Loss of H2A.Z Is Not Sufficient to Determine Transcriptional Activity of Snf2-Related CBP Activator Protein or p400 Complexes.

The p400 and SRCAP (Snf2-related CBP activator protein) complexes remodel chromatin by catalyzing deposition of histone H2A.Z into nucleosomes. This remodeling activity has been proposed as a basis for regulation of transcription by these complexes. Transcript levels of p21 or Sp1 mRNAs after knockdown of p400 or SRCAP reveals that each regulates transcription of these promoters differently. In this study, we asked whether deposition of H2A.Z within specific nucleosomes by p400 or SRCAP dictates transcriptional activity. Our data indicates that nucleosome density at specific p21 or Sp1 promoter positions is not altered by the loss of either remodeling complex. However, knockdown of SRCAP or p400 reduces deposition of H2A.Z∼50% into all p21 and Sp1 promoter nucleosomes. Thus, H2A.Z deposition is not targeted to specific nucleosomes. These results indicate that the deposition of H2A.Z by the p400 or SRCAP complexes is not sufficient to determine how each regulates transcription. This conclusion is further supported by studies that demonstrate a SRCAP(ΔATP ) mutant unable to deposit H2A.Z has similar transcriptional activity as wild-type SRCAP.

Activation of Cdk2/Cyclin E complexes is dependent on the origin of replication licensing factor Cdc6 in mammalian cells.

Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.

Phosphatidylinositol 3-kinase regulates macrophage responses to double-stranded RNA and encephalomyocarditis virus.

Virus infection of macrophages stimulates the expression of proinflammatory and antiviral genes interleukin-1 (IL-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In this study, we show that phosphatidylinositol 3-kinase (PI3K) is required for the inflammatory response of macrophages to virus infection. When macrophages are infected with encephalomyocarditis virus (EMCV) there is a rapid and transient activation of PI3K and phosphorylation of its downstream target Akt. Inhibitors of PI3K attenuate EMCV- and double-stranded RNA-induced iNOS, COX-2 and IL-1 beta expression in RAW264.7 cells and mouse peritoneal macrophages. The attenuation of inflammatory gene expression in response to PI3K inhibition correlates with the induction of macrophage apoptosis. The morphology of macrophages shifts from activation in response to EMCV infection to apoptosis in the cells treated with PI3K inhibitors and EMCV. These morphological changes are accompanied by the activation of caspase-3. These findings suggest that PI3K plays a central role in the regulation of macrophage responses to EMCV infection. When PI3K is activated, it participates in the regulation of inflammatory gene expression; however, if PI3K is inhibited macrophages are unable to mount an inflammatory antiviral response and die by apoptosis.

Receptor type protein tyrosine phosphatase-kappa mediates cross-talk between transforming growth factor-beta and epidermal growth factor receptor signaling pathways in human keratinocytes.

Epidermal growth factor receptor (EGFR) signaling pathways promote human keratinocyte survival and proliferation. In contrast, transforming growth factor-beta (TGF-beta) signaling pathways are strongly anti-proliferative. Receptor type protein tyrosine phosphatase-kappa (RPTP-kappa) specifically dephosphorylates EGFR, thereby blocking EGFR-dependent signaling, and inhibiting proliferation. We report here that RPTP-kappa mediates functional integration of EGFR and TGF-beta signaling pathways in human keratinocytes. TGF-beta up-regulates RPTP-kappa mRNA and protein, in a dose and time dependent manner. Induction of RPTP-kappa by TGF-beta significantly decreases basal and EGF-stimulated EGFR tyrosine phosphorylation. shRNA-mediated reduction of TGF-beta-induced RPTP-kappa significantly attenuates the ability of TGF-beta to inhibit proliferation. RPTP-kappa induction is dependent on activation of transcription factors Smad3 and Smad4. Inhibition of TGF-beta receptor kinase completely prevents induction of RPTP-kappa. Chromatin immunoprecipitation assays reveal that TGF-beta stimulates Smad3 and Smad4 binding to RPTP-kappa gene promoter. Smad3/4 binding is localized to an 186-base pair region, which contains a consensus Smad3-binding element. These data describe a novel mechanism of cross-talk between EGFR and TGF-beta pathways, in which RPTP-kappa functions to integrate growth-promoting and growth-inhibiting signaling pathways.

The rapid activation of N-Ras by alpha-thrombin in fibroblasts is mediated by the specific G-protein Galphai2-Gbeta1-Ggamma5 and occurs in lipid rafts.

alpha-thrombin is a potent mitogen for fibroblasts and initiates a rapid signal transduction pathway leading to the activation of Ras and the stimulation of cell cycle progression. While the signaling events downstream of Ras have been studied in significant detail and appear well conserved across many species and cell types, the precise molecular events beginning with thrombin receptor activation and leading to the activation of Ras are not as well understood. In this study, we examined the immediate events in the rapid response to alpha-thrombin, in a single cell type, and found that an unexpected degree of specificity exists in the pathway linking alpha-thrombin to Ras activation. Specifically, although IIC9 cells express all three Ras isoforms, only N-Ras is rapidly activated by alpha-thrombin. Further, although several Galpha subunits associate with PAR1 and are released following stimulation, only Galpha(i2) couples to the rapid activation of Ras. Similarly, although IIC9 cells express many Gbeta and Ggamma subunits, only a subset associates with Galpha(i2), and of those, only a single Gbetagamma dimer, Gbeta(1)gamma(5), participates in the rapid activation of N-Ras. We then hypothesized that co-localization into membrane microdomains called lipid rafts, or caveolae, is at least partially responsible for this degree of specificity. Accordingly, we found that all components localize to lipid rafts and that disruption of caveolae abolishes the rapid activation of N-Ras by alpha-thrombin. We thus report the molecular elucidation of an extremely specific and rapid signal transduction pathway linking alpha-thrombin stimulation to the activation of Ras.

The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F.

The retinoblastoma protein pRb plays a pivotal role in G(1)- to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and COOH-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the NH(2)-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using "reverse mutational analysis" of Rb(DeltaCDK) (a dominantly active repressive allele of Rb), we have previously shown that restoration of Thr-373 is sufficient to render Rb(DeltaCDK) sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the NH(2)-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we have expanded this analysis to include additional residues in the NH(2)-terminal region of pRb and further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further have found that removal of the COOH-terminal domain of either RbDeltaCDK(+T373) or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Our data demonstrate a novel function for the NH(2)-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.

Cyclin A-CDK activity during G1 phase impairs MCM chromatin loading and inhibits DNA synthesis in mammalian cells.

Progression through the mammalian cell division cycle is regulated by the sequential activation of cyclin-dependent kinases, CDKs, at specific phases of the cell cycle. Cyclin A-CDK2 and cyclin A-CDK1 phosphorylate nuclear substrates during S and G(2) phases, respectfully. However, the DNA helicase complex, MCM2-7, is loaded onto the origin of replications in G(1), prior to the normally scheduled induction of cyclin A. It has previously been shown that cyclin A-CDKs phosphorylate MCM2 and MCM4 in vitro, thereby diminishing helicase activity. Thus, in this study we hypothesize that, in vivo, cyclin A-CDK activity during G(1) would result in an inhibition of progression into the S phase. To test this, we establish an in vivo method of inducing cyclin A-CDK activity in G(1) phase and observe that activation of cyclin A-CDK, but not cyclin E-CDK complexes, inhibit DNA synthesis without affecting other G(1) events such as cyclin D synthesis, E2F activation and cdc6 loading onto chromatin. We further report that the mechanism of this S phase inhibition occurs, at least in part, through impaired loading of MCM onto chromatin, presumably due to decreased levels of cdt1 and premature phosphorylation of MCM by cyclin A-CDK. In addition to providing in vivo confirmation of in vitro predictions regarding cyclin A-CDK phosphorylation of the MCM complex, our results provide insight into the cellular effects of unscheduled cyclin A-CDK activity in mammalian cells.

Identification and characterization of a novel Mdm2 splice variant acutely induced by the chemotherapeutic agents adriamycin and actinomycin D.

Mdm2, as the most important negative regulator of p53, plays an important homeostatic role in regulating cell division and the cellular response to DNA damage, oncogenic insult and other forms of cellular stress. We discovered that the DNA damaging agent adriamycin (doxorubicin) induces a novel aberrantly spliced Mdm2 mRNA which incorporates 108 bp of intronic sequence not normally found in the Mdm2 mature mRNA. Accordingly, we term this Mdm2 splice variant Mdm2(+108). Importantly, this insertion introduces in-frame nonsense codons, thus encoding a profoundly truncated mdm2 protein lacking the C-terminal RING finger domain and the E3 ubiquitin ligase activity. A wide range of pharmacological testing revealed that Mdm2(+108) is induced, in mouse and rat cells, in specific response to Adriamycin and actinomycin D, but not other modes of DNA damage. Meanwhile, antibodies against the N-terminal region of mdm2 reveal a marked reduction in detectable mdm2 protein upon Adriamycin treatment, while p53 accumulates to strikingly high levels. We thus conclude that this alternative spicing of Mdm2 may be an important mechanism to facilitate massive accumulation of p53 in response to genotoxic agents.

Zinc induces ERK-dependent cell death through a specific Ras isoform.

The effect of Zn on p53-independent cell death was examined in IIC9 embryonic fibroblasts. Despite the fact that these cells are p53-minus, Zn-mediated death occurs via an apoptotic mechanism. Death is facilitated by the presence of the Zn ionophore, pyrithione, indicating that intracellular Zn initiates the death response. Our investigations of the mechanism of Zn action demonstrate that Zn induces the death of IIC9 cells in a manner that is ERK-dependent. Expression of dn-(dominant negative)Ras attenuates ERK1/2 activation by Zn, and correspondingly reduces its cytotoxic effects. Raf-RBD pull-down experiments confirm that Zn treatment activates Ras and identified H-Ras as the specific isoform activated. This contrasts the activation of N-Ras that occurs when IIC9 cells are stimulated with thrombin. Thus, although the prolonged activation of the Ras/ERK pathway by Zn is similar to that seen when induced by mitogen, the distinguishing feature appears to be the isoform specificity of Ras activation.

Reverse mutational analysis reveals threonine-373 as a potentially sufficient phosphorylation site for inactivation of the retinoblastoma tumor suppressor protein (pRB).

Previous studies in our laboratory have shown that constitutive cyclin E expression can alleviate the requirement for cyclin D-CDK activity in the inactivation of the retinoblastoma protein (pRb). Rb(DeltaCDK), a mutant construct of pRb with 15 of the 16 CDK phosphorylation sites mutated to alanine represses activation of E2F by mitogen, despite cyclin E overexpression. However, restoration of the four cyclin E-CDK2 phosphorylation sites to Rb(DeltaCDK) renders this construct sensitive to inactivation by CDK phosphorylation. In the present study, we engage a "reverse mutational analysis" by restoring cyclin E-CDK2 phosphorylation sites to Rb(DeltaCDK) individually and in combinations in an attempt to discover phosphorylation sites on Rb that are critical for inactivation. Surprisingly, we report that, in both rodent and human cells, restoration of threonine-373 to Rb(DeltaCDK), alone or in combination with other phospho-restorations, results in a loss of the constitutively repressive effect of this construct on E2F activation. Further, induction of endogenous cyclin A protein is blocked by Rb(DeltaCDK), but not by mutants of Rb(DeltaCDK) containing a restored threonine-373. Finally, while S phase entry is blocked by expression of Rb(DeltaCDK), restoration of threonine-373 largely attenuates this effect. These findings reveal that phosphorylation of threonine-373 by CDK2-cyclin E represent a potentially crucial event in the inactivation of the pRb protein.

RNA interference takes flight: a new RNAi screen reveals cell cycle regulators in Drosophila cells.

UNLABELLED: In a new study, a systematic screen for genes necessary for normal cell cycle progression has been completed in Drosophila S2 cells. THE RESULTS: some familiar faces and some new faces add to our appreciation of the staggering complexity of cellular growth and proliferation. The apparent utility of genome-wide RNA interference screens is validated once again.

A new lipase in regulating lipid mobilization: hormone-sensitive lipase is not alone.

Three papers recently appeared simultaneously that identify a new triacylglycerol lipase. Zimmerman et al. identified this lipase as adipocyte triacylglycerol lipase (ATGL). Jenkins et al. identified it as the zeta-isoform of a calcium-independent phospholipase A(2) (iPLA(2)), whereas Villena et al. found this enzyme and called it desnutrin. Interestingly, if, as implied, this lipase works in concert with hormone-sensitive lipase, then our concept of regulated fatty acid mobilization from triacylglycerols will have to be revised.

Killing two birds with one RKIP.

Analysis of metastatic prostate cancers has identified the Raf kinase inhibitory protein (RKIP) as a suppressor of metastases. Previous studies demonstrated that RKIP binds to Raf-1 and prevents the activation of the extracellular regulated kinase (ERK) cascade. New work shows that phosphorylation of RKIP by protein kinase C disassociates RKIP from Raf-1 and stimulates its binding to, and inhibition of G-protein-coupled receptor kinase 2 (GRK2). This switching enhances signaling by activation of the ERK pathway and by decreased receptor desensitization.

CDK2 and cyclin E knockout mice: lessons from breast cancer.

The recent discovery that murine embryos can develop normally in spite of ablation of either CDK2 or cyclin E challenges the previously held dogma that cyclin E-CDK2 activity is strictly required for the cell-division cycle. However, genetic, cellular, biochemical and clinical evidence correlate aberrant cyclin E expression with tumorigenesis and poor patient prognosis, particularly in breast cancer. Thus cyclin E is a crucial regulator of estrogen-mediated growth signaling in breast tissue and, in spite of the apparent dispensability of cyclin E-CDK2 in development, the relationship between cyclin E and the development of breast cancer is convincing.

Alpha-thrombin-mediated phosphatidylinositol 3-kinase activation through release of Gbetagamma dimers from Galphaq and Galphai2.

Chinese hamster embryonic fibroblasts (IIC9 cells) express the Galpha subunits Galphas, Galphai2, Galphai3, Galphao, Galpha(q/11), and Galpha13. Consistent with reports in other cell types, alpha-thrombin stimulates a subset of the expressed G proteins in IIC9 cells, namely Gi2, G13, and Gq as measured by an in vitro membrane [35S]guanosine 5'-O-(3-thio)triphosphate binding assay. Using specific Galpha peptides, which block coupling of G-protein receptors to selective G proteins, as well as dominant negative xanthine nucleotide-binding Galpha mutants, we show that activation of the phosphatidylinositol 3-kinase/Akt pathway is dependent on Gq and Gi2. To examine the role of the two G proteins, we examined the events upstream of PI 3-kinase. The activation of the PI 3-kinase/Akt pathway by alpha-thrombin in IIC9 cells is blocked by the expression of dominant negative Ras and beta-arrestin1 (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053, and Goel, R., Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2002) J. Biol. Chem. 277, 18640-18648), indicating a role for Ras and beta-arrestin1. Interestingly, inhibition of Gi2 and Gq activation blocks Ras activation and beta-arrestin1 membrane translocation, respectively. Furthermore, expression of the Gbetagamma sequestrant, alpha-transducin, inhibits both Ras activation and membrane translocation of beta-arrestin1, suggesting that Gbetagamma dimers from Galphai2 and Galphaq activate different effectors to coordinately regulate the PI 3-kinase/Akt pathway.

Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression.

The activation of CDK2-cyclin E in late G1 phase has been shown to play a critical role in retinoblastoma protein (pRb) inactivation and G1-S phase progression of the cell cycle. The phosphatidylinositol 3-OH-kinase inhibitor LY294002 has been shown to block cyclin D1 accumulation, CDK4 activity and, thus, G1 progression in alpha-thrombin-stimulated IIC9 cells (Chinese hamster embryonic fibroblasts). Our previous results show that expression of cyclin E rescues S phase progression in alpha-thrombin-stimulated IIC9 cells treated with LY294002, arguing that cyclin E renders CDK4 activity dispensable for G1 progression. In this work we investigate the ability of alpha-thrombin-induced CDK2-cyclin E activity to inactivate pRb in the absence of prior CDK4-cyclin D1 activity. We report that in the absence of CDK4-cyclin D1 activity, CDK2-cyclin E phosphorylates pRb in vivo on at least one residue and abolishes pRb binding to E2F response elements. We also find that expression of cyclin E rescues E2F activation and cyclin A expression in cyclin D kinase-inhibited, alpha-thrombin-stimulated cells. Furthermore, the rescue of E2F activity, cyclin A expression, and DNA synthesis by expression of E can be blocked by the expression of either CDK2(D145N) or RbDeltaCDK, a constitutively active mutant of pRb. However, restoring four known cyclin E-CDK2 phosphorylation sites to RbDeltaCDK renders it susceptible to inactivation in late G1, as assayed by E2F activation, cyclin A expression, and S phase progression. These data indicate that CDK2-cyclin E, without prior CDK4-cyclin D activity, can phosphorylate and inactivate pRb, activate E2F, and induce DNA synthesis.

The ethanol metabolite, linolenic acid ethyl ester, stimulates mitogen-activated protein kinase and cyclin signaling in hepatic stellate cells.

Chronic ethanol consumption can result in hepatic fibrosis and cirrhosis. In addition to oxidative metabolism, ethanol can be metabolized by esterification with fatty acids to form fatty acid ethyl esters (FAEE) such as linolenic acid ethyl ester (LAEE). We have previously demonstrated that LAEE has promitogeinc and activating effects on hepatic stellate cells (HSC), but the mechanisms of these actions are not known. Intracellular signaling through MAP kinase pathways, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) can influence the activity of the transcription factor AP-1, while cell-cycle regulatory proteins such as cyclin E and cyclin-dependent kinase (CDK), play an important role in cell proliferation. In this study, we demonstrate that treatment of HSC with LAEE increases cyclin E expression and cyclin E/CDK2 activity, which may underlie the promitogenic effects of this compound. In addition, LAEE increases ERK and JNK activity, and these pathways play an important role in the activation of AP-1-dependent gene expression by LAEE. The stimulation of intracellular signaling pathways in HSC by this well-characterized ethanol metabolite may contribute to ethanol-induced hepatic fibrogenesis.

beta-Arrestin 1 couples thrombin to the rapid activation of the Akt pathway.

In a variety of cell types including chinese hamster embryonic fibroblasts (IIC9 cells), alpha-thrombin is a potent mitogen. Thrombin irreversibly activates Par 1, a member of the seven membrane-spanning superfamily of G protein-coupled receptors (GPCRs). This, in turn, activates several heterotrimeric G proteins and induces signaling pathways that are critical for cell cycle reentry and proliferation. In IIC9 cells, alpha-thrombin activates the phosphatidylinositol-3-OH kinase (PI 3-Kinase)/Akt pathway, which is essential for G1 cell cycle progression. At present the mechanism for activation and regulation of the PI 3-kinase/Akt pathway is not fully understood. My preliminary data demonstrates a role for beta-arrestin 1 in the regulation of alpha-thrombin-induced Akt activity. In addition to their importance in receptor down-regulation, beta-arrestins are now known to scaffold proteins involved in stimulating specific signaling pathways. My preliminary data show that equal to or precedes -thrombin activates a rapid Akt activity in a beta-arrestin 1-dependent manner in IIC9 cells.

alpha-Thrombin activates Akt via a nonreceptor tyrosine kinase in IIC9 cells.

Previous data from our laboratory show that PI 3-kinase is required for alpha-thrombin-stimulated G(1) progression in IIC9 cells. In IIC9 cells, PI 3-kinase acts downstream of Ras to activate Akt, in a pathway parallel to ERK1. Here we show that alpha-thrombin does not transactivate either the EGF receptor or the PDGF receptor as measured by tyrosine phosphorylation, suggesting that activation of PI 3-kinase by alpha-thrombin is not the result of an RTK. Interestingly, both genistein and PP1 block alpha-thrombin-stimulated Akt phosphorylation, suggesting the involvement of a member of the Src family of nonreceptor tyrosine kinases.