THE PML GENE ENCODES A PHOSPHOPROTEIN ASSOCIATED WITH THE NUCLEAR MATRIX

The t(15;17)(q22;q12) translocation is the cytogenetic hallmark of acu te promyelocytic leukemia (APL). The PML and retinoic acid receptor-al pha (RAR alpha) transcription factor genes are involved at the translo cation breakpoint. To elucidate the biologic function of PML, antipept ide antibody against PML protein was raised in rabbits. This antibody was able to detect a 90-kD PML protein and a 110-kD PML-RAR alpha fusi on protein by Western blotting and a nuclear speckled pattern in all c ell lines by immunofluorescent staining. In K562 and NIH/3T3 cells tra nsfected with a PML expression plasmid, we found PML to be associated with the nuclear matrix. Our results also showed that PML is a phospho protein. A weak signal was detected in a Western blot containing the i mmunoprecipitated PML protein using the phosphotyrosine-specific monoc lonal antibody. Therefore, at least one of the sites was phosphorylate d by a tyrosine kinase. From our analysis of the phosphoamino acids of the PML protein by complete hydrolysis and thin-layer chromatography, we concluded that both tyrosine and serine residues of PML are phosph orylated. To investigate whether expression of the PML protein is cell -cycle related, HeLa cells synchronized at various phases of the cell cycle were analyzed by immunofluorescence staining and confocal micros copy for PML expression. We found that PML was expressed at a lower le vel in S, G2, and M phases and at a significantly higher level in G1 p hase. Our study showed that PML has many similar properties compared w ith the tumor suppressor, eg, Rb. These findings further support the i mportant role of PML in APL pathogenesis. (C) 1995 by The American Soc iety of Hematology.