Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA damage responsive checkpoint complex.

DNA damage activates cell cycle checkpoint signaling pathways that coordinate cell cycle arrest and DNA repair. Three of the proteins involved in checkpoint signaling, Rad1, Hus1, and Rad9, have been shown to interact by immunoprecipitation and yeast two-hybrid studies. However, it is not known how these proteins interact and assemble into a complex. In the present study we demonstrated that in human cells all the hRad9 and hHus1 and approximately one-half of the cellular pool of hRad1 interacted as a stable, biochemically discrete complex, with an apparent molecular mass of 160 kDa. This complex was reconstituted by co-expression of all three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical chromatographic behavior as the endogenous complex. Interaction studies using differentially tagged proteins demonstrated that the proteins did not self-multimerize. Rather, each protein had a binding site for the other two partners, with the N terminus of hRad9 interacting with hRad1, the N terminus of hRad1 interacting with hHus1, and the N terminus of hHus1 interacting with the C terminus of hRad9's predicted PCNA-like region. Collectively, these analyses suggest a model of how these three proteins assemble to form a functional checkpoint complex, which we dubbed the 9-1-1 complex.

A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells.

The microbially derived antiproliferative agent rapamycin inhibits cell growth by interfering with the signaling functions of the mammalian target of rapamycin (mTOR). In this study, we demonstrate that interleukin-3 stimulation induces a wortmannin-sensitive increase in mTOR kinase activity in a myeloid progenitor cell line. The involvement of phosphoinositide 3'-kinase (PI3K) in the regulation of mTOR activity was further suggested by findings that mTOR was phosphorylated in vitro and in vivo by the PI3K-regulated protein kinase, AKT/PKB. Although AKT phosphorylated mTOR at two COOH-terminal sites (Thr2446 and Ser2448) in vitro, Ser2448 was the major phosphorylation site in insulin-stimulated or -activated AKT-expressing human embryonic kidney cells. Transient transfection assays with mTOR mutants bearing Ala substitutions at Ser2448 and/or Thr2446 indicated that AKT-dependent mTOR phosphorylation was not essential for either PHAS-I phosphorylation or p70S6K activation in HEK cells. However, a deletion of amino acids 2430-2450 in mTOR, which includes the potential AKT phosphorylation sites, significantly increased both the basal protein kinase activity and in vivo signaling functions of mTOR. These results demonstrate that mTOR is a direct target of the PI3K-AKT signaling pathway in mitogen-stimulated cells, and that the identified AKT phosphorylation sites are nested within a "repressor domain" that negatively regulates the catalytic activity of mTOR. Furthermore, the activation status of the PI3K-AKT pathway in cancer cells may be an important determinant of cellular sensitivity to the cytostatic effect of rapamycin.

The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9.

DNA damage activates cell cycle checkpoints that prevent progression through the cell cycle. In yeast, the DNA damage checkpoint response is regulated by a series of genes that have mammalian homologs, including rad1, rad9, hus1, and rad17. On the basis of sequence homology, yeast and human Rad1, Rad9, and Hus1 protein homologs are predicted to structurally resemble the sliding clamp PCNA. Likewise, Rad17 homologs have extensive homology with replication factor C (RFC) subunits (p36, p37, p38, p40, and p140), which form a clamp loader for PCNA. These observations predict that Rad1, Hus1, and Rad9 might interact with Rad17 as a clamp-clamp loader pair during the DNA damage response. In this report, we demonstrate that endogenous human Rad17 (hRad17) interacts with the PCNA-related checkpoint proteins hRad1, hRad9, and hHus1. Mutational analysis of hRad1 and hRad17 demonstrates that this interaction has properties similar to the interaction between RFC and PCNA, a well characterized clamp-clamp loader pair. Moreover, we show that DNA damage affects the association of hRad17 with the clamp-like checkpoint proteins. Collectively, these data provide the first experimental evidence that hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9 in manner similar to the interaction between RFC and PCNA.

Retention of the human Rad9 checkpoint complex in extraction-resistant nuclear complexes after DNA damage.

Studies in yeasts and mammals have identified many genes important for DNA damage-induced checkpoint activation, including Rad9, Hus1, and Rad1; however, the functions of these gene products are unknown. In this study we show by immunolocalization that human Rad9 (hRad9) is localized exclusively in the nucleus. However, hRad9 was readily released from the nucleus into the soluble extract upon biochemical fractionation of un-irradiated cells. In contrast, DNA damage promptly converted hRad9 to an extraction-resistant form that was retained at discrete sites within the nucleus. Conversion of hRad9 to the extraction-resistant nuclear form occurred in response to diverse DNA-damaging agents and the replication inhibitor hydroxyurea but not other cytotoxic stimuli. Additionally, extraction-resistant hRad9 interacted with its binding partners, hHus1 and an inducibly phosphorylated form of hRad1. Thus, these studies demonstrate that hRad9 is a nuclear protein that becomes more firmly anchored to nuclear components after DNA damage, consistent with a proximal function in DNA damage-activated checkpoint signaling pathways.

The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade.

Liver injury during cholestasis reflects a balance between the effects of toxic and nontoxic bile acids. However, the critical distinction between a toxic and nontoxic bile acid remains subtle and unclear. For example, the glycine conjugate of chenodeoxycholate (GCDC) induces hepatocyte apoptosis, whereas the taurine conjugate (TCDC) does not. We hypothesized that the dissimilar cellular responses may reflect differential activation of a phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. In the bile acid-transporting McNtcp.24 rat hepatoma cell line, TCDC, but not GCDC, stimulated PI3K activity. Consistent with this observation, inhibition of PI3K rendered TCDC cytotoxic, and constitutive activation of PI3K rendered GCDC nontoxic. Both Akt and the atypical protein kinase C isoform zeta (PKCzeta) have been implicated in PI3K-dependent survival signaling. However, TCDC activated PKCzeta, but not Akt. Moreover, inhibition of PKCzeta converted TCDC into a cytotoxic agent, whereas overexpression of wild-type PKCzeta blocked GCDC-induced apoptosis. We also demonstrate that TCDC activated nuclear factor kappaB (NF-kappaB) in a PI3K- and PKCzeta-dependent manner. Moreover, inhibition of NF-kappaB by an IkappaB super-repressor rendered TCDC cytotoxic, suggesting that NF-kappaB is also necessary to prevent the cytotoxic effects of TCDC. Collectively, these data suggest that some hydrophobic bile acids such as TCDC activate PI3K-dependent survival pathways, which prevent their otherwise inherent toxicity.

The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1.

The investigational anticancer agent 7-hydroxystaurosporine (UCN-01) abrogates the G2 checkpoint in tumor cells and sensitizes them to the lethal effects of genotoxic anticancer agents. On the basis of the role of the Cdc25C phosphatase in maintenance of this damage-inducible checkpoint, we hypothesized that UCN-01 inhibits a component of the signal transduction pathway that modulates Cdc25C phosphorylation. Of the three kinases known to phosphorylate Cdc25C on Ser216, both checkpoint kinase 1 (hChk1) and Cdc25C-associated protein kinase 1 (cTAK1) were potently inhibited by UCN-01 with IC50s of 11 and 27 nM, respectively. Treatment of K562 erythroblastoid leukemia cells with similar drug concentrations resulted in decreased levels of Ser216 phosphorylation of Cdc25C and complete disruption of the y-radiation-induced G2 checkpoint. In contrast to hChk1, the hChk2 kinase was 100-fold more resistant to inhibition by UCN-01 (IC50, 1040 nM). These results suggest that disruption of the DNA damage-induced G2 checkpoint by UCN-01 is mediated through the inhibition of the Cdc25C kinases, hChk1 and cTAK1, and that hChk2 activity is not sufficient to enforce the G2 checkpoint in cells treated with a pharmacological inhibitor of hChk1.

Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 in the activation loop of protein kinase C-zeta.

Previous studies have suggested that 1) atypical protein kinase C (PKC) isoforms are required for insulin stimulation of glucose transport, and 2) 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is required for activation of atypical PKCs. Presently, we evaluated the role of PDK-1, both in the activation of PKC-zeta, and the translocation of epitope-tagged glucose transporter 4 (GLUT4) to the plasma membrane, during insulin action in transiently transfected rat adipocytes. Overexpression of wild-type PDK-1 provoked increases in the activity of cotransfected hemagglutinin (HA)-tagged PKC-zeta and concomitantly enhanced HA-tagged GLUT4 translocation. Expression of both kinase-inactive PDK-1 and an activation-resistant form of PKC-zeta that is mutated at Thr-410, the immediate target of PDK-1 in the activation loop of PKC-zeta, inhibited insulin-induced increases in both HA-PKC-zeta activity and HA-GLUT4 translocation to the same extent as kinase-inactive PKC-zeta. Moreover, the inhibitory effects of kinase-inactive PDK-1 were fully reversed by cotransfection of wild-type PDK-1 and partly reversed by wild-type PKC-zeta, but not by wild-type PKB. In contrast to the T410A PKC-zeta mutant, an analogous double mutant of PKB (T308A/S473A) that is resistant to PDK-1 activation had only a small effect on insulin-stimulated HA-GLUT4 translocation and did not inhibit HA-GLUT4 translocation induced by overexpression of wild-type PDK-1. Our findings suggest that both PDK-1 and its downstream target, Thr-410 in the activation loop of PKC-zeta, are required for insulin-stimulated glucose transport.

Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine.

Caffeine exposure sensitizes tumor cells to ionizing radiation and other genotoxic agents. The radiosensitizing effects of caffeine are associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints. The similarity of these checkpoint defects to those seen in ataxia-telangiectasia (A-T) suggested that caffeine might inhibit one or more components in an A-T mutated (ATM)-dependent checkpoint pathway in DNA-damaged cells. We now show that caffeine inhibits the catalytic activity of both ATM and the related kinase, ATM and Rad3-related (ATR), at drug concentrations similar to those that induce radiosensitization. Moreover, like ATM-deficient cells, caffeine-treated A549 lung carcinoma cells irradiated in G2 fail to arrest progression into mitosis, and S-phase-irradiated cells exhibit radioresistant DNA synthesis. Similar concentrations of caffeine also inhibit gamma- and UV radiation-induced phosphorylation of p53 on Ser15, a modification that may be directly mediated by the ATM and ATR kinases. DNA-dependent protein kinase, another ATM-related protein involved in DNA damage repair, was resistant to the inhibitory effects of caffeine. Likewise, the catalytic activity of the G2 checkpoint kinase, hChk1, was only marginally suppressed by caffeine but was inhibited potently by the structurally distinct radiosensitizer, UCN-01. These data suggest that the radiosensitizing effects of caffeine are related to inhibition of the protein kinase activities of ATM and ATR and that both proteins are relevant targets for the development of novel anticancer agents.

Gi proteins use a novel beta gamma- and Ras-independent pathway to activate extracellular signal-regulated kinase and mobilize AP-1 transcription factors in Jurkat T lymphocytes.

Receptors coupled to pertussis toxin (PTX)-sensitive Gi proteins regulate T lymphocyte cytokine secretion, proliferation, and chemotaxis, yet little is known about the molecular mechanisms of Gi protein signaling in mammalian lymphocytes. Using the Jurkat T lymphocyte cell line, we found that a stably expressed Gi protein-coupled receptor (the delta-opioid receptor (DOR1)) stimulates MEK-1 and extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) and transcriptional activity by an ERK target, Elk-1, via a mechanism requiring a PTX-sensitive Gi protein. Levels of beta-adrenergic receptor kinase-1 C-terminal fragment that inhibited signaling by Gi protein beta gamma subunits in these cells had no effect on DOR1 stimulation of either MEK-1- or Elk-1-dependent transcription, indicating that this pathway is independent of beta gamma. Analysis of this betagamma-independent pathway indicates a role for a herbimycin A-sensitive tyrosine kinase. Unlike beta gamma-mediated pathways, the beta gamma-independent pathway was insensitive to RasN17, inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), and constitutive PI 3-kinase activity. The beta gamma-independent pathway regulates downstream events, since blocking it abrogated both Elk-1-dependent transcription and mobilization of the mitogenic transcription factor, AP-1, in response to DOR1 signaling. These results characterize a novel, Ras- and PI 3kinase-independent pathway for ERK activation by Gi protein signaling that is distinct from ERK activation by beta gamma and may therefore be mediated by the alphai subunit.

A phosphatidylinositol 3-kinase-dependent pathway that differentially regulates c-Raf and A-Raf.

Cytokines trigger the rapid assembly of multimolecular signaling complexes that direct the activation of downstream protein kinase cascades. Two protein kinases that have been linked to growth factor-regulated proliferation and survival are mitogen-activated protein/ERK kinase (MEK) and its downstream target Erk, a member of the mitogen-activated protein kinase family. Using complementary pharmacological and genetic approaches, we demonstrate that MEK and Erk activation requires a phosphatidylinositol 3-kinase (PI3-K)-generated signal in an interleukin (IL)-3-dependent myeloid progenitor cell line. Analysis of the upstream pathway leading to MEK activation revealed that inhibition of PI3-K did not block c-Raf activation, whereas MEK activation was effectively blocked under these conditions. Furthermore, agents that elevated cAMP suppressed IL-3-induced c-Raf activation but did not inhibit MEK activation. Because c-Raf activation and MEK activation were inversely affected by PI3-K- and cAMP-dependent pathways, we examined whether IL-3 activated the alternative Raf isoforms A-Raf and B-Raf. Although IL-3 did not activate B-Raf, A-Raf was activated by the cytokine. Moreover, A-Raf activation, like MEK activation, was blocked by inhibition of PI3-K but was insensitive to cAMP. Experiments with dominant negative mutants of the Raf isoforms showed that overexpression of dominant negative c-Raf did not prevent MEK activation. However, dominant negative A-Raf effectively blocked MEK activation, suggesting that activation of the MEK-Erk signaling cascade is mediated through A-Raf. Taken together, these results suggest that IL-3 receptors engage and activate both c-Raf and A-Raf in hemopoietic cells. However, these intermediates are differentially regulated by upstream signaling cascades and selectively coupled to downstream signaling pathways.

Human homologs of Schizosaccharomyces pombe rad1, hus1, and rad9 form a DNA damage-responsive protein complex.

DNA damage activates cell cycle checkpoints in yeast and human cells. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe checkpoint-deficient mutants have been characterized, and the corresponding genes have been cloned. Searches for human homologs of S. pombe rad1, rad9, and hus1 genes identified the potential human homologs hRad1, hRad9, and hHus1; however, little is known about the roles of these proteins in human cells. The present studies demonstrate that hRad1 and hHus1 associate in a complex that interacts with a highly modified form of hRad9, but hHus1 and hRad1 do not associate with hRad17. In addition to being a key participant in complex formation, hRad9 is phosphorylated in response to DNA damage. Together, these results suggest that hRad9, hRad1, and hHus1 are central components of a DNA damage-responsive protein complex in human cells.

Ionizing radiation-induced MEK and Erk activation does not enhance survival of irradiated human squamous carcinoma cells.

PURPOSE: Ionizing radiation (IR) triggers several intracellular signaling cascades that have commonly been regarded as mitogenic, including the Raf-MEK-Erk kinase cascade. In addition to promoting proliferation, activated MEK and Erk may also prevent cell death induced by cytotoxic stimuli. Because Raf, MEK, and Erk are activated by IR in some tumor cell lines, this suggests that IR-induced activation of the kinase cascade may enhance the survival of irradiated cells. METHODS AND MATERIALS: IR-induced activation of MEK and Erk was assessed in irradiated UM-SCC-6 cells, a human squamous carcinoma cell line. Activation of MEK and Erk was blocked with the pharmacological inhibitor of MEK activation, PD098059. Clonogenic survival was assessed in irradiated UM-SCC-6 cells that were pretreated with nothing or with the MEK inhibitor. RESULTS: In UM-SCC-6 cells, IR doses as low as 2 Gy rapidly activated MEK and Erk. Pretreatment of the cells with the pharmacological inhibitor of MEK activation, PD098059, effectively blocked IR-induced activation of MEK and Erk. However, inhibition of the kinase cascade did not affect the clonogenic survival of irradiated cells in either early or delayed-plating experiments. CONCLUSION: Taken together, these results suggest that although MEK and Erk are rapidly activated by IR treatment, these protein kinases do not affect the clonogenic survival of irradiated UM-SCC6 cells.

Interaction between lck and syk family tyrosine kinases in Fc gamma receptor-initiated activation of natural killer cells.

Ligation of the Fc gamma R on natural killer (NK) cells results in the tyrosine phosphorylation of multiple substrates critical for intracellular signaling and activation of NK cell effector functions. However, it remains unclear which nonreceptor protein-tyrosine kinases (PTK) participate in this process. In this report we demonstrate that Fc gamma R ligation induced the tyrosine phosphorylation and increased the catalytic activities of both syk family PTKs, ZAP-70, and syk. The phosphorylation of ZAP-70 and syk was enhanced markedly by overexpression of wild-type lck but not by a kinase-inactive mutant, suggesting that early Fc gamma R-initiated activation of lck results in the subsequent regulation of syk family PTKs. The regulatory interplay between src and syk family PTKs was emphasized further by the observation that lck overexpression enhanced the association of ZAP-70 with the zeta chain of the Fc gamma R complex. Additional analyses indicated that lck induced the subsequent tyrosine phosphorylation of phospholipase C (PLC)-gamma 2. Interestingly, the regulatory effects of lck on ZAP-70, syk, and PLC-gamma 2 could not be replaced by overexpression of either fyn or src, demonstrating a selective role for lck in effectively coupling Fc gamma R stimulation to critical downstream signaling events. Taken together, our results suggest not only that Fc gamma R stimulation on NK cells is coupled to the intracellular activation of both ZAP-70 and syk, but that the src family member, lck, can selectively regulate this tyrosine kinase cascade.

Interleukin-2 triggers a novel phosphatidylinositol 3-kinase-dependent MEK activation pathway.

Phosphatidylinositol 3-kinase (PI3-K) has been implicated as a signal-transducing component in interleukin-2 (IL-2)-induced mitogenesis. However, the function of this lipid kinase in regulating IL-2-triggered downstream events has remained obscure. Using the potent and specific PI3-K inhibitor, wortmannin, we assessed the role of PI3-K in IL-2-mediated signaling and proliferation in the murine T-cell line CTLL-2. Addition of the drug to exponentially growing cells resulted in an accumulation of cells in the G0/G1 phase of the cell cycle. Furthermore, wortmannin also partially suppressed IL-2-induced S-phase entry in G1-synchronized cells. Analysis of IL-2-triggered signaling pathways revealed that wortmannin pretreatment resulted in complete inhibition of IL-2-provoked p70 S6 kinase activation and also attenuated IL-2-induced MAP kinase activation at drug concentrations identical to those required for inhibition of PI3-K catalytic activity. Wortmannin also diminished the IL-2-triggered activation of the MAP kinase activator, MEK, but did not inhibit activation of Raf, the canonical upstream activator of MEK. These results suggest that a novel wortmannin-sensitive activation pathway regulates MEK and MAP kinase in IL-2-stimulated T lymphocytes.

Cytokine receptor signaling mechanisms.

Recent studies have dramatically advanced our understanding of the post-receptor signaling machinery engaged by members of the cytokine receptor superfamily. Proximal components of this signaling machinery are members of a novel class of protein tyrosine kinases termed JAKs. A working model proposes that ligand-induced dimerization of cytokine receptor subunits triggers the catalytic activation of the associated JAKs, which then couple the receptor to downstream signaling pathways. Ongoing research efforts are also yielding information regarding two potentially interactive pathways of nuclear signaling activated by cytokine receptor occupancy. These pathways are initiated by the activation of the Ras to mitogen-activated protein (MAP) kinase cascade and by the tyrosine phosphorylation of a newly defined set of transcription factors, the signal transducers and activators of transcription (STATs). Collectively, these results begin to provide a molecular basis for the long-observed phenomena of cytokine pleiotropy and redundancy.

Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity.

Although diverse signaling events are initiated by stimulation of multichain immune recognition receptors on lymphocytes, it remains unclear as to which specific signal transduction pathways are functionally linked to granule exocytosis and cellular cytotoxicity. In the case of natural killer (NK) cells, it has been presumed that the rapid activation of protein kinase C (PKC) enables them to mediate antibody-dependent cellular cytotoxicity (ADCC) and "natural" cytotoxicity toward tumor cells. However, using cloned human NK cells, we determined here that Fc receptor stimulation triggers granule release and ADCC through a PKC-independent pathway. Specifically, pretreatment of NK cells with the selective PKC inhibitor, GF109203X (using concentrations that fully blocked phorbol myristate acetate/ionomycin-induced secretion) had no effect on FcR-initiated granule release or ADCC. In contrast, FcR ligation led to the rapid activation of phosphatidylinositol 3-kinase (PI 3-kinase), and inhibition of this enzyme with the selective inhibitor, wortmannin, blocked FcR-induced granule release and ADCC. Additional experiments showed that, whereas FcR-initiated killing was wortmannin sensitive and GF109203X insensitive, natural cytotoxic activity toward the tumor cell line K562 was wortmannin insensitive and GF109203X sensitive. Taken together, these results suggest that: (a) PI 3-kinase activation induced by FcR ligation is functionally coupled to granule exocytosis and ADCC; and (b) the signaling pathways involved in ADCC vs natural cytotoxicity are distinct.

The Src-family kinase, Fyn, regulates the activation of phosphatidylinositol 3-kinase in an interleukin 2-responsive T cell line.

The proliferation of antigen-activated T cells is mediated by the T cell-derived growth factor, interleukin 2 (IL-2). The biochemical signaling cascades initiating IL-2-induced growth are dependent upon protein tyrosine kinase (PTK) activity. One IL-2-regulated PTK implicated in this cascade is the Src-family kinase, Fyn. Previous studies have described a physical association between Fyn and a potential downstream substrate, phosphatidylinositol 3-kinase (PI3-kinase) as well as the IL-2-dependent activation of PI3-kinase in T cells; however, the role of Fyn in IL-2-induced PI3-kinase activation remains unclear. In this report, we demonstrate that IL-2 stimulation triggers tyrosine phosphorylation of the p85 subunit of PI3-kinase in the murine T cell line, CTLL-2. Lysates prepared from growth factor-deprived and IL-2-stimulated T cells reconstituted both the binding of CTLL-2 cell-derived Fyn to and the IL-2-inducible tyrosine phosphorylation of exogenously added recombinant p85. Furthermore, overexpression of wild-type Fyn in these cells enhanced both the basal and IL-2-mediated activation of PI3-kinase. Additional studies of the Fyn-PI3-kinase interaction demonstrated that the Src homology 3 (SH3) domain of Fyn constitutes a direct binding site for the p85 subunit of PI3-kinase. These results support the notion that Fyn may be directly involved in the activation of the downstream signaling enzyme, PI3-kinase, in IL-2-stimulated T cells.