Mechanisms of action of differentiation inducers: Detection of inducer binding protein(s) in murine erythroleukemia cells

Abstract

We have shown previously that murine erythroleukemia (MEL) and human neuroectodermal RD/TE-671 cells are induced to differentiate by ureido derivatives of pyridine (UDPs) and may contain inducer binding protein(s). In the present study, we prepared radiolabeled [3H]UDP {2-(3-ethylureido)-6-[3H]-acetylamino-pyridine) as ligand and investigated whether it interacts selectively with novel binding proteins. MEL and RD/TE-671 cells, incubated with the inducer [3H]UDP and subsequently fractionated, yielded a radiolabeled postmitochondrial soluble fraction containing the [3H]UDP-protein complex. We purified the UDP binding protein by using UDP-sepharose affinity chromatography, gel filtration, and SDS-PAGE electrophoresis and analyzed its structure. The data presented here indicate for the first time that the inducer UDP interacts with a 38,333 ± 30 Da binding protein(s) (p38), of unknown function, in both cell lines. Microsequencing and sequence alignment search revealed that the p38 protein(s) contains at least two homologous domains, one being part of ABC-type transporters and another found in the Wingless-type (Wnt) proteins. Kinetic analysis revealed that the p38 forms a relatively stable protein complex with [3H]UDP that accumulates within the cytosol and nucleus of MEL cells during the precommitment period. This complex, however, decays later on after commitment to erythroid maturation has been initiated. De novo protein and mRNA synthesis is needed for the UDP-p38 complex to form, as shown by the use of metabolic inhibitors. Purified p38 was used to develop an anti-p38 polyclonal serum, and Western blot analysis revealed that the level of p38 was quite similar in both UDP-inducible and -resistant MEL subclones that we developed. Although only a portion of the primary structure of the p38 is known from microsequencing, the mechanism by which the UDP-p38 complex contributes to induction of differentiation in both UDP-responsive mouse MEL and human RD/TE-671 cells is discussed. Copyright © 2005 Cognizant Comm. Corp.