The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.

The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Here, we show that Chk1, a component of the DNA damage and replication checkpoints, protects vertebrate cells against spontaneous chromosome missegregation and is required to sustain anaphase delay when spindle function is disrupted by taxol, but not when microtubules are completely depolymerized by nocodazole. Spindle checkpoint failure in Chk1-deficient cells correlates with decreased Aurora-B kinase activity and impaired phosphorylation and kinetochore localization of BubR1. Furthermore, Chk1 phosphorylates Aurora-B and enhances its catalytic activity in vitro. We propose that Chk1 augments spindle checkpoint signaling and is required for optimal regulation of Aurora-B and BubR1 when kinetochores produce a weakened signal. In addition, Chk1-deficient cells exhibit increased resistance to taxol. These results suggest a mechanism through which Chk1 could protect against tumorigenesis through its role in spindle checkpoint signaling.

A simple perifusion system for in vitro studies on the rate of insulin secretion of isolated rat islets is described. A biphasic pattern of insulin secretion was produced by glucose whereas tolbutamide stimulated only the first phase of insulin secretion. The perifusion system was used to determine the effect of anti-mitotic agents on the biphasic pattern of insulin secretion. Yinblastine and colchicine destroy microtubules whereas deuterium oxide (D Q 0) produces stabilization and interference with the function of microtubules. Vinblastine (10' 4 M) and D 2 O (100 per cent) inhibited completely the first and second phase of glucose-induced insulin secretion. The inhibitory effect of D 2 O was reversible and a biphasic pattern of secretion occurred following the replacement of D 2 O with water. Colchicine (10" 3 M) produced significant inhibition of only the second phase of glucose-induced insulin secretion. The first phase of tolbutamide-induced insulin secretion was inhibited by vinblastine and D 2 O and the inhibitory effect of D. 2 O was reversible. Colchicine did not inhibit the first phase of tolbutamide-induced insulin release. These findings indicate that the microtubular system is involved in both phases of insulin secretion. The first phase of secretion may be due to the release of beta granules already associated with the microtubular system and the second phase could be the result of stored and newlysynthesized granules becoming associated with the system. DIABETES 21:987-98, October, 1972.In previous studies, we proposed that the microtubular-microfilament system was involved in the intracellular transport of beta granules following glucose stimulation of insulin secretion. 1 This hypothesis was based upon the ultrastructural demonstration of a microtubular-microfilament system in beta cells and the finding that mitotic spindle-inhibitors would inhibit the secretion of insulin by isolated rat islets maintained in vitro. A subsequent series of studies by Malaisse et ai B>3

Centrosomal abnormalities are frequently observed in cancers and in cells with defective DNA repair. Here, we used light and electron microscopy to show that DNA damage induces centrosome amplification, not fragmentation, in human cells. Caffeine abrogated this amplification in both ATM (ataxia telangiectasia, mutated)‐ and ATR (ATM and Rad3‐related)‐defective cells, indicating a complementary role for these DNA‐damage‐responsive kinases in promoting centrosome amplification. Inhibition of checkpoint kinase 1 (Chk1) by RNA‐mediated interference or drug treatment suppressed DNA‐damage‐induced centrosome amplification. Radiation‐induced centrosome amplification was abrogated in Chk1−/− DT40 cells, but occurred at normal levels in Chk1−/− cells transgenically expressing Chk1. Expression of kinase‐dead Chk1, or Chk1S345A, through which the phosphatidylinositol‐3‐kinase cannot signal, failed to restore centrosome amplification, showing that signalling to Chk1 and Chk1 catalytic activity are necessary to promote centrosome overduplication after DNA damage.

Chk1 is phosphorylated within its C-terminal regulatory domain by the upstream ATM/ ATR kinases during checkpoint activation, however how this modulates Chk1 function is poorly understood. Here, we show that Chk1 kinase activity is rapidly stimulated in a cell cycle phase-specific manner in response to both DNA damage and replication arrest, and that the extent and duration of activation correlates closely with regulatory phosphorylation at serines (S) S317, S345, and S366. Despite their evident co-regulation, substitutions of individual Chk1 regulatory sites with alanine (A) residues have differential effects on checkpoint proficiency and kinase activation. Thus, whereas Chk1 S345 is essential for all functions tested, mutants lacking S317 or S366 retain partial proficiency for G2/ M and S/ M checkpoint arrests triggered by DNA damage or replication arrest. These phenotypes reflect defects in Chk1 kinase induction, since the mutants are either partially (317A, 366A) or completely (345A) resistant to kinase activation. Importantly, S345 phosphorylation is impaired in Chk1 S317A and S366A mutants, suggesting that modification of adjacent SQ sites promotes this key regulatory event. Finally, we provide biochemical evidence that Chk1 catalytic activity is stimulated via a de-repression mechanism.