THE CHIMERIC PROTEIN, PEBP2-BETA CBF-BETA-SMMHC, DISORGANIZES CYTOPLASMIC STRESS FIBERS AND INHIBITS TRANSCRIPTIONAL ACTIVATION/

The chromosomal inversion 16(p13;q22) associated with human acute myel oid leukemia generates the chimeric PEBP2 beta/CBF beta-SMMHC gene. Th e PEBP2 beta/CBF beta portion of the chimeric polypeptide harbors most of the amino acid sequence of the PEBP2 beta/CBF beta protein, the no n-DNA. binding subunit of the heterodimeric transcription factor, PEBP 2/CBF, whereas the SMMHC portion of the chimera consists of the rod do main of the smooth muscle myosin heavy chain molecule. In this study w e examined the subcellular localization of the chimeric protein land i ts effect both on stress fibers and transcriptional activation by tran sfecting cDNA into tissue culture cells. The localization of the chime ra was investigated by immunocytochemical staining of cells and was fo und to be both cytoplasmic and nuclear, One aspect of the effect of ex pression of the chimera was a drastic alteration of cell morphology, T he cells appeared elongated and possessed long cytoplasmic processes. Double fluorescent labeling revealed disorganization of the stress fib ers and an altered F-actin staining pattern in the transfected cells. Studies using a deletion mutant showed that both the PEBP2 beta/CBF be ta and SMMHC domains are necessary for the induction of the morphologi cal alteration, A significant proportion of the chimeric protein was r etained in the cytoskeleton after detergent extraction of the cells an d could be recuperated as a membrane fraction, suggesting that this is one of the probable sites of action of the PEBP2 beta/CBF beta-SMMHC protein. Another effect of the chimeric protein was inhibition of tran scriptional activation dependent on the PEBP2/CBF binding DNA sequence . However, deregulation of PEBP2/CBF site dependent transcription by i tself was not sufficient to induce cell morphological changes. Taken t ogether, these results indicate that the PEBP2 beta/CBF beta-SMMHC chi meric protein acts at two levels, at the level of stress fiber organiz ation and at the level of transcriptional activation. We suggest that the action of PEBP2 beta/CBF beta-SMMHC depends to a great extent on w hether it is located in the cytoplasm or in the nucleus.