Abstract
Microtubule-associated protein 2c (MAP2c) is involved in neuronal development and is less characterized than its homolog Tau, which has various roles in neurodegeneration. Using NMR methods providing single-residue resolution and quantitative comparison, we investigated molecular interactions important for the regulatory roles of MAP2c in microtubule dynamics. We found that MAP2c and Tau significantly differ in the position and kinetics of sites that are phosphorylated by cAMP-dependent protein kinase (PKA), even in highly homologous regions. We determined the binding sites of unphosphorylated and phosphorylated MAP2c responsible for interactions with the regulatory protein 14-3-3ζ. Differences in phosphorylation and in charge distribution between MAP2c and Tau suggested that both MAP2c and Tau respond to the same signal (phosphorylation by PKA) but have different downstream effects, indicating a signaling branch point for controlling microtubule stability. Although the interactions of phosphorylated Tau with 14-3-3ζ are supposed to be a major factor in microtubule destabilization, the binding of 14-3-3ζ to MAP2c enhanced by PKA-mediated phosphorylation is likely to influence microtubule-MAP2c binding much less, in agreement with the results of our tubulin co-sedimentation measurements. The specific location of the major MAP2c phosphorylation site in a region homologous to the muscarinic receptor-binding site of Tau suggests that MAP2c also may regulate processes other than microtubule dynamics.
Highlights
Microtubule-associated protein 2c (MAP2c) is involved in neuronal development and is less characterized than its homolog Tau, which has various roles in neurodegeneration
We found that the same regions of unphosphorylated MAP2c and Tau interact with 14-3-3 and that the binding affinity of MAP2c is greatly increased by phosphorylation, as described previously for Tau [25, 39]
The key role of phosphorylation in regulating the interactions of microtubule-associated proteins (MAPs) with microtubules has been well-known for decades [12,13,14,15,16,17]. 14-3-3 proteins are proposed to be involved in the phosphorylation-dependent control of microtubule dynamics by competing for phosphorylated Tau with tubulin [24, 39, 40]
Summary
Microtubule-associated protein 2c (MAP2c) is involved in neuronal development and is less characterized than its homolog Tau, which has various roles in neurodegeneration. Using NMR methods providing single-residue resolution and quantitative comparison, we investigated molecular interactions important for the regulatory roles of MAP2c in microtubule dynamics. We found that MAP2c and Tau significantly differ in the position and kinetics of sites that are phosphorylated by cAMP-dependent protein kinase (PKA), even in highly homologous regions. The specific location of the major MAP2c phosphorylation site in a region homologous to the muscarinic receptor-binding site of Tau suggests that MAP2c may regulate processes other than microtubule dynamics. The 14-3-3 family includes highly conserved ubiquitous proteins [22] mostly expressed in the brain, and in particular in regions exhibiting neuroplasticity, and having important roles in neuronal development [23]. 14-3-3 interacts with unphosphorylated Tau; the phosphorylation of Tau increases its affinity for 14-3-3 (24 –27)
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