Fanconi anemia (FA) 1 is a genetic disease of cancer susceptibility marked by congenital defects, bone marrow failure, and myeloid leukemia (1-4). To date at least seven complementation groups have been defined (5, 6). Six genes accounting for six groups have been cloned (7-15). However, all the gene products resemble no known proteins and have no identifiable functional protein motif.Cells derived from patients with the disease exhibit characteristic hypersensitivity caused by DNA cross-linking agents and generalized decreased survival (16 -20). In addition, a well described G 2 phase cell cycle delay has also been described that is thought to be secondary to a defective S or G 2 checkpoint (21-23). However, no defined biochemical mechanism for this hypersensitivity has been elucidated. Patient and cellular phenotypes across all the complementation groups are similar, suggesting an interrelatedness or cooperativity between the FA proteins.This cooperativity has been borne out by work we have done in showing binding of FancA and FancC in a protein complex in both nucleus and cytoplasm (24 -26). Recent work has found the FancG and FancF proteins in the complex as well (27)(28)(29)(30). A large complex is suggested by our recent work 2 , and binding does not occur in any of the complementation groups except the FA-D group.One clue to FA function lies in the study of the FancA protein, which contains a classic bipartite nuclear localization signal and is phosphorylated. FancA nuclear localization, phosphorylation, and binding to FancC are abolished in all complementation groups except the FA-D group (24 -26, 29). This suggests that a nuclear event is critical to the normal function of the FA proteins, and the aberrant protein in the FA-D group may have a role downstream of the FA complex in the nucleus.To date, other than the alteration of binding, nuclear localization, and FancA phosphorylation in mutant FA complementation groups, no other consistent biochemical change has been described, although some have described variations in FancC levels in the cell cycle (31,32). In our study, we have found that the FA proteins not only reside in the nucleus but also are closely associated with the nuclear matrix and chromatin.The nuclear matrix is a loose mechanical framework of proteins that has also been implicated in enzymatic activities in the regulation of transcription, replication, and DNA repair. The nuclear matrix is intimately associated with chromatin. BRG and brm, the human swi-snf homologues, are examples of nuclear matrix proteins involved in transcriptional regulation that change phosphorylation state, shift to chromatin, and become distinct from the nuclear matrix during mitosis (33-35). Immunofluorescence of FancA reveals a similar shift away from the condensed chromosomes of mitosis, suggesting that they also interact with chromatin and the nuclear matrix. Our studies demonstrate that the FA proteins associate with chromatin and the nuclear matrix in an inducible fashion. MATERIALS AND METHODSCell Cultu...
Fanconi anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and high incidence of leukemia and solid tumors. Eight genes have been cloned, with the accompanying protein products participating in at least two complexes, which appear to be functionally dependent upon one another. Previous studies have described chromatin localization of the FA core complex, except at mitosis, which is associated with phosphorylation of the FANCG protein (F. Qiao, A. Moss, and G. M. Kupfer, J. Biol. Chem. 276:23391-23396, 2001). The phosphorylation of FANCG at serine 7 by using mass spectrometry was previously mapped. The purpose of this study was to map the phosphorylation sites of FANCG at mitosis and to assess their functional importance. Reasoning that a potential kinase might be cdc2, which was previously reported to bind to FANCC, we showed that cdc2 chiefly phosphorylated a 14-kDa fragment of the C-terminal half of FANCG. Mass spectrometry analysis demonstrated that this fragment contains amino acids 374 to 504. Kinase motif analysis demonstrated that three amino acids in this fragment were leading candidates for phosphorylation. By using PCR-directed in vitro mutagenesis we mutated S383, S387, and T487 to alanine. Mutation of S383 and S387 abolished the phosphorylation of FANCG at mitosis. These results were confirmed by use of phosphospecific antibodies directed against phosphoserine 383 and phosphoserine 387. Furthermore, the ability to correct FA-G mutant cells of human or hamster (where S383 and S387 are conserved) origin was also impaired by these mutations, demonstrating the functional importance of these amino acids. S387A mutant abolished FANCG fusion protein phosphorylation by cdc2. The FA pathway, of which FANCG is a part, is highly regulated by a series of phosphorylation steps that are important to its overall function.
Summary. Uterine samples were either rapidly frozen in liquid nitrogen or placed in Bouin's fixative. A commercial primary polyclonal antibody made in rabbits against human recombinant basic fibroblast growth factor (bFGF) was used. Western blot analysis indicated that the antibody was specific for bFGF and did not react with acidic FGF. The primary antibody was followed by either goat anti-rabbit immunoglobulin G (IgG) conjugated to the fluorescent phycobiliprotein tracer phycoerythrin or biotinylated goat anti-rabbit IgG and a biotin\p=n-\avidin\p=n-\peroxidase complex. Specificity controls using adjacent sections were carried out by (i) substituting normal rabbit sera for the primary antisera, (ii) omitting the primary antisera or (iii) extracting sections with NaCl (2 mol l \ m=-\ 1) prior to the immunochemical procedures. No binding of the antibody was observed with any of the specificity control sections. The connective tissue stroma and the basal lamina associated with uterine glandular and surface epithelial layers were positive for bFGF. Localization was not observed within surface or glandular epithelial cells. The basal lamina and endothelial cells associated with blood vessels within the uterus and the smooth muscle cells of the myometrium were positive for bFGF. There were no differences in uterine localization patterns or intensity during the oestrous cycle or after ovariectomy and steroid hormone supplementation. These studies demonstrate the specific localization of bFGF within the mouse uterus.
While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds speci®cally to the a subunits of Gs (stimulatory GTP-binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitteroccupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the b-adrenergic receptor. Although 5-guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulinGppNHp) activated adenylyl cyclase with an EC 50 of 30 nM. Desensitization of b-adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin-GppNHp to activate Gs and adenylyl cyclase. When the photoaf®nity GTP analog, azidoanilido GTP (AAGTP; P3(4-azidoanilido)-P1-5'-GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by Gas in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to Gas, activating the latter species. Similar preferential' activation of Gas by tubulin-AAGTP versus the free nucleotide was seen using puri®ed components. Thus, membrane-associated tubulin may serve to activate Gas, independent of signals not normally coupled to that protein.Tubulin may act as an agent to link a variety of membraneassociated signalling systems.
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