(1/8836) Elevated DNA double strand breaks and apoptosis in the CNS of scid mutant mice.

Genetic approaches have provided evidence that DNA end-joining problems serve an essential role in neuronal survival during development of mammalian embryos. In the present study, we tested whether the DNA repair enzyme, DNA dependent protein kinase, plays an important role in the survival of cerebral cortical neurons in mice. DNA-PK is comprised of a DNA-binding subunit called Ku and a catalytic subunit called DNA-PKcs. In mice with the scid mutation, DNA-PKcs is truncated near the kinase domain, which causes loss of kinase activity. We compared the spatial and temporal aspects of neuronal cell death in scid versus isogenic wild-type embryos and found a significant increase in dying cells in scid mice, as assessed by nuclear changes, DNA fragmentation and caspase-3 activity. Additional biochemical and immunocytochemical studies indicated that of several DNA repair enzymes investigated, only PARP was increased in scid mice, possibly in response to elevated DNA strand breaks.  (+info)

(2/8836) Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade.

BACKGROUND: The AMP-activated protein kinase (AMPK) cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs) that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRAD alpha/beta and MO25 alpha/beta. RESULTS: We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRAD alpha and MO25 alpha, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRAD alpha/beta and MO25 alpha/beta activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRAD alpha or STRAD beta and MO25 alpha or MO25 beta are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1), but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. CONCLUSIONS: These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.  (+info)

(3/8836) Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA.

The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17-Rfc2-Rfc3-Rfc4-Rfc5) complex and an RHR heterotrimer (Rad1-Hus1-Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5' recessed ends whereas RFC preferred 3' recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5' recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.  (+info)

(4/8836) The Drosophila sterile-20 kinase slik controls cell proliferation and apoptosis during imaginal disc development.

Cell proliferation and programmed cell death are closely controlled during animal development. Proliferative stimuli generally also induce apoptosis, and anti-apoptotic factors are required to allow net cell proliferation. Genetic studies in Drosophila have led to identification of a number of genes that control both processes, providing new insights into the mechanisms that coordinate cell growth, proliferation, and death during development and that fail to do so in diseases of cell proliferation. We present evidence that the Drosophila Sterile-20 kinase Slik promotes cell proliferation and controls cell survival. At normal levels, Slik provides survival cues that prevent apoptosis. Cells deprived of Slik activity can grow, divide, and differentiate, but have an intrinsic survival defect and undergo apoptosis even under conditions in which they are not competing with normal cells for survival cues. Like some oncogenes, excess Slik activity stimulates cell proliferation, but this is compensated for by increased cell death. Tumor-like tissue overgrowth results when apoptosis is prevented. We present evidence that Slik acts via Raf, but not via the canonical ERK pathway. Activation of Raf can compensate for the lack of Slik and support cell survival, but activation of ERK cannot. We suggest that Slik mediates growth and survival cues to promote cell proliferation and control cell survival during Drosophila development.  (+info)

(5/8836) T cell receptor-independent basal signaling via Erk and Abl kinases suppresses RAG gene expression.

Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.  (+info)

(6/8836) Identification and characterization of human VCY2-interacting protein: VCY2IP-1, a microtubule-associated protein-like protein.

VCY2 is a testis-specific protein that locates in a frequently deleted azoospermia factor c region on chromosome Yq. Although its genomic structure has been characterized, the function of VCY2 is still unknown. To gain insight regarding the likely function of VCY2, we investigated the proteins that interact with VCY2 using the yeast two-hybrid system. We identified a novel VCY2 interaction partner, named VCY2IP-1, that encodes an open reading frame of 1059 amino acids. The amino acid sequence of VCY2IP-1 shows 59.3% and 41.9% homology to two human microtubule-associated proteins (MAPs), MAP1B and MAP1A, respectively. VCY2IP-1 has an extensive homology to the N-terminus and C-terminus regions of MAP1B and MAP1A, placing it within a large family of MAPs. We mapped VCY2IP-1 to chromosome 19p13.11. The VCY2IP-1 gene spans 15 kilobases (kb) and consists of seven exons. Northern blot analysis identified a single, intense band of approximately 3.2-kb VCY2IP-1 transcript, predominantly expressed in human testis. In situ hybridization of human testicular sections showed the localization of VCY2IP-1 transcripts in germ cells, and reverse transcription-polymerase chain reaction analysis demonstrated the presence of VCY2 and VCY2IP-1 transcripts in human ejaculated spermatozoa. Our expression data support the involvement of VCY2 and VCY2IP-1 in spermatogenesis. Based on the high homology of VCY2IP-1 with MAPs, we propose the involvement of VCY2 in the cytoskeletal network via interaction with VCY2IP-1.  (+info)

(7/8836) Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer.

The nonapeptide hormone oxytocin exerts many important biological functions, including uterine contractions during parturition and milk ejection during lactation. The manifold effects of oxytocin are mediated by a single oxytocin receptor (OTR) type, a member of the super-family of G-protein-coupled receptors. There is accumulating recent evidence that certain G-protein-coupled receptors exist in the form of oligomeric complexes. Here we demonstrate, using two different co-immunoprecipitation strategies as well as bioluminescence resonance energy transfer techniques, that the OTR is capable of forming oligomeric complexes in vivo and that these complexes exist at the cell surface membrane. The human OTR was N-terminally tagged with either a Myc or Flag epitope and transiently expressed in COS-7 cells. Cell lysates were immunoprecipitated using an anti-Flag antibody and analyzed by SDS-PAGE and Western blotting using an anti-Myc antibody, or vice versa. Either strategy provided evidence for the co-precipitation of Myc- or Flag-tagged OTR respectively. Biochemical characterization of OTR dimers showed that homodimer formation is not dependent on the establishment of disulfide bonds. The existence of OTR dimers and oligomers at the level of the cell surface was demonstrated by exposing intact living cells to an anti-Flag antibody and analyzing the immunoprecipitate by Western blotting with an anti-Myc antibody. This approach demonstrated furthermore that the presence of receptor oligomers at the cell surface is modulated by ligand in a time-dependent fashion. Finally, we obtained evidence that the OTR is forming oligomeric structures in intact living cells by observing the occurrence of bioluminescence resonance energy transfer in cells co-transfected with OTR constructs bearing at their C-terminus either a Renilla luciferase or the yellow fluorescent protein. Taken together, these data show that the OTR can form homodimers and oligomers in the cell model used and that these oligomers are present at the cell surface.  (+info)

(8/8836) Impaired renal clearance explains elevated troponin T fragments in hemodialysis patients.

BACKGROUND: Patients with severe renal dysfunction often have unexplained elevated serum concentrations of cardiac troponin T (cTnT). We investigated whether in vivo fragmentation of cTnT could explain these increases. METHODS AND RESULTS: cTnT, creatine kinase isoenzyme MB, and myoglobin serum concentrations were measured in all 63 dialysis patients of our in-hospital dialysis department. A highly sensitive immunoprecipitation assay, followed by electrophoresis and Western blotting, was used to extract and concentrate cTnT and its possible fragments from serum of these 63 hemodialysis patients. Although creatine kinase isoenzyme MB values excluded recent ischemic myocardial events in 55 of the 63 cases, cTnT fragments ranging in size from 8 to 25 kDa were present in the serum samples of all dialysis patients. CONCLUSIONS: cTnT is fragmented into molecules small enough to be cleared by the kidneys of healthy subjects. Impaired renal function causes accumulation of these cTnT fragments and is very likely the cause of the unexplained elevations of serum cTnT found in patients with severe renal failure.  (+info)