The chimeric protein, PEBP2β/CBFβ-SMMHC, disorganizes cytoplasmic stress fibers and inhibits transcriptional activation

Abstract

The chromosomal inversion 16(p13;q22) associated with human acute myeloid leukemia generates the chimeric PEBP2b/CBFb-SMMHC gene. The PEBP2b/CBFb portion of the chimeric polypeptide harbors most of the amino acid sequence of the PEBP2b/CBFb protein, the non-DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF, whereas the SMMHC portion of the chimera consists of the rod domain of the smooth muscle myosin heavy chain molecule. In this study we examined the subcellular localization of the chimeric protein and its e ect both on stress ®bers and transcriptional activation by transfecting cDNA into tissue culture cells. The localization of the chimera was investigated by immunocytochemical staining of cells and was found to be both cytoplasmic and nuclear. One aspect of the e ect of expression of the chimera was a drastic alteration of cell morphology. The cells appeared elongated and possessed long cytoplasmic processes. Double¯uorescent labeling revealed disorganization of the stress ®bers and an altered F-actin staining pattern in the transfected cells. Studies using a deletion mutant showed that both the PEBP2b/CBFb and SMMHC domains are necessary for the induction of the morphological alteration. A signi®cant proportion of the chimeric protein was retained in the cytoskeleton after detergent extraction of the cells and could be recuperated as a membrane fraction, suggesting that this is one of the probable sites of action of the PEBP2b/ CBFb-SMMHC protein. Another e ect of the chimeric protein was inhibition of transcriptional activation dependent on the PEBP2/CBF binding DNA sequence. However, deregulation of PEBP2/CBF site dependent transcription by itself was not su cient to induce cell morphological changes. Taken together, these results indicate that the PEBP2b/CBFb-SMMHC chimeric protein acts at two levels, at the level of stress ®ber organization and at the level of transcriptional activation. We suggest that the action of PEBP2b/CBFb-SMMHC depends to a great extent on whether it is located in the cytoplasm or in the nucleus.

The PEBP2β/CBFβ-SMMHC chimeric protein is localized both in the cell membrane and nuclear subfractions of leukemic cells carrying chromosomal inversion 16

“…We previously reported that the transient expression of the PEBP2␤/CBF␤-SMMHC protein in the C2C12 myoblast cell line resulted in drastic morphological changes and the presence of protein in the cell membrane and nuclear subfractions. 23 We suggested the disorganization of the cytoplasmic stress fibers observed in the transfected C2C12 cells to be somehow related to the membrane localization of chimeric protein. The dual functions exerted by the membrane-and nuclear-located PEBP2␤/CBF␤-SMMHC protein that we proposed previously may be applicable to leukemic cells carrying inversion 16 and may provide a clue to unraveling the molecular mechanisms of myeloid leukemogenesis.…”

“…23 Briefly, the cells were suspended in a buffer containing 10 mM Hepes-KOH, pH 7.6, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell suspension was homogenized in a Dounce homogenizer and the mixture was centrifuged at 1000 g for 10 min at 4°C.…”

Indispensable role of the transcription factor PEBP2/CBF in angiogenic activity of a murine endothelial cell MSS31

“…The PEBP2b-MYH11 gene is observed in the inverted chromosome 16(p13;q22) associated with human acute myeloid leukemia and produces a chimeric protein of PEBP2b/CBFb and smooth muscle myosin heavy chain. This chimeric protein interacts with the runt domain of the a subunit and deregulates PEBP2/CBF in a dominant interfering manner (Liu et al, 1993Claxton et al, 1994;Cao et al, 1997;Tanaka et al, 1998). PEBP2b/ CBFb knock-out mice as well as PEBP2b-MYH11 knocked-in mice showed essentially the same phenotype as the AML1/PEBP2a/CBFa2 knock-out mice (Castilla et al, 1996;Sasaki et al, 1996;Wang et al, 1996b;Niki et al, 1997).…”

True

“…59,83,84 This may result from increased affinity of CBF␤-SMMHC for the cytoskeleton, compared with CBF␤, perhaps as a result of interaction of its SMMHC segment with cytoskeletal-associated non-muscle myosins. 85 CBF␤-SMMHC:CBF␣ complexes retain the ability to bind DNA. 77,81 Therefore, CBF␤-SMMHC might also interfere with CBF␣ trans-activation via local effects on promoter/enhancer transcription complexes.…”