HOXA9 forms triple complexes with PBX2 and MEIS1 in myeloid cells.
(33/8966)
Aberrant activation of the HOX, MEIS, and PBX homeodomain protein families is associated with leukemias, and retrovirally driven coexpression of HOXA9 and MEIS1 is sufficient to induce myeloid leukemia in mice. Previous studies have demonstrated that HOX-9 and HOX-10 paralog proteins are unique among HOX homeodomain proteins in their capacity to form in vitro cooperative DNA binding complexes with either the PBX or MEIS protein. Furthermore, PBX and MEIS proteins have been shown to form in vivo heterodimeric DNA binding complexes with each other. We now show that in vitro DNA site selection for MEIS1 in the presence of HOXA9 and PBX yields a consensus PBX-HOXA9 site. MEIS1 enhances in vitro HOXA9-PBX protein complex formation in the absence of DNA and forms a trimeric electrophoretic mobility shift assay (EMSA) complex with these proteins on an oligonucleotide containing a PBX-HOXA9 site. Myeloid cell nuclear extracts produce EMSA complexes which appear to contain HOXA9, PBX2, and MEIS1, while immunoprecipitation of HOXA9 from these extracts results in coprecipitation of PBX2 and MEIS1. In myeloid cells, HOXA9, MEIS1, and PBX2 are all strongly expressed in the nucleus, where a portion of their signals are colocalized within nuclear speckles. However, cotransfection of HOXA9 and PBX2 with or without MEIS1 minimally influences transcription of a reporter gene containing multiple PBX-HOXA9 binding sites. Taken together, these data suggest that in myeloid leukemia cells MEIS1 forms trimeric complexes with PBX and HOXA9, which in turn can bind to consensus PBX-HOXA9 DNA targets. (+info)
The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle.
(34/8966)
The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100-150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability. (+info)
Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis.
(35/8966)
Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xL and Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xL dissociation from Bak occurred on exposure of Bak's NH2 terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death. (+info)
Activation of membrane-associated procaspase-3 is regulated by Bcl-2.
(36/8966)
The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2-inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases. (+info)
Stimulus-dependent translocation of kappa opioid receptors to the plasma membrane.
(37/8966)
We examined the cellular and subcellular distribution of the cloned kappa opioid receptor (KOR1) and its trafficking to the presynaptic plasma membrane in vasopressin magnocellular neurosecretory neurons. We used immunohistochemistry to show that KOR1 immunoreactivity (IR) colocalized with vasopressin-containing cell bodies, axons, and axon terminals within the posterior pituitary. Ultrastructural analysis revealed that a major fraction of KOR1-IR was associated with the membrane of peptide-containing large secretory vesicles. KOR1-IR was rarely associated with the plasma membrane in unstimulated nerve terminals within the posterior pituitary. A physiological stimulus (salt-loading) that elicits vasopressin release also caused KOR1-IR to translocate from these vesicles to the plasma membrane. After stimulation, there was a significant decrease in KOR1-IR associated with peptide-containing vesicles and a significant increase in KOR1-IR associated with the plasma membrane. This stimulus-dependent translocation of receptors to the presynaptic plasma membrane provides a novel mechanism for regulation of transmitter release. (+info)
Similarities between a dipeptide hydrolase from brush-border and cytosol fractions of guinea-pig intestinal mucosa.
(38/8966)
A dipeptide hydrolase from the brush border of guinea-pig intestinal mucosa was purified. The enzyme resembles another dipeptide hydrolase isolated from the cytosol fraction of intestinal mucosa. Studies on the binding of cytosol peptide hydrolase to brush-border membranes indicate that the enzyme found in the brush border may be a cytoplasmic contaminant. (+info)
Subcellular localization of oestrogen-induced uterine peroxidase.
(39/8966)
The distribution of oestrogen-induced peroxidase in the resuspended 8000g pellet of rat uterine homogenates was examined by centrifugation in a sucrose density gradient. Within 10h of treatment with oestradiol, peroxidase activity was found in a region devoid of catalase or urate oxidase (peroxisomal markers) which did not overlap the fractions containing succinate dehydrogenase (mitochondrial marker) or acid phosphatase (lysosomal marker). The induced uterine enzyme was localized in reticular membrane-bound vesicles with isopycnic density of 1.28g/ml from which it could be released by treatment with detergent. (+info)
Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis.
(40/8966)
It has been suggested that the ratio of Bcl-2 family proapoptotic proteins to antiapoptotic proteins determines the sensitivity of leukemic cells to apoptosis. However, it is believed that Bcl-2 family proteins exert their function on apoptosis only when they target to the mitochondrial outer membrane. The vinblastine-resistant T-lymphoblastic leukemic cell line CEM/VLB100 has increased sensitivity to tumor necrosis factor-alpha (TNF-alpha)-induced cytochrome c release, mitochondrial respiratory inhibition, and consequently apoptosis, compared with parental CEM cells. However, there was no difference between the two cell lines in the expression of Bcl-2 family proteins Bcl-2, Bcl-XL, Bcl-XS, Bad, and Bax at the whole cell level, as analyzed by Western blotting. Bcl-2 mainly located to mitochondria and light membrane as a membrane-bound protein, whereas Bcl-XL was located in both mitochondria and cytosol. Similar levels of both Bcl-2 and Bcl-XL were present in the resting mitochondria of the two cell lines. Although the proapoptotic proteins Bcl-XS, Bad, and Bax were mainly located in the cytosol, CEM/VLB100 mitochondria expressed higher levels of these proapoptotic proteins. Subcellular redistribution of the Bcl-2 family proteins was detected in a cell-free system by both Western blotting and flow cytometry after exposure to TNF-alpha. The levels of Bcl-2 family proteins were not altered at the whole cell level by TNF-alpha. However, after exposure to TNF-alpha, Bax, Bad, and Bcl-XS translocated from the cytosol to the mitochondria of both cell lines. An increase in Bcl-2 levels was observed in CEM mitochondria, which showed resistance to TNF-alpha-induced cytochrome c release. By contrast, decreased mitochondrial Bcl-2 was observed in CEM/VLB100 cells, which released cytochrome c from the mitochondria and underwent apoptosis as detected by fluorescence microscopy. We conclude that mitochondrial levels of Bcl-2 family proteins may determine the sensitivity of leukemic cells to apoptosis and that, furthermore, these levels may change rapidly after exposure of cells to toxic stimuli. (+info)