Biochemical analysis of Prospero protein during asymmetric cell division: Cortical Prospero is highly phosphorylated relative to nuclear Prospero

Drosophila neuroblasts are a model system for studying asymmetric eel divis ion. Neuroblasts bud off a series of smaller progeny, called ganglion mothe r cells (GMCs). An essential regulator of GMC development is the Prospero h omeodomain transcription factor: Prospero is asymmetrically localized to th e basal cortex of the mitotic neuroblast and partitioned into the newborn G MC. Prospero is translocated into the GMC nucleus, where it is necessary to establish GMC-specific gene expression. Cortical localization of Prospero protein is observed only during mitosis; cortical localization requires ent ry into mitosis and cortical delocalization requires exit from mitosis. The tight correlation and functional requirement between mitosis and cortical Prospero localization suggests that mitosis-specific posttranslational modi fications may be involved in regulating Prospero subcellular localization. Here we use monoclonals recognizing the N-terminal or C-terminal region of Prospero to explore its posttranslational regulation. One- and two-dimensio nal Western analysis reveal a complex pattern of Prospero isoforms; phospha tase assays show that there are several phosphoisoforms of Prospero. Develo pmental 2D Western blots, cell fractionation assays, and analysis of a miss ense prospero mutation show that cortical Prospero protein is highly phosph orylated compared to nuclear Prospero protein. Our results are consistent w ith two functions of Prospero phosphorylation: (i) phosphorylation may be r equired for Prospero cortical localization; or (ii) phosphorylation may be a consequence of Prospero cortical localization, in which case it may facil itate subsequent events, such as Prospero cortical release or nuclear local ization. (C) 1998 Academic Press.