Phosphorylation of BAD at Ser-128 during mitosis and paclitaxel-induced apoptosis

Phosphorylation of BCL-2 family member BAD at different residues triggers different physiological effects, either inhibiting or promoting apoptosis. The recently identified phosphorylation site at Ser-128 enhances the apoptotic activity of BAD. We here show that BAD becomes phosphorylated at Ser-128 in the mitotic phase of the cell cycle in NIH3T3 cells. We also show that BAD-S128 is phosphorylated in taxol-treated mouse fibroblasts and MDA-MB-231 human breast cancer cells. However, expression of a phosphorylation-defective dominant negative BAD mutant did not block taxol-induced apoptosis. These data support the view that the phosphorylation of BAD Serine 128 exerts cell-specific effects on apoptosis. Whereas the BAD Serine 128 phosphorylation induces apoptosis in neuronal cells, it does not appear to promote apoptosis in proliferating non-neural cells during mitosis or upon exposure to the antineoplastic agent taxol.     

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation

The Bcl-2 homology 3 (BH3) domain of prodeath Bcl-2 family members mediates their interaction with prosurvival Bcl-2 family members and promotes apoptosis. We report that survival factors trigger the phosphorylation of the proapoptotic Bcl-2 family member BAD at a site (Ser-155) within the BAD BH3 domain. When BAD is bound to prosurvival Bcl-2 family members, BAD Ser-155 phosphorylation requires the prior phosphorylation of Ser-136, which recruits 14-3-3 proteins that then function to increase the accessibility of Ser-155 to survival-promoting kinases. Ser-155 phosphorylation disrupts the binding of BAD to prosurvival Bcl-2 proteins and thereby promotes cell survival. These findings define a mechanism by which survival signals inactivate a proapoptotic Bcl-2 family member, and suggest a role for 14-3-3 proteins as cofactors that regulate sequential protein phosphorylation events.     

Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery

A mechanism that triggers neuronal apoptosis has been characterized. We report that the cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and that Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. Our results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions.     

Exogenously added fibroblast growth factor 2 (FGF-2) to NIH3T3 cells interacts with nuclear ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner

Fibroblast growth factor 2 (FGF-2) has been detected in the nuclei of many tissues and cell lines. Here we demonstrate that FGF-2 added exogenously to NIH3T3 cells enters the nucleus and interacts with the nuclear active 90-kDa ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner. By using purified proteins, FGF-2 is shown to directly interact through two separate domains with two RSK2 domains on both sides of the hydrophobic motif, namely the NH2-terminal kinase domain (residues 360-381) by amino acid Ser-117 and the COOH-terminal kinase domain (residues 388-400) by amino acids Leu-127 and Lys-128. Moreover, this interaction leads to maintenance of the sustained activation of RSK2 in G1 phase of the cell cycle. FGF-2 mutants (FGF-2 S117A, FGF-2 L127A, and FGF-2 K128A) that fail to interact in vitro with RSK2 fail to maintain a sustained RSK2 activity in vivo.     

BAD Ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival

The BH3 domain of BAD mediates its death-promoting activities via heterodimerization to the Bcl-XL family of death regulators. Growth and survival factors inhibit the death-promoting activity of BAD by stimulating phosphorylation at multiple sites including Ser-112 and Ser-136. Phosphorylation at these sites promotes binding of BAD to 14-3-3 proteins, sequestering BAD away from the mitochondrial membrane where it dimerizes with Bcl-XL to exert its killing effects. We report here that the phosphorylation of BAD at Ser-155 within the BH3 domain is a second phosphorylation-dependent mechanism that inhibits the death-promoting activity of BAD. Protein kinase A, RSK1, and survival factor signaling stimulate phosphorylation of BAD at Ser-155, blocking the binding of BAD to Bcl-XL. RSK1 phosphorylates BAD at both Ser-112 and Ser-155 and rescues BAD-mediated cell death in a manner dependent upon phosphorylation at both sites.     

Mitotic effects of a constitutively active mutant of the Xenopus polo-like kinase Plx1

During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, we demonstrate that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes induced directly the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delayed the timing of deactivation of Cdc25C during exit from M phase and accelerated Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a "trigger" kinase for the activation of Cdc25C during the G(2)/M transition. In addition, during anaphase T201D Plx1 reduced preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 was found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis.     

Studies of Schizosaccharomyces pombe DNA polymerase alpha at different stages of the cell cycle.

The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.     

Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimer formation and ligand binding

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3zeta. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3zeta in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3 proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3zeta at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3zeta to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3zeta dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3zeta functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.     

Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G(2)/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3beta. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G(2)/M arrest. HCT116 human colon cancer-derived Chk2(-/-) cells were significantly more resistant to G(2)/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G(2)/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.     

Cell cycle differences in DNA damage-induced BRCA1 phosphorylation affect its subcellular localization

Phosphorylation of BRCA1 tumor suppressor protein is regulated during the cell cycle and in response to DNA damage. Several Ser/Thr kinases have been implicated in BRCA1 phosphorylation, including ATM/ATR, cdk2, and hChk2 kinases. In this study, phospho-Ser-specific antibodies recognizing Ser-988, -1423, -1497, and -1524 residues of BRCA1 were employed to study BRCA1 phosphorylation during the S and G2/M phases under conditions of DNA damage. We observed that IR (ionizing radiation) treatment induced phosphorylation of Ser-988/Ser-1524 during the S phase and of Ser-988/Ser-1423 during the G2/M phase. UV treatment induced phosphorylation of Ser-988 during the S phase and of Ser-1423 during the G2/M phase. Phosphorylation of serines 1423 and -1524 was not induced in HCC1937 breast cancer cells, which contain mutant BRCA1 protein. Confocal microscopy revealed that unphosphorylated BRCA1 localizes on chromosomes from metaphase through telophase, whereas Ser-988-phosphorylated BRCA1 resides in the inner chromosomal structure, centrosome, and the cleavage furrow during prophase through telophase. We also found that Ser-988-phosphorylated BRCA1 relocalizes to the perinuclear region when cells are subjected to IR or UV radiation in the S phase. These results reinforce a model wherein phosphorylation of specific residues of BRCA1 after DNA damage affects its localization and function.     

The small heat-shock protein alpha B-crystallin promotes FBX4-dependent ubiquitination

AlphaB-crystallin is a small heat-shock protein in which three serine residues (positions 19, 45, and 59) can be phosphorylated under various conditions. We describe here the interaction of alphaB-crystallin with FBX4, an F-box-containing protein that is a component of the ubiquitin-protein isopeptide ligase SCF (SKP1/CUL1/F-box). The interaction with FBX4 was enhanced by mimicking phosphorylation of alphaB-crystallin at both Ser-19 and Ser-45 (S19D/S45D), but not at other combinations. Ser-19 and Ser-45 are preferentially phosphorylated during the mitotic phase of the cell cycle. Also alphaB-crystallin R120G, a mutant found to co-segregate with a desmin-related myopathy, displayed increased interaction with FBX4. Both alphaB-crystallin S19D/S45D and R120G specifically translocated FBX4 to the detergent-insoluble fraction and stimulated the ubiquitination of one or a few yet unknown proteins. These findings implicate the involvement of alphaB-crystallin in the ubiquitin/proteasome pathway in a phosphorylation- and cell cycle-dependent manner and may provide new insights into the alphaB-crystallin-induced aggregation in desmin-related myopathy.     

Caffeine induces G2/M arrest and apoptosis via a novel p53-dependent pathway in NB4 promyelocytic leukemia cells

Methylxantine derivative, caffeine, is known to prevent the p53-dependent apoptosis pathway via inhibition of ATM (ataxia telangiectasia mutated) kinase, which activates p53 by phosphorylation of the Ser-15 residue. In contrast, it has been reported that caffeine induces p53-mediated apoptosis through Bax protein in non-small-cell lung cancer cells. Therefore, the effects of caffeine on cellular growth in malignant cells are controversial. We investigated the effects of caffeine on cell proliferation, cell cycle progression, and induction of apoptosis in NB4 promyelocytic leukemia cells containing wild-type p53. Caffeine suppressed the cellular growth of NB4 cells in a dose- and time-dependent manner. Caffeine induced G(2)/M phase cell cycle arrest in NB4 cells in association with the induction of phosphorylation at the Ser-15 residue of p53 and induction of tyrosine phosphorylation of cdc2. Expression of Bax protein was increased in NB4 cells after treatment with caffeine. Interestingly, the antisense oligonucleotides for p53 significantly reduced p53 expression and caffeine-induced G(2)/M phase cell cycle arrest in NB4 cells. These results suggest that caffeine induces cell cycle arrest and apoptosis in association with activation of p53 by a novel pathway to phosphorylate the Ser-15 residue and induction of phosphorylation of cdc 2 in leukemic cells with normal p53.     

Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel

We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-Akt-BAD cascade, ERK-BAD cascade, and Akt-Raf-1 cascade in the paclitaxel-resistant SW626 human ovarian cancer cell line, which lacks functional p53. Treatment of SW626 cells with paclitaxel activates Akt and ERK with different time frames. Interference with the Akt cascade either by treatment with PI-3K inhibitor (wortmannin or LY294002) or by exogenous expression of a dominant negative Akt in SW626 cells caused decreased cell viability following treatment with paclitaxel. Interference with the ERK cascade by treatment with an MEK inhibitor, PD98059, in SW626 cells also caused decreased cell viability following treatment with paclitaxel. Treatment of cells with paclitaxel also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. The phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin or cotransfection with the dominant negative Akt. On the other hand, the phosphorylation of BAD at Ser-112 was blocked by PD98059. We further examined the role of BAD in the viability following paclitaxel treatment using BAD mutants. Exogenous expression of doubly substituted BAD2SA in SW626 cells caused decreased viability following treatment with paclitaxel. Moreover, because paclitaxel-induced apoptosis is mediated by activated Raf-1 and the region surrounding Ser-259 in Raf-1 conforms to a consensus sequence for phosphorylation by Akt, the regulation of Raf-1 by Akt was examined. We demonstrated an association between Akt and Raf-1 and showed that the phosphorylation of Raf-1 on Ser-259 induced by paclitaxel was blocked by treatment with wortmannin or LY294002. Furthermore, interference with the Akt cascade induced by paclitaxel up-regulated Raf-1 activity, and expression of constitutively active Akt inhibited Raf-1 activity, suggesting that Akt negatively regulates Raf-1. Our findings suggest that paclitaxel induces the phosphorylation of BAD Ser-112 via the ERK cascade, and the phosphorylation of both BAD Ser-136 and Raf-1 Ser-259 via the PI-3K-Akt cascade, and that inhibition of either of these cascades sensitizes ovarian cancer cells to paclitaxel.     

Growth factors inactivate the cell death promoter BAD by phosphorylation of its BH3 domain on Ser155

The Bcl-2 family protein BAD promotes apoptosis by binding through its BH3 domain to Bcl-x(L) and related cell death suppressors. When BAD is phosphorylated on either Ser(112) or Ser(136), it forms a complex with 14-3-3 in the cytosol and no longer interacts with Bcl-x(L) at the mitochondria. Here we show that phosphorylation of a distinct site Ser(155), which is at the center of the BAD BH3 domain, directly suppressed the pro-apoptotic function of BAD by eliminating its affinity for Bcl-x(L). Protein kinase A functioned as a BAD Ser(155) kinase both in vitro and in cells. BAD Ser(155) was found to be a major site of phosphorylation induced following stimulation by growth factors and prevented by protein kinase A inhibitors but not by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Growth factors inhibited BAD-induced apoptosis in both a Ser(112)/Ser(136)- and a Ser(155)-dependent fashion. Thus, growth factors engage an anti-apoptotic signaling pathway that inactivates BAD by direct modification of its BH3 cell death effector domain.     

Immortalized mouse cell lines that lack a functional Rev3 gene are hypersensitive to UV irradiation and cisplatin treatment

The catalytic subunit of polymerase zeta is encoded from the Rev3 gene. The enzyme is conserved through eukaryotic evolution and its main function appears to be translesion synthesis (TLS) over damaged bases that stall DNA replication. In non-vertebrate cells, inactivation of polymerase zeta results in a moderate hypersensitivity to DNA damage but no proliferative defect in the absence of exogenous damage. Mouse embryos that lack Rev3 however have a severe growth defect and are aborted at midgestation. This has suggested that polymerase zeta may be involved in vital processes in mammalian cells. Here we describe the establishment of immortalized mouse fibroblast cell lines that lack a functional Rev3 gene. These were established from homozygously Rev3-targeted mouse embryos that were also heterozygously targeted at the p53 locus, but the cell lines lost the wild type p53 allele during transformation. Cell lines in which the Rev3 gene is targeted on both alleles grow more slowly than control lines and the deficiency is also associated with an increased frequency of cells at the G2/M phase of the cell cycle and augmented apoptosis. Targeted cells are hypersensitive to UV irradiation and cisplatin treatment and arrest at the S or G2/M phase of the cell cycle if exposed to these treatments. Thus, although vital for murine embryonic development, polymerase zeta activity is not essential for continuous proliferation of transformed mammalian cells that lack p53. It does, however, appear to play an important role in allowing mammalian cells to tolerate DNA damage.     

Synthesis and biological evaluation of dimeric cinnamaldehydes as potent antitumor agents

It has been reported that 2-hydroxycinnamaldehyde and 2-benzoyl-oxycinnamaldehyde inhibited the activity of farnesyl protein transferase, angiogenesis, cell-cell adhesion, and tumor growth in vivo model. In order to improve its anti-tumor activity, dimeric cinnamaldehydes have been synthesized based on 2-hydroxycinnamaldehyde. The synthesized compounds strongly inhibited the growth of human colon tumor cells with GI50 values of 0.6-10 microM. Especially, 2-piperazine derivative blocked in vivo growth of human colon tumor xenograft in nude mice at 10 mg/kg. It was found that their anti-tumor effects induce apoptosis and cell cycle arrest at G2/M phase by the compounds. It was confirmed by detection of apoptosis markers such as activated caspase-3 and cleaved PARP, and cell cycle analysis. The dimeric compounds also inhibited Cdc25B phosphatase which is essential for preinitiating G2/M transition and S phase progression.     

Citron kinase is a cell cycle-dependent,nuclear protein required for G2/M transition of hepatocytes

Citron Kinase (Citron-K) is a cell cycle-dependent protein regulating the G(2)/M transition in hepatocytes. Synchronization studies demonstrated that expression of the Citron-K protein starts at the late S and/or the early G(2) phase after that of cyclin B1. Expression of Citron-K is developmentally regulated. Levels of Citron-K mRNA and protein are highest in embryonic liver and gradually decrease after birth. Citron-K exists in interphase nuclei and begins to disperse into the cytoplasm at prophase. It concentrates at the cleavage furrow and midbody during anaphase, telophase, and cytokinesis, implicating a role in the control of cytokinesis. However, studies with knockouts show that Citron-K is not essential for cytokinesis in hepatocytes. Instead, loss of Citron-K causes a significant increase of G(2) tetraploid nuclei in one-week-old rat and mouse liver. In addition, Citron-K deficiency triggers apoptosis in a small subset of embryonic liver cells. In summary, our data demonstrate that Citron-K has a distinct cell cycle-dependent expression pattern and cellular localization as a downstream target of Rho-GTPase and functions in the control of G(2)/M transition in the hepatocyte cell cycle.     

SIRT2 is a negative regulator of anoxia-reoxygenation tolerance via regulation of 14-3-3 zeta and BAD in H9c2 cells

Knockdown or inhibition of SIRT2 enhances biological stress-tolerance. We extend this phenotype showing that SIRT2 knockdown reduces anoxia-reoxygenation injury in H9c2 cells. Gene array analysis following SIRT2 siRNA knockdown identifies 14-3-3 zeta as the most robustly induced gene. SIRT2 knockdown evokes induction of this chaperone, facilitating cytosolic sequestration of BAD with a corresponding reduction in mitochondrial BAD localization. Concurrent siRNA against SIRT2 and 14-3-3 zeta abolishes the SIRT2-depleted cytoprotective phenotype. SIRT2 functions to moderate cellular stress-tolerance, in part, by modulating the levels of 14-3-3 zeta with the concordant control of BAD subcellular localization.