Myeloid Leukemia Factor 1 Associates with a Novel Heterogeneous Nuclear Ribonucleoprotein U-like Molecule

Abstract

Myeloid leukemia factor 1 (MLF1) is an oncoprotein associated with hemopoietic lineage commitment and acute myeloid leukemia. Here we show that Mlf1 associated with a novel binding partner, Mlf1-associated nuclear protein (Manp), a new heterogenous nuclear ribonucleoprotein (hnRNP) family member, related to hnRNP-U. Manp localized exclusively in the nucleus and could redirect Mlf1 from the cytoplasm into the nucleus. The nuclear content of Mlf1 was also regulated by 14-3-3 binding to a canonical 14-3-3 binding motif within the N terminus of Mlf1. Significantly Mlf1 contains a functional nuclear export signal and localized primarily to the nuclei of hemopoietic cells. Mlf1 was capable of binding DNA, and microarray analysis revealed that it affected the expression of several genes, including transcription factors. In summary, this study reveals that Mlf1 translocates between nucleus and cytoplasm, associates with a novel hnRNP, and influences gene expression. Myeloid leukemia factor 1 (MLF1)4 is a gene involved in hemopoietic lineage commitment and acute myeloid leukemia (1, 2). The gene was originally identified in a (3;5) translocation associated with acute myeloid leukemia that generated an abnormal fusion with nucleophosmin (NPM), i.e. NPM-MLF1 (1). The t(3;5) is found mainly in the M6 erythroleukemic subtype of acute myeloid leukemia (3). Significantly the fusion protein NPM-MLF1 is almost exclusively nuclear (1).The murine orthologue of MLF1 was isolated independently as hemopoietic lineage switch 7 (Hls7), a gene up-regulated when J2E erythroleukemic cells spontaneously developed a monoblastoid appearance (2, 4). Upon reintroduction into parental J2E cells, Hls7/Mlf1 induced a dramatic phenotypic change as the proerythroblastoid cells now displayed an immature, blast-like appearance (2). Hls7/Mlf1 has also been shown to influence hemopoietic lineage commitment by altering the balance between erythroid and myeloid progenitors (2). In contrast with the nuclear localization of the NPM-MLF1 fusion protein, Mlf1 is located primarily in the cytoplasm with some punctate nuclear staining (1, 2).Apart from a classical 14-3-3 binding site, Mlf1 has no recognizable motifs (1, 2, 5, 6). Therefore, yeast two-hybrid screens have been conducted to identify partner proteins that may elucidate the function of Mlf1. One molecule shown to bind Mlf1 was Mlf1 adaptor molecule (Madm), an adaptor protein involved in nuclear-cytoplasmic shuttling (7). Another Mlf1-binding protein is Mlf1-interacting protein/KSHV latent nuclear antigen-interacting protein (Mlf1IP/KLIP1), a novel nuclear molecule that may function as a transcriptional regulator (8, 9). Interestingly in Drosophila, Mlf1 associates with DNA replication-related element binding factor (DREF) (5), a DNAbinding protein that regulates genes involved in proliferation, including E2f (10). It has been suggested that "the DREF system may occupy an intersection in growth and differentiation signaling pathways" (11).Recently we have shown that Mlf1 interferes with erythropoieti...

Subcellular localization of full-length human myeloid leukemia factor 1 (MLF1) is independent of 14-3-3 proteins

“…In contrast to the previous research on the mouse homologue [11], we found that there is no direct influence of 14-3-3 proteins on the nucleo-cytoplasmic distribution of human MLF1 in HEK293T, Cos-7 and K-562 cells. These results indicate a more complex regulation mechanism of human MLF1 than the one suggested for the mouse homologue and imply that the subcellular localization of human MLF1 is not directly 14-3-3-dependent.…”

“…Interestingly, although MLF1 localizes to the cytoplasm in non-hemopoietic cells [8], it preferentially resides in the nucleus of hemopoietic cells [8][9][10][11]. Beyond these reports, information on the physiological function of MLF1 is limited and is derived mostly from studies identifying MLF1 interaction partners, such as CSN3 [12], MLF1IP [13,14], MADM [15], Manp, and the 14-3-3 proteins [11,[5][6][7][8][9][10][11][12][13][14][15][16][17]]. 14-3-3 proteins are eukaryotic proteins of 25 to 30 kDa that influence a variety of physiological processes [18].…”

Identification of Novel Nuclear Localization Signals of Drosophila Myeloid Leukemia Factor

“…It has been reported recently that mammalian MLF1 protein contains both NLS and nuclear export signal (NES), and that MLF1 translocates between the nucleus and cytoplasm (Winteringham et al, 2006;Yoneda-Kato and Kato, 2008). In Drosophila, dMLF interacts with Su(fu) and consequently translocates Su(fu) from cytoplasm to nucleus (Martin-Lannerée et al, 2006).…”

“…It has been reported that four isoforms of the dMLF protein are present that are probably the result of alternative splicing and that two forms of dMLF (dMLFA and dMLFB) differ in their subcellular localizations (Martin-Lannerée et al, 2006). In addition, MLF1 has been reported to translocate between the nucleus and cytoplasm (Winteringham et al, 2006;Yoneda-Kato and Kato, 2008).…”

“…The human MLF1, was first found in the form of a fusion protein with nucleophosmin (NPM) generated by the t(3;5)(q25.1;q34) chromosomal translocation, which is associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) . It has been reported that MLF1 is associated with several proteins such as 14-3-3ζ, Madm, MLF1IP/KLIP1, Manp and CSN3, and that hMLF1 regulates the cell cycle via the CSN3/COP1 pathway (Hanissian et al, 2004;Lim et al, 2002;Winteringham et al, 2006;Yoneda-Kato et al, 2005), although the biochemical activity of MLF1 has not been well characterized.…”