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Mutations at phosphorylation sites of Xenopus microtubule-associated protein 4 affect its microtubule-binding ability
1Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto 600-8813, Japan.
Abstract:
Microtubule-associated proteins (MAPs) bind to and stabilize microtubules (MTs) both in vitro and in vivo and are thought to regulate MT dynamics during the cell cycle. It is known that p220, a major MAP of Xenopus, is phosphorylated by p34(cdc2) kinase as well as MAP kinase in mitotic cells, and that the phosphorylated p220 loses its MT-binding and -stabilizing abilities in vitro. We cloned a full-length cDNA encoding p220, which identified p220 as a Xenopus homologue of MAP4 (XMAP4). To examine the physiological relevance of XMAP4 phosphorylation in vivo, Xenopus A6 cells were transfected with cDNAs encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein. Mutations of serine and threonine residues at p34(cdc2) kinase-specific phosphorylation sites to alanine interfered with mitosis-associated reduction in MT affinity of XMAP4, and their overexpression affected chromosome movement during anaphase A. These findings indicated that phosphorylation of XMAP4 (probably by p34(cdc2) kinase) is responsible for the decrease in its MT-binding and -stabilizing abilities during mitosis, which are important for chromosome movement during anaphase A.
Insights
Phosphorylation of Xenopus MAP4 (XMAP4) by p34(cdc2) kinase reduces its microtubule-binding ability during mitosis. This is crucial for proper chromosome movement during anaphase A in cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule-associated proteins (MAPs) regulate microtubule dynamics during the cell cycle.
- Xenopus p220, a major MAP, is phosphorylated by p34(cdc2) and MAP kinase during mitosis, losing its microtubule-binding ability.
- p220 was identified as the Xenopus homologue of MAP4, termed XMAP4.
Purpose of the Study:
- To investigate the in vivo physiological relevance of XMAP4 phosphorylation.
- To determine the role of XMAP4 phosphorylation in microtubule dynamics and chromosome segregation during mitosis.
Main Methods:
- Cloning of full-length cDNA encoding XMAP4.
- Transfection of Xenopus A6 cells with wild-type and mutant XMAP4-green fluorescent protein constructs.
- Analysis of microtubule binding affinity and chromosome movement in transfected cells.
Main Results:
- Mutations at p34(cdc2) kinase-specific phosphorylation sites prevented the mitosis-associated reduction in XMAP4's microtubule affinity.
- Overexpression of these mutants disrupted chromosome movement during anaphase A.
- Phosphorylation of XMAP4 by p34(cdc2) kinase decreases its microtubule-binding and -stabilizing abilities.
Conclusions:
- XMAP4 phosphorylation, likely by p34(cdc2) kinase, is essential for regulating microtubule dynamics during mitosis.
- This phosphorylation event is critical for accurate chromosome segregation during anaphase A.