Quantitative mapping of microtubule-associated protein 2c (MAP2c) phosphorylation and regulatory protein 14-3-3ζ-binding sites reveals key differences between MAP2c and its homolog Tau

Abstract

Edited by Norma AllewellMicrotubule-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 14-3-3 . 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 14-3-3 are supposed to be a major factor in microtubule destabilization, the binding of 14-3-3 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.Cytoskeletal microtubule-associated proteins (MAPs) 3 are proteins of critical importance for regulating the stability and dynamics of microtubules (1). MAP2 and Tau represent MAP subfamilies expressed in neurons; MAP2 is localized in dendrites, whereas Tau is found mainly in axons (2). Tau and MAP2 belong to the class of intrinsically disordered proteins (IDPs), which lack a unique structure and which exist in multiple, quickly interconverting conformations (3-7). NMR is the method of choice for structural investigation of this type of protein. Tau and MAP2 differ in their N-terminal projection domains, which contain acidic and proline-rich subdomains, whereas the C-terminal parts, containing the microtubulebinding domain (MTBD) and the C-terminal region, are homologous (8). Tau is expressed in several splice variants. The human brain isoforms differ in the number of microtubulebinding regions (MTBRs) in the C-terminal portion and in the presence of two inserts near the N terminus. For the sake of simplicity, only the 441-residue variant lacking exons 6, 8, and 10 (9) is discussed in this paper. This variant has been studied in detail and was shown to form paired helical filaments and neurofibillary tangles in brains of patients suffering from Alzheimer's disease (10). The MAP2 family is composed of two high-molecular-weight proteins, MAP2a and MAP2b, each consisting of 1830 amino acids, and two low-molecular-weight proteins, MAP2c and MAP2d, consisting of 467 and 498 amino acids, respectively. The ...

Functionally specific binding regions of microtubule-associated protein 2c exhibit distinct conformations and dynamics

“…In order to achieve sufficient resolution, relaxation rates were measured using 13 C, 15 Nlabeled MAP2c and non-uniformly sampled 3D relaxation experiments based on the HNCO correlation (39). The R 1 , R 2 relaxation rates and steadystate heteronuclear Overhauser enhancement were measured at 600 MHz and 950 MHz spectrometers.…”

“…NMR has proved to be a reliable tool to study their dynamics and structural features (11,13,(21)(22)(23)(24)(25). Despite their highly flexible nature, Tau (23) and MAP2c (25) adopt transient secondary structures, documenting that the structures of Tau and MAP2c are far from a random coil.…”

“…3d) Conformational analysis -In order to convert the obtained experimental data into a structural model, conformational analysis was performed using the ASTEROIDS algorithm (34). Assigned chemical shifts (13, 25), SAXS, and PRE data served as an experimental input, reflecting local conformation, long-range contacts, and overall shape of the molecule, respectively. Potential presence of dimers (or higher oligomers) has a negligible effect on description of local conformational behavior but may influence analysis of long-range interactions.…”

“…1) (5). The sequence diversity is further magnified by different phosphorylation patterns (6)(7)(8)(9)(10)(11)(12)(13).…”

“…The result of mutations was confirmed by sequencing. MAP2c expression, purification and phosphorylation by PKA were performed as described earlier (13, 25,65). The SR4-SR5-SH3 plectin fragment was expressed in Escherichia coli using a DNA construct including exons 16-21 (30).…”