β-TrCP, the F-box protein of the SCFβ - TrCPubiquitin ligase (SCF, Skp1/Cul1/F-box protein), recognizes the doubly phosphorylated DSG motif (DpSGΦXpS) in various SCFβ - TrCPtarget proteins. The ...Cdc25A phosphatase, a key cell-cycle regulator in vertebrate cells, undergoes a rapid ubiquitin-dependent degradation in response to genotoxic stress. β-TrCP binds to the DSG motif of human Cdc25A in a manner dependent on Chk1 and other unknown kinases. However, Xenopus Cdc25A does not have a DSG motif at the corresponding site of human Cdc25A. Here, we report that both Xenopus Cdc25A and human Cdc25A have a previously undescribed nonphosphorylated DDG motif (DDGΦXD) for recognition by β-TrCP. When analyzed by using Xenopus eggs, the binding of β-TrCP to the DDG motif is essential for the Chk1-induced ubiquitination and degradation of Xenopus Cdc25A and also plays a role in the degradation of human Cdc25A. The DDG motif also exists in human Cdc25B phosphatase (another key cell-cycle regulator), binds β-TrCP strongly, and is essential for the ubiquitination and degradation of the (labile) phosphatase in normal conditions. We provide strong evidence that, in both Cdc25A and Cdc25B, the binding (efficiency) of β-TrCP to the DDG motif is regulated by nearby residues, while ubiquitination is regulated by other events in addition to the β-TrCP binding. Finally, our additional data suggest that β-TrCP may recognize nonphosphorylated DDG-like motifs in many other proteins, including X11L (a putative suppressor of β-amyloid production) and hnRNP-U (a pseudosubstrate of SCFβ - TrCP).
Spleen tyrosine kinase (Syk) is a key regulatory factor in the IgE-mediated allergic signal transduction pathway in mast cells and basophils. Syk is phosphorylated on a number of tyrosines following ...the binding of IgE/allergen complexes to FcɛRI receptors leading to initiation of inflammatory signaling via downstream enzymes and scaffolding proteins. We examined the kinases responsible for the phosphorylation of key Syk tyrosines in rat RBL-2H3 basophilic cells and bone marrow-derived mast cells (BMMCs). The phosphorylation of Syk tyrosine 346 was completely blocked by the novel Src family kinase inhibitor BIRA766, suggesting this tyrosine is a pure substrate for Src family kinases. This was supported by the findings that kinase-dead (KD) Syk was efficiently phosphorylated on this tyrosine and that a specific Syk inhibitor BAY61-3606 was without effect. The phosphorylation of other Syk tyrosines 317, 342, 519 and 520 was reduced by Syk and Src family inhibitors, suggesting a role for auto- and trans-phosphorylation. Lyn was the predominant Src family kinase expressed and activated in RBL-2H3 cells, meanwhile Lyn knockdown with a specific siRNA interfered with the phosphorylation of all Syk tyrosines and the Syk substrates SLP-76 and LAT. Pharmacological inhibition of Syk completely blocked the degranulation of RBL-2H3 and BMMCs. However, Lyn knockdown sensitized RBL-2H3 cells to FcɛRI-induced degranulation. We showed that whilst interference with Lyn expression disrupts FcɛRI proximal signaling via Syk and its direct substrates including SLP-76 and LAT, distal activation of downstream proteins including Erk is enhanced. This study identifies the responsible kinases for the phosphorylation of key Syk tyrosines and the propagation of FcɛRI receptor mediated signal transduction in allergic responses.
Meiotic cells undergo two successive divisions without an intervening S phase. However, the mechanism of S-phase omission between the two meiotic divisions is largely unknown. Here we show that Wee1, ...a universal mitotic inhibitor, is absent in immature (but not mature) Xenopus oocytes, being down-regulated specifically during oogenesis; this down-regulation is most likely due to a translational repression. Even the modest ectopic expression of Wee1 in immature (meiosis I) oocytes can induce interphase nucleus reformation and DNA replication just after meiosis I. Thus, the presence of Wee1 during meiosis I converts the meiotic cell cycle into a mitotic-like cell cycle having S phase. In contrast, Myt1, a Wee1-related kinase, is present and directly involved in G(2) arrest of immature oocytes, but its ectopic expression has little effect on the meiotic cell cycle. These results strongly indicate that the absence of Wee1 in meiosis I ensures the meiotic cell cycle in Xenopus oocytes. Based on these results and the data published previously in other organisms, we suggest that absence of Wee1 may be a well-conserved mechanism for omitting interphase or S phase between the two meiotic divisions.
Burrowing, an ethologically relevant rodent behaviour, has been proposed as a novel outcome measure to assess the global impact of pain in rats. In a prospective multicentre study using male rats ...(Wistar, Sprague-Dawley), replication of suppressed burrowing behaviour in the complete Freund adjuvant (CFA)-induced model of inflammatory pain (unilateral, 1 mg/mL in 100 µL) was evaluated in 11 studies across 8 centres. Following a standard protocol, data from participating centres were collected centrally and analysed with a restricted maximum likelihood-based mixed model for repeated measures. The total population (TP-all animals allocated to treatment; n = 249) and a selected population (SP-TP animals burrowing over 500 g at baseline; n = 200) were analysed separately, assessing the effect of excluding "poor" burrowers. Mean baseline burrowing across studies was 1113 g (95% confidence interval: 1041-1185 g) for TP and 1329 g (1271-1387 g) for SP. Burrowing was significantly suppressed in the majority of studies 24 hours (7 studies/population) and 48 hours (7 TP, 6 SP) after CFA injections. Across all centres, significantly suppressed burrowing peaked 24 hours after CFA injections, with a burrowing deficit of -374 g (-479 to -269 g) for TP and -498 g (-609 to -386 g) for SP. This unique multicentre approach first provided high-quality evidence evaluating suppressed burrowing as robust and reproducible, supporting its use as tool to infer the global effect of pain on rodents. Second, our approach provided important informative value for the use of multicentre studies in the future.
Cdc25 phosphatases activate cyclin‐dependent kinases (Cdks) and thereby promote cell cycle progression. In vertebrates, Chk1 and Chk2 phosphorylate Cdc25A at multiple N‐terminal sites and target it ...for rapid degradation in response to genotoxic stress. Here we show that Chk1, but not Chk2, phosphorylates Xenopus Cdc25A at a novel C‐terminal site (Thr504) and inhibits it from C‐terminally interacting with various Cdk–cyclin complexes, including Cdk1–cyclin A, Cdk1–cyclin B, and Cdk2–cyclin E. Strikingly, this inhibition, rather than degradation itself, of Cdc25A is essential for the Chk1‐induced cell cycle arrest and the DNA replication checkpoint in early embryos. 14‐3‐3 proteins bind to Chk1‐phosphorylated Thr504, but this binding is not required for the inhibitory effect of Thr504 phosphorylation. A C‐terminal site presumably equivalent to Thr504 exists in all known Cdc25 family members from yeast to humans, and its phosphorylation by Chk1 (but not Chk2) can also inhibit all examined Cdc25 family members from C‐terminally interacting with their Cdk–cyclin substrates. Thus, Chk1 but not Chk2 seems to inhibit virtually all Cdc25 phosphatases by a novel common mechanism.
Nek2, a NIMA‐related kinase, has been postulated to play a role in both the meiotic and mitotic cell cycles in vertebrates. Xenopus has two Nek2 splice variants, Nek2A and Nek2B, which are zygotic ...and maternal forms, respectively. Here we have examined the role of Nek2B in oocyte meiosis and early embryonic mitosis. Specific inhibition of Nek2B function does not interfere with the oscillation of Cdc2 activity in either the meiotic or mitotic cell cycles; however, it does cause abortive cleavage of early embryos, in which bipolar spindle formation is severely impaired due to fragmentation or dispersal of the centrosomes, to which endogenous Nek2B protein localizes. In contrast, inhibition of Nek2B function does not affect meiotic spindle formation in oocytes, in which functional centrosomes are absent. Thus, strikingly, Nek2B is specifically required for centrosome assembly and/or maintenance (and hence for normal bipolar spindle formation and cleavage) in early Xenopus embryos. Finally, (ectopic) Nek2A but not Nek2B is very labile in cleaving embryos, suggesting that Nek2A cannot replace the centrosomal function of Nek2B in early embryos.
In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication ...checkpoint is thought to be activated by the exponentially increased nucleo‐cytoplasmic ratio. Here we show that the checkpoint kinase Chk1, but not Cds1 (Chk2), is activated transiently at the MBT in a maternal/zygotic gene product‐regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre‐MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1‐independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chk1, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development.
Spleen tyrosine kinase (Syk) is a key regulatory factor in the IgE-mediated allergic signal transduction pathway in mast cells and basophils. Syk is phosphorylated on a number of tyrosines following ...the binding of IgE/allergen complexes to Fcinline imageRI receptors leading to initiation of inflammatory signaling via downstream enzymes and scaffolding proteins. We examined the kinases responsible for the phosphorylation of key Syk tyrosines in rat RBL-2H3 basophilic cells and bone marrow-derived mast cells (BMMCs). The phosphorylation of Syk tyrosine 346 was completely blocked by the novel Src family kinase inhibitor BIRA766, suggesting this tyrosine is a pure substrate for Src family kinases. This was supported by the findings that kinase-dead (KD) Syk was efficiently phosphorylated on this tyrosine and that a specific Syk inhibitor BAY61-3606 was without effect. The phosphorylation of other Syk tyrosines 317, 342, 519 and 520 was reduced by Syk and Src family inhibitors, suggesting a role for auto- and trans-phosphorylation. Lyn was the predominant Src family kinase expressed and activated in RBL-2H3 cells, meanwhile Lyn knockdown with a specific siRNA interfered with the phosphorylation of all Syk tyrosines and the Syk substrates SLP-76 and LAT. Pharmacological inhibition of Syk completely blocked the degranulation of RBL-2H3 and BMMCs. However, Lyn knockdown sensitized RBL-2H3 cells to Fcinline imageRI-induced degranulation. We showed that whilst interference with Lyn expression disrupts Fcinline imageRI proximal signaling via Syk and its direct substrates including SLP-76 and LAT, distal activation of downstream proteins including Erk is enhanced. This study identifies the responsible kinases for the phosphorylation of key Syk tyrosines and the propagation of Fcinline imageRI receptor mediated signal transduction in allergic responses.
Burrowing, an ethologically relevant rodent behaviour, has been proposed as a novel outcome measure to assess the global impact of pain in rats. In a prospective multicentre study using male rats ...(Wistar, Sprague-Dawley), replication of suppressed burrowing behaviour in the complete Freund adjuvant (CFA)-induced model of inflammatory pain (unilateral, 1 mg/mL in 100 µL) was evaluated in 11 studies across 8 centres. Following a standard protocol, data from participating centres were collected centrally and analysed with a restricted maximum likelihood-based mixed model for repeated measures. The total population (TP-all animals allocated to treatment; n = 249) and a selected population (SP-TP animals burrowing over 500 g at baseline; n = 200) were analysed separately, assessing the effect of excluding "poor" burrowers. Mean baseline burrowing across studies was 1113 g (95% confidence interval: 1041-1185 g) for TP and 1329 g (1271-1387 g) for SP. Burrowing was significantly suppressed in the majority of studies 24 hours (7 studies/population) and 48 hours (7 TP, 6 SP) after CFA injections. Across all centres, significantly suppressed burrowing peaked 24 hours after CFA injections, with a burrowing deficit of -374 g (-479 to -269 g) for TP and -498 g (-609 to -386 g) for SP. This unique multicentre approach first provided high-quality evidence evaluating suppressed burrowing as robust and reproducible, supporting its use as tool to infer the global effect of pain on rodents. Second, our approach provided important informative value for the use of multicentre studies in the future.
Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to ...negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes.
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