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Phosphorylation states of microtubule-associated protein 2 (MAP2) determine the regulatory role of MAP2 in
T J Itoh1, S Hisanaga, T Hosoi
1Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan.
Abstract:
Phosphorylation-dependent regulation of microtubule-stabilizing activities of microtubule-associated protein 2 (MAP2) was examined using optical microscopy. MAP2, purified from mammalian brain, was phosphorylated by either cAMP-dependent protein kinase (PKA) or cyclin B-dependent cdc2 kinase. Using PKA, 15 mol of phosphoryl groups was incorporated per mole of MAP2, but about 70% of the phosphates was distributed to the projection region. Using cdc2 kinase, 7-10 mol of phosphoryl groups was incorporated per mole of MAP2, and more than 60% of the phosphates was distributed to the microtubule-binding region. Both types of phosphorylation similarly reduced binding activity of MAP2 onto microtubules. Direct observation of individual microtubules using dark-field microscopy showed that interconversion between the polymerization phase and the depolymerization phase was repeated in both unphosphorylated and PKA-phosphorylated MAP2. In cdc2 kinase-phosphorylated MAP2, however, the phase transition from depolymerization to polymerization occurred with difficulty, with the result being that the half-life of individual microtubules was as short as in the absence of MAP2. Examination of spontaneous polymerization of microtubules using dark-field microscopy showed that the microtubule-nucleating activity of MAP2 was reduced by PKA-dependent phosphorylation and was completely abolished by cdc2 kinase-dependent phosphorylation. These observations show that cdc2 kinase-dependent phosphorylation inhibits both the microtubule-stabilizing activity and the microtubule-nucleating activity of MAP2, while PKA-dependent phosphorylation affects only the microtubule-nucleating activity of MAP2.
Insights
Phosphorylation by cdc2 kinase inhibits microtubule stabilization and nucleation by microtubule-associated protein 2 (MAP2). cAMP-dependent protein kinase (PKA) phosphorylation only affects MAP2
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Microtubule-associated protein 2 (MAP2) plays a crucial role in microtubule stabilization.
- Phosphorylation is a key post-translational modification regulating protein function.
- Understanding MAP2 regulation is vital for comprehending cellular dynamics.
Purpose of the Study:
- To investigate the phosphorylation-dependent regulation of MAP2's microtubule-stabilizing and nucleating activities.
- To compare the effects of cAMP-dependent protein kinase (PKA) and cdc2 kinase phosphorylation on MAP2 function.
Main Methods:
- Purification of MAP2 from mammalian brain.
- In vitro phosphorylation of MAP2 using PKA and cdc2 kinase.
- Optical microscopy, including dark-field microscopy, to observe microtubule dynamics and MAP2 binding.
- Quantification of phosphate incorporation and distribution.
Main Results:
- Both PKA and cdc2 kinase phosphorylation reduced MAP2 binding to microtubules.
- PKA phosphorylation (15 mol/mol MAP2) primarily affected the projection region, while cdc2 kinase phosphorylation (7-10 mol/mol MAP2) targeted the microtubule-binding region.
- cdc2 kinase phosphorylation significantly impaired microtubule stabilization and nucleation, shortening microtubule half-life.
- PKA phosphorylation reduced microtubule nucleation but did not impede stabilization.
- cdc2 kinase phosphorylation completely abolished MAP2's microtubule-nucleating activity.
Conclusions:
- cdc2 kinase-dependent phosphorylation inhibits both microtubule-stabilizing and -nucleating activities of MAP2.
- PKA-dependent phosphorylation primarily affects the microtubule-nucleating activity of MAP2.
- Differential phosphorylation by distinct kinases leads to distinct functional outcomes for MAP2.