Mechanism Of Phosphorylation Of Miranda Regulating Drosophila Neuroblast Asymmetric Cell Division

Basal localization of cell fate determinants is one of the characteristic features of neuroblast (NB) asymmetric division in Drosophila. Basally enriched component Miranda (Mira), the cargo protein of cell fate determinats Prospero (Pros) and Brain tumor (Brat), acts as a marker protein of NB asymmetric division. We observed Mira dynamic localization in larval brain NBs with time-lapse confocal and declared Mira processed cortex and cytoplasmic translocation before its basal enrichment.For seeking regulators of Mira localization in NBs, we found Tyrosine kinase dWeel combined with Mira. Our evidence proved Mira was the new substrate of dWeel, which could phosphorylate Mira Tyr (Y) 487. Further study suggested hyper-phosphorylated Mira, which was activated by dWeel, was cytoplasmic in NBs during transition from G2 to mitosis.We further identified evolutionarily conserved Phosphotyrosyl Phosphatase Activator (PTPA) as a novel mediator specifically for effective cortex localization of Mira. In ptpa NBs, Mira is cytoplasmic in early mitosis (prophase and metaphase) and its basal localization is delayed until anaphase. Altered 2D Western blot pattern of Mira from ptpa brain extracts, as well as from ptpa knockdown S2 cells, suggests hyper-phosphorylation of Mira in the absence of PTPA. Our data indicate that dephosphorylation of Mira Thr(T)519 is defective in ptpa mutant. Further study suggests that PTPA forms a complex with Protein Phosphatase 4 (PP4). In addition PP4-19C levels are upregulated in ptpa mutant and prevention of this increase enhances phenotype.In summary, our data suggest that sequential phosphorylation statu of Mira temporally regulates Mira dynamic localization in mitosis; dWeel kinase activated phosphorylation of Mira Y487 is in charge of Mira cytoplasmic location in transition from late G2 to mitosis; then PTPA/PP4 mediated dephosporylation of Mira T591 indicates Mira cortical translocation in early mitosis stage (prophase and metaphase), followed immediately in basal by aPKC kinase function. We reveal a new regulation pathway of Mira localization and Drosophila larval NB asymmetric cell division.