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Results for “Checkpoint Kinase 1”

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PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗

PubMed2001

p53, CHK2, and CHK1 genes in Finnish families with Li-Fraumeni syndrome: further evidence of CHK2 in inherited cancer predisposition.

Germ-line mutations in the p53 gene predispose individuals to Li-Fraumeni syndrome (LFS). The cell cycle checkpoint kinases CHK1 and CHK2 act upstream of p53 in DNA damage responses, and recently rare germ-line mutations in CHK2 were reported in LFS families. We have analyzed CHK1, CHK2, and p53 genes for mutations in 44 Finnish families with LFS, Li-Fraumeni-like syndrome, or families phenotypically suggestive of LFS with conformation-sensitive gel electrophoresis. Five different disease-causing mutations were observed in 7 families (7 of 44 families; 15.9%): 4 in the p53 gene (5 of 44 families; 11.4%) and 1 in the CHK2 gene (2 of 44 families; 4.5%). Interestingly, the other CHK2-mutation carrier also has a mutation in the MSH6 gene. The cancer phenotype in the CHK2-families was not characteristic of LFS, and may indicate variable phenotypic expression in the rare families with CHK2 mutations. No mutations in the CHK1 gene were identified. Additional work is necessary to completely unravel the molecular background of LFS.

Adult↗

PubMed2005

Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1.

Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2005

Ablation of the spindle assembly checkpoint by a compound targeting Mps1.

The spindle assembly checkpoint ensures accurate chromosome segregation by delaying anaphase initiation until all chromosomes are properly attached to the mitotic spindle. Here, we show that the previously reported c-Jun amino-terminal kinase (JNK) inhibitor SP600125 effectively disrupts spindle checkpoint function in a JNK-independent fashion. SP600125 potently inhibits activity of the mitotic checkpoint kinase monopolar spindle 1 (Mps1) in vitro and triggers efficient progression through a mitotic arrest imposed by spindle poisons. Importantly, expression of an Mps1 mutant protein refractory to SP600125-mediated inhibition restores spindle checkpoint function in the presence of SP600125, showing that its mitotic phenotype is induced by Mps1 inhibition in vivo. Remarkably, primary human cells are largely resistant to the checkpoint-inactivating action of SP600125, suggesting the existence of Mps1-independent checkpoint pathways that are compromised in tumour cells.

Amino Acid Sequence↗

PubMed2006

Claspin: timing the cell cycle arrest when the genome is damaged.

DNA damage checkpoints maintain genomic integrity by delaying cell cycle progression in response to genotoxic stress and stalled replication forks. One central pathway in the checkpoint response is the ATR-Chk1 pathway, in which, upon DNA damage, ATR phosphorylates and activates the effector kinase Chk1. This process depends on the adaptor protein Claspin that bridges ATR and Chk1. Once the damage is repaired, this pathway must somehow be switched off to allow the cell to continue the cell division process, an event known as checkpoint recovery. Polo-like kinase 1 (Plk1) plays a central role during checkpoint recovery. Interestingly, the Xenopus homologue of Plk1, Plx1, is able to bind and phosphorylate Claspin, releasing it from DNA and thereby contributing to Chk1 inactivation. Moreover, it was recently demonstrated that Claspin levels are controlled by proteasomal degradation, and this is regulated by Plk1. Importantly, Plk1-mediated proteosomal degradation of Claspin appears to be essential for check-point recovery. Here we review these recent findings and discuss the mechanisms of checkpoint regulation by Claspin.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Metaphase arrest by cyclin E-Cdk2 requires the spindle-checkpoint kinase Mps1.

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest. Here, we demonstrate that metaphase arrest with cyclin E-Cdk2, but not with Mos, requires the spindle-checkpoint kinase monopolar spindles 1 (Mps1), a cyclin E-Cdk2 target that is also implicated in centrosome duplication. xMps1 is synthesized and activated during oocyte maturation and inactivated upon CSF release. In egg extracts, CSF release by calcium was inhibited by constitutively active cyclin E-Cdk2 and delayed by wild-type xMps1. Ablation of cyclin E by antisense oligonucleotides blocked accumulation of xMps1, suggesting that cyclin E-Cdk2 controls Mps1 levels. During meiosis II, activated cyclin E-Cdk2 significantly inhibited the APC/C even in the absence of the Mos-MAPK pathway, but this inhibition was not sufficient to suppress S phase between meiosis I and II. These results uniquely place xMps1 downstream of cyclin E-Cdk2 in mediating a pathway of APC/C inhibition and metaphase arrest.

Animals↗

PubMed2000

Chromosome instability in fibroblasts derived from Li-Fraumeni syndrome families without TP53 mutations.

The mean in vitro lifespan of dermal fibroblast strains derived from cancer-affected individuals belonging to families conforming to the classical Li-Fraumeni-syndrome or the Li-Fraumeni-like syndrome (LF strains), but in whom no TP53 mutation has been found, was not significantly different to that of normal strains. This was in contrast to LF strains that carry TP53 mutations. Cytogenetic observations of numerical and structural chromosome abnormalities were made on Giemsa stained metaphases prepared at different times during the lifespan of strains. Five strains from different LF families showed significantly increased frequencies of abnormal cells during the last 10% of their lifetime compared with seven normal strains and three other LF strains fell outside the normal range but did not reach significance. Two LF strains fell within the normal range indicating heterogeneity of the phenotype in this subset of LF fibroblasts. Numerical aberrations were the major aberration type observed. These observations of genetic instability are similar, but generally less strongly expressed, to those seen in LF strains with TP53 mutations. The basis for genetic instability in LF strains without TP53 mutations is not known, but appears not to involve defects in either the G(1)checkpoint or the checkpoint kinase hChk2.

Adolescent↗

PubMed2004

Roles of polo-like kinase 1 in the assembly of functional mitotic spindles.

BACKGROUND: The stable association of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation. RESULTS: We have analyzed Plk1 functions in synchronized mammalian cells by RNA interference (RNAi). Plk1-depleted cells enter mitosis after a short delay, accumulate in a preanaphase state, and subsequently often die by apoptosis. Spindles in Plk1-depleted cells lack focused poles and are not associated with centrosomes. Chromosomes attach to these spindles, but the checkpoint proteins Mad2, BubR1, and CENP-E are enriched at many kinetochores. When Plk1-depleted cells are treated with the Aurora B inhibitor Hesperadin, which silences the spindle checkpoint by stabilizing microtubule-kinetochore interactions, cells degrade APC/C substrates and exit mitosis without chromosome segregation and cytokinesis. Experiments with monopolar spindles that are induced by the kinesin inhibitor Monastrol indicate that Plk1 is required for the assembly of spindles that are able to generate poleward pulling forces. CONCLUSIONS: Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2006

Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint.

In the presence of unattached/weakly attached kinetochores, the spindle assembly checkpoint (SAC) delays exit from mitosis by preventing the anaphase-promoting complex (APC)-mediated proteolysis of cyclin B, a regulatory subunit of cyclin-dependent kinase 1 (Cdk1). Like all checkpoints, the SAC does not arrest cells permanently, and escape from mitosis in the presence of an unsatisfied SAC requires that cyclin B/Cdk1 activity be inhibited. In yeast , and likely Drosophila, this occurs through an "adaptation" process involving an inhibitory phosphorylation on Cdk1 and/or activation of a cyclin-dependent kinase inhibitor (Cdki). The mechanism that allows vertebrate cells to escape mitosis when the SAC cannot be satisfied is unknown. To explore this issue, we conducted fluorescence microscopy studies on rat kangaroo (PtK) and human (RPE1) cells dividing in the presence of nocodazole. We find that in the absence of microtubules (MTs), escape from mitosis occurs in the presence of an active SAC and requires cyclin B destruction. We also find that cyclin B is progressively destroyed during the block by a proteasome-dependent mechanism. Thus, vertebrate cells do not adapt to the SAC. Rather, our data suggest that in normal cells, the SAC cannot prevent a slow but continuous degradation of cyclin B that ultimately drives the cell out of mitosis.

Animals↗

PubMed2003

Negative regulation of mitotic promoting factor by the checkpoint kinase chk1 in simian virus 40 lytic infection.

Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G(1)/S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G(1)/S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G(1)/S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G(2). Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.

Alkaloids↗

PubMed2004

The oncogenic serine/threonine kinase Pim-1 phosphorylates and inhibits the activity of Cdc25C-associated kinase 1 (C-TAK1): a novel role for Pim-1 at the G2/M cell cycle checkpoint.

The Pim-1 oncogene encodes a serine-threonine kinase that relays signals from cytokine receptors and contributes to the formation of lymphoid tumors when expressed at high levels. Here we show that the protein kinase Cdc25 C-associated kinase 1 (C-TAK1) is a binding partner and a substrate of Pim-1. A physical interaction of Pim-1 and C-TAK1 could be shown biochemically and in yeast two-hybrid assays. Immunofluorescence experiments suggested that Pim-1.C-TAK1 complexes are predominantly cytoplasmic. When transiently transfected, Pim-1 was also found in the nucleus and could recruit C-TAK1 to this compartment. Both Pim-1 and C-TAK1 underwent autophosphorylation, but only Pim-1 was able to phosphorylate C-TAK1 but not vice versa. Mass spectrometry analysis of C-TAK1 suggested that the sites of autophosphorylation and Pim-1-mediated phosphorylation are distinct and not overlapping. Phosphorylation by Pim-1 decreased C-TAK1 kinase activity significantly, in particular its ability to phosphorylate and inactivate Cdc25C, a protein that actively promotes cell cycle progression at the G(2)/M phase. Hence our findings directly suggest a novel role for Pim-1 as a positive regulator at the G(2)/M transition of the cell cycle.

Animals↗

PubMed2002

A discrete cell cycle checkpoint in late G(1) that is cytoskeleton-dependent and MAP kinase (Erk)-independent.

Cell spreading on extracellular matrix and associated changes in the actin cytoskeleton (CSK) are necessary for progression through G(1) and entry into S phase of the cell cycle. Pharmacological disruption of CSK integrity inhibits early mitogenic signaling to the extracellular signal-regulated kinase (Erk) subfamily of the mitogen-activated protein kinases (MAPKs) and arrests the cell cycle in G(1). Here we show that this block of G(1) progression is not simply a consequence of inhibition of the MAPK/Erk pathway but instead it reveals the existence of a discrete CSK-sensitive checkpoint. Use of PD98059 to inhibit MAPK/Erk and cytochalasin D (Cyto D) to disrupt the actin CSK at progressive time points in G(1) revealed that the requirement for MAPK/Erk activation lasts only to mid-G(1), while the actin CSK must remain intact up to late G(1) restriction point, R, in order for capillary endothelial cells to enter S phase. Additional analysis using Cyto D pulses defined a narrow time window of 3 h just prior to R in which CSK integrity was shown to be critical for the G(1)/S transition. Cyto D treatment led to down-regulation of cyclin D1 protein and accumulation of the cdk inhibitor, p27(Kip1), independent of cell cycle phase, suggesting that these changes resulted directly from CSK disruption rather than from a general cell cycle block. Together, these data indicate the existence of a distinct time window in late G(1) in which signals elicited by the CSK act independently of early MAPK/Erk signals to drive the cell cycle machinery through the G(1)/S boundary and, hence, promote cell growth.

Actin Cytoskeleton↗

PubMed1998

Ectopic expression of cdc2/cdc28 kinase subunit Homo sapiens 1 uncouples cyclin B metabolism from the mitotic spindle cell cycle checkpoint.

Primary human fibroblasts arrest growth in response to the inhibition of mitosis by mitotic spindle-depolymerizing drugs. We show that the mechanism of mitotic arrest is transient and implicates a decrease in the expression of cdc2/cdc28 kinase subunit Homo sapiens 1 (CKsHs1) and a delay in the metabolism of cyclin B. Primary human fibroblasts infected with a retroviral vector that drives the expression of a mutant p53 protein failed to downregulate CKsHs1 expression, degraded cyclin B despite the absence of chromosomal segregation, and underwent DNA endoreduplication. In addition, ectopic expression of CKsHs1 interfered with the control of cyclin B metabolism by the mitotic spindle cell cycle checkpoint and resulted in a higher tendency to undergo DNA endoreduplication. These results demonstrate that an altered regulation of CKsHs1 and cyclin B in cells that carry mutant p53 undermines the mitotic spindle cell cycle checkpoint and facilitates the development of aneuploidy. These data may contribute to the understanding of the origin of heteroploidy in mutant p53 cells.

CDC2 Protein Kinase↗

PubMed2006

Regulation of Polo-like kinase 1 by DNA damage in mitosis. Inhibition of mitotic PLK-1 by protein phosphatase 2A.

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Polo-like kinase 1 (Plk1) is an important regulator of several events during mitosis. In addition to Plk1 functions in cell cycle, Plk1 is involved in DNA damage check-point in G2 phase. Normally, ataxia telangiectasia-mutated kinase (ATM) is a key enzyme involved in G2 phase cell cycle arrest following DNA damage, and inhibition of Plk1 by DNA damage during G2 occurs in a ATM/ATR-dependent manner. However, it is still unclear how Plk1 is regulated in response to DNA damage in mitosis in which Plk1 is already activated. Here, we show that treatment of mitotic cells with doxorubicin and gamma-irradiation inhibits Plk1 activity through dephosphorylation of Plk1, and cells were arrested in G2 phase. Treatments of the phosphatase inhibitors and siRNA experiments suggested that PP2A pathway might be involved in regulating mitotic Plk1 activity in mitotic DNA damage. Finally, we propose a novel pathway, which is connected between ATM/ATR/Chk and protein phosphatase-Plk1 in DNA damage response in mitosis.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase.

S(N)1-type alkylating agents represent an important class of chemotherapeutics, but the molecular mechanisms underlying their cytotoxicity are unknown. Thus, although these substances modify predominantly purine nitrogen atoms, their toxicity appears to result from the processing of O(6)-methylguanine ((6Me)G)-containing mispairs by the mismatch repair (MMR) system, because cells with defective MMR are highly resistant to killing by these agents. In an attempt to understand the role of the MMR system in the molecular transactions underlying the toxicity of alkylating agents, we studied the response of human MMR-proficient and MMR-deficient cells to low concentrations of the prototypic methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We now show that MNNG treatment induced a cell cycle arrest that was absolutely dependent on functional MMR. Unusually, the cells arrested only in the second G(2) phase after treatment. Downstream targets of both ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases were modified, but only the ablation of ATR, or the inhibition of CHK1, attenuated the arrest. The checkpoint activation was accompanied by the formation of nuclear foci containing the signaling and repair proteins ATR, the S(*)/T(*)Q substrate, gamma-H2AX, and replication protein A (RPA). The persistence of these foci implied that they may represent sites of irreparable damage.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2000

Loss of G(1)/S checkpoint in human immunodeficiency virus type 1-infected cells is associated with a lack of cyclin-dependent kinase inhibitor p21/Waf1.

Productive high-titer infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile reverse transcripts and lack of viral progeny formation. An interplay between Tat and p53 has previously been reported, where Tat inhibited the transcription of the p53 gene, which may aid in the development of AIDS-related malignancies, and p53 expression inhibited HIV-1 long terminal repeat transcription. Here, by using a well-defined and -characterized stress signal, gamma irradiation, we find that upon gamma irradiation, HIV-1-infected cells lose their G(1)/S checkpoints, enter the S phase inappropriately, and eventually apoptose. The loss of the G(1)/S checkpoint is associated with a loss of p21/Waf1 protein and increased activity of a major G(1)/S kinase, namely, cyclin E/cdk2. The p21/Waf1 protein, a known cyclin-dependent kinase inhibitor, interacts with the cdk2/cyclin E complex and inhibits progression of cells into S phase. We find that loss of the G(1)/S checkpoint in HIV-1-infected cells may in part be due to Tat's ability to bind p53 (a known activator of the p21/Waf1 promoter) and sequester its transactivation activity, as seen in both in vivo and in vitro transcription assays. The loss of p21/Waf1 in HIV-1-infected cells was specific to p21/Waf1 and did not occur with other KIP family members, such as p27 (KIP1) and p57 (KIP2). Finally, the advantage of a loss of the G(1)/S checkpoint for HIV-1 per se may be that it pushes the host cell into the S phase, which may then allow subsequent virus-associated processes, such as RNA splicing, transport, translation, and packaging of virion-specific genes, to occur.

CDC2-CDC28 Kinases↗

PubMed2005

Polo-like kinase 1 creates the tension-sensing 3F3/2 phosphoepitope and modulates the association of spindle-checkpoint proteins at kinetochores.

BACKGROUND: In mitosis, a mechanochemical system recognizes tension that is generated by bipolar microtubule attachment to sister kinetochores. This is translated into multiple outputs including the stabilization of microtubule attachments, changes in kinetochore protein dynamics, and the silencing of the spindle checkpoint. How kinetochores sense tension and translate this into various signals represent critical unanswered questions. The kinetochores of chromosomes not under tension are specifically phosphorylated at an epitope recognized by the 3F3/2 monoclonal antibody. Determining the kinase that generates the 3F3/2 phosphoepitope at kinetochores should reveal an important component of this system that regulates mitotic progression. RESULTS: We demonstrate that Polo-like kinase 1 (Plk1) creates the 3F3/2 phosphoepitope on mitotic kinetochores. In a permeabilized in vitro cell system, the depletion of Xenopus Plk1 from M phase extract leads to the loss of 3F3/2 kinase activity. Purified recombinant Plk1 is sufficient to generate the 3F3/2 phosphoepitope in this system. Using siRNA, we show that the reduction of Plk1 protein levels significantly diminishes 3F3/2 phosphoepitope expression at kinetochores. The consensus phosphorylation sites of Plk1 show strong similarity to the 3F3/2 phosphoepitope sequence determined by phosphopeptide mapping. The inhibition of Plk1 by siRNA alters the normal kinetochore association of Mad2, Cenp-E, Hec1/Ndc80, Spc24, and Cdc20 and induces a spindle-checkpoint-mediated mitotic arrest. CONCLUSIONS: Plk1 generates the 3F3/2 phosphoepitope at kinetochores that are not under tension and contributes to the normal kinetochore association of several key proteins important in checkpoint signaling. Mechanical tension regulates Plk1 accumulation at kinetochores and possibly its kinase activity.

Amino Acid Motifs↗

PubMed2004

Chronic HIV type 1 infection down-regulates expression of DAP kinase and p19ARF-p53 checkpoint and is associated with resistance to CD95-mediated apoptosis in HUT78 T cells.

Death-associated protein kinase (DAP kinase) is a proapoptotic serine/threonine kinase that has been shown to play a role in both death-receptor signaling and mitochondrial signaling pathways of apoptosis. DAP kinase activates the p19ARF-p53 apoptotic checkpoint. In this study we report that the expression of DAP kinase, p19ARF, p53, and p21WAF1 was significantly down-regulated in the chronically HIV-1SF2-infected HUT78 T cells (HUT78/HIV-1SF2) as compared to uninfected HUT78 cells. An increased proportion of HUT78/HIV1SF2 cells was detected in S phase and a decreased proportion in G0/G1 phase indicating that more HUT78/HIV1SF2 cells progressed through the G1/S transition. Furthermore, HUT78/HIV-1SF2 cells showed increased resistance to CD95-mediated apoptosis as compared to HIV-1SF2-uninfected HUT78 cells and activation of caspase-3, -8, and -9 was significantly reduced in HUT78/HIV-1SF2 cells. These data suggest that down-regulation of DAP kinase and downstream signaling factors may be one of the mechanism that HIV-1 may employ to protect the infected host cells from cell death and to allow persistent HIV-1 replication.

Apoptosis↗