Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain. Monomerization of 14-3-3ζ leads to the loss of affinity for the unphosphorylated residues. In neuroblastoma cell extract, MAP2c is heavily phosphorylated by PKA and the proline kinase ERK2. Although 14-3-3ζ dimer or monomer do not interact with the residues phosphorylated by ERK2, ERK2 phosphorylation of MAP2c in the C-terminal domain reduces the binding of MAP2c to both oligomeric variants of 14-3-3ζ.

• Keywords
• 14‐3‐3 proteins, extracellular signal‐regulated kinase 2, microtubule‐associated protein, nuclear magnetic resonance, protein kinase A,
• MeSH
• Phosphorylation MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Mitogen-Activated Protein Kinase 1 metabolism   genetics   MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   genetics   MeSH
• 14-3-3 Proteins * metabolism   chemistry   genetics   MeSH
• Microtubule-Associated Proteins * metabolism   chemistry   genetics   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• MAPK1 protein, human MeSH Browser
• Mitogen-Activated Protein Kinase 1 MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH
• 14-3-3 Proteins * MeSH
• Microtubule-Associated Proteins * MeSH
• YWHAZ protein, human MeSH Browser

Protein phosphorylation is a critical mechanism that biology uses to govern cellular processes. To study the impact of phosphorylation on protein properties, a fully and specifically phosphorylated sample is required although not always achievable. Commonly, this issue is overcome by installing phosphomimicking mutations at the desired site of phosphorylation. 14-3-3 proteins are regulatory protein hubs that interact with hundreds of phosphorylated proteins and modulate their structure and activity. 14-3-3 protein function relies on its dimeric nature, which is controlled by Ser58 phosphorylation. However, incomplete Ser58 phosphorylation has obstructed the detailed study of its effect so far. In the present study, we describe the full and specific phosphorylation of 14-3-3ζ protein at Ser58 and we compare its characteristics with phosphomimicking mutants that have been used in the past (S58E/D). Our results show that in case of the 14-3-3 proteins, phosphomimicking mutations are not a sufficient replacement for phosphorylation. At physiological concentrations of 14-3-3ζ protein, the dimer-monomer equilibrium of phosphorylated protein is much more shifted towards monomers than that of the phosphomimicking mutants. The oligomeric state also influences protein properties such as thermodynamic stability and hydrophobicity. Moreover, phosphorylation changes the localization of 14-3-3ζ in HeLa and U251 human cancer cells. In summary, our study highlights that phosphomimicking mutations may not faithfully represent the effects of phosphorylation on the protein structure and function and that their use should be justified by comparing to the genuinely phosphorylated counterpart.

• Keywords
• 14-3-3, dissociation constant, oligomeric state, phosphomimicking mutation, phosphorylation,
• Publication type
• Journal Article MeSH

Mutations of cysteine are often introduced to e.g. avoid formation of non-physiological inter-molecular disulfide bridges in in-vitro experiments, or to maintain specificity in labeling experiments. Alanine or serine is typically preferred, which usually do not alter the overall protein stability, when the original cysteine was surface exposed. However, selecting the optimal mutation for cysteines in the hydrophobic core of the protein is more challenging. In this work, the stability of selected Cys mutants of 14-3-3ζ was predicted by free-energy calculations and the obtained data were compared with experimentally determined stabilities. Both the computational predictions as well as the experimental validation point at a significant destabilization of mutants C94A and C94S. This destabilization could be attributed to the formation of hydrophobic cavities and a polar solvation of a hydrophilic side chain. A L12E, M78K double mutant was further studied in terms of its reduced dimerization propensity. In contrast to naïve expectations, this double mutant did not lead to the formation of strong salt bridges, which was rationalized in terms of a preferred solvation of the ionic species. Again, experiments agreed with the calculations by confirming the monomerization of the double mutants. Overall, the simulation data is in good agreement with experiments and offers additional insight into the stability and dimerization of this important family of regulatory proteins.

• Keywords
• 14-3-3 protein, Differential scanning calorimetry, Free energy calculation, Molecular dynamics simulation, Protein stability, Thermodynamic integration,
• MeSH
• Cysteine chemistry   genetics   metabolism   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Multimerization * MeSH
• Mutation MeSH
• Computer Simulation MeSH
• 14-3-3 Proteins chemistry   genetics   metabolism   MeSH
• Protein Stability MeSH
• Thermodynamics * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cysteine MeSH
• 14-3-3 Proteins MeSH
• YWHAZ protein, human MeSH Browser

The 14-3-3 protein family performs regulatory functions in eukaryotic organisms by binding to a large number of phosphorylated protein partners. Whilst the binding mode of the phosphopeptides within the primary 14-3-3 binding site is well established based on the crystal structures of their complexes, little is known about the binding process itself. We present a computational study of the process by which phosphopeptides bind to the 14-3-3ζ protein. Applying a novel scheme combining Hamiltonian replica exchange molecular dynamics and distancefield restraints allowed us to map and compare the most likely phosphopeptide-binding pathways to the 14-3-3ζ protein. The most important structural changes to the protein and peptides involved in the binding process were identified. In order to bind phosphopeptides to the primary interaction site, the 14-3-3ζ adopted a newly found wide-opened conformation. Based on our findings we additionally propose a secondary interaction site on the inner surface of the 14-3-3ζ dimer, and a direct interference on the binding process by the flexible C-terminal tail. A minimalistic model was designed to allow for the efficient calculation of absolute binding affinities. Binding affinities calculated from the potential of mean force along the binding pathway are in line with the available experimental estimates for two of the studied systems.

• MeSH
• Phosphorylation MeSH
• Protein Conformation * MeSH
• Models, Molecular * MeSH
• 14-3-3 Proteins metabolism   MeSH
• Molecular Dynamics Simulation * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 14-3-3 Proteins MeSH

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.

• Keywords
• 14-3-3 protein, mass spectrometry (MS), microtubule-associated protein (MAP), nuclear magnetic resonance (NMR), protein kinase A (PKA),
• MeSH
• Amino Acid Motifs MeSH
• Phosphorylation MeSH
• Mass Spectrometry MeSH
• Kinetics MeSH
• Rats MeSH
• Magnetic Resonance Spectroscopy MeSH
• Microtubules metabolism   MeSH
• Neurons metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• 14-3-3 Proteins chemistry   MeSH
• Microtubule-Associated Proteins chemistry   MeSH
• tau Proteins chemistry   MeSH
• Signal Transduction MeSH
• Tubulin metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• MAP2 protein, rat MeSH Browser
• Mapt protein, rat MeSH Browser
• Cyclic AMP-Dependent Protein Kinases MeSH
• 14-3-3 Proteins MeSH
• Microtubule-Associated Proteins MeSH
• tau Proteins MeSH
• Tubulin MeSH