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Microtubule binding of the drosophila DMAP-85 protein is regulated by phosphorylation in vitro
V Cambiazo1, E Logarinho, H Pottstock
1Laboratorio de Biologia Celular, INTa, Universidad de Chile, Macul 5540 Santiago, Chile. vcambiaz@uec.inta.uchile.cl
Abstract:
The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule (MT) stability. Previously we isolated DMAP-85, a Drosophila MAP shown to be associated with stable MTs. In this work we show that DMAP-85 phosphorylated in cell-free early embryo extracts is released from MTs. MPM-2 antibodies recognize the phosphorylated protein. In vitro, DMAP-85 can be phosphorylated by the mitotic kinase Polo affecting its binding to MTs and creating MPM-2 epitopes on the protein. The results suggest that phosphorylation of DMAP-85 might affect its MT stabilizing activity during early mitotic cycles.
Insights
Phosphorylation releases Drosophila microtubule-associated protein 85 (DMAP-85) from microtubules (MTs). This phosphorylation, recognized by MPM-2 antibodies and mediated by Polo kinase, may alter DMAP-85
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule-associated proteins (MAPs) regulate microtubule (MT) stability.
- DMAP-85, a Drosophila MAP, is associated with stable MTs.
- Phosphorylation is a key regulatory mechanism for MAP function.
Purpose of the Study:
- To investigate the effect of DMAP-85 phosphorylation on its association with microtubules.
- To identify kinases that phosphorylate DMAP-85 and the functional consequences.
- To understand the role of DMAP-85 phosphorylation in early mitotic cycles.
Main Methods:
- Isolation of DMAP-85 from Drosophila early embryo extracts.
- In vitro phosphorylation assays using purified DMAP-85 and mitotic kinases.
- Analysis of DMAP-85 binding to MTs following phosphorylation.
- Detection of phosphorylated DMAP-85 using MPM-2 antibodies.
Main Results:
- Phosphorylation of DMAP-85 in cell-free extracts leads to its release from MTs.
- MPM-2 antibodies specifically recognize the phosphorylated form of DMAP-85.
- In vitro, Polo kinase phosphorylates DMAP-85, affecting its MT binding and creating MPM-2 epitopes.
- These findings indicate a dynamic regulation of DMAP-85-MT interactions.
Conclusions:
- Phosphorylation of DMAP-85 by mitotic kinases, such as Polo, regulates its interaction with microtubules.
- This phosphorylation event may modulate the MT-stabilizing activity of DMAP-85 during early mitosis.
- The study provides insights into the dynamic regulation of microtubule stability through MAP phosphorylation.