a) associates with the proteins mediating its receptor activity, suggesting a complex-controlled function of mGluR1a. Here, using glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence assays in vitro and in vivo, we have found CFTR-associated ligand (CAL) to be a novel binding partner of mGluR1a, through its PSD95/discslarge/ZO1homology domain. Deletion of mGluR1a-carboxyl terminus (CT) or mutation of Leu to Ala in the CT of mGluR1a reduces the association, indicating the essential binding region of mGluR1a for CAL. Functionally, the interaction of mGluR1a with CAL was shown to inhibit mGluR1a-mediated ERK1/2 activation, without an apparent effect, via the C-terminal-truncated receptor. These findings might provide a novel mechanism for the regulation of mGluR1a-mediated signaling through the interaction with CAL.

Disassembly of MDC1 foci is controlled by ubiquitin-proteasome-dependent degradation.

The orderly recruitment, retention, and disassembly of DNA damage response proteins at sites of damaged DNA is a conserved process throughout eukaryotic evolution. The recruitment and retention of DNA repair factors in foci is mediated by a complex network of protein-protein interactions; however, the mechanisms of focus disassembly remain to be defined. Mediator of DNA damage checkpoint protein 1 (MDC1) is an early and key component of the genome surveillance network activated by DNA double-strand breaks (DSBs). Here, we investigated the disassembly of MDC1 foci. First, we show that ubiquitylation directs the MDC1 protein for proteasome-dependent degradation. Ubiquitylated MDC1 associates with chromatin before and after exposure of cells to ionizing radiation (IR). In addition, increased MDC1 ubiquitylation in the chromatin fraction is observed in response to IR, which is correlated with a reduction in total MDC1 protein levels. We demonstrate that blocking MDC1 degradation by proteasome inhibitors leads to a persistence of MDC1 foci. Consistent with this observation, chromatin immunoprecipitation experiments reveal increased MDC1 protein at site-specific DSBs. Interestingly, we show that the persistence of MDC1 foci is associated with an abrogated recruitment of the downstream factor BRCA1 in a manner that is RNF8 independent. Collectively, the evidence presented here supports a novel mechanism for the disassembly of MDC1 foci via ubiquitin-proteasome dependent degradation, which appears to be a key step for the efficient assembly of BRCA1 foci.