Most cited article - PubMed ID 29925592
Functionally specific binding regions of microtubule-associated protein 2c exhibit distinct conformations and dynamics
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
Isoforms of microtubule-associated protein 2 (MAP2) differ from their homolog Tau in the sequence and interactions of the N-terminal region. Binding of the N-terminal region of MAP2c (N-MAP2c) to the dimerization/docking domains of the regulatory subunit RIIα of cAMP-dependent protein kinase (RIIDD2) and to the Src-homology domain 2 (SH2) of growth factor receptor-bound protein 2 (Grb2) have been described long time ago. However, the structural features of the complexes remained unknown due to the disordered nature of MAP2. Here, we provide structural description of the complexes. We have solved solution structure of N-MAP2c in complex with RIIDD2, confirming formation of an amphiphilic α-helix of MAP2c upon binding, defining orientation of the α-helix in the complex and showing that its binding register differs from previous predictions. Using chemical shift mapping, we characterized the binding interface of SH2-Grb2 and rat MAP2c phosphorylated by the tyrosine kinase Fyn in their complex and proposed a model explaining differences between SH2-Grb2 complexes with rat MAP2c and phosphopeptides with a Grb2-specific sequence. The results provide the structural basis of a potential role of MAP2 in regulating cAMP-dependent phosphorylation cascade via interactions with RIIDD2 and Ras signaling pathway via interactions with SH2-Grb2.
- Keywords
- A-kinase anchoring protein (AKAP), growth factor receptor-bound protein 2 (GRB2), microtubule associated protein (MAP) 2, nuclear magnetic resonance (NMR), protein kinase A (PKA),
- MeSH
- GRB2 Adaptor Protein * metabolism chemistry MeSH
- Humans MeSH
- Protein Domains MeSH
- Microtubule-Associated Proteins * metabolism chemistry genetics MeSH
- Proto-Oncogene Proteins c-fyn metabolism chemistry genetics MeSH
- Signal Transduction MeSH
- src Homology Domains MeSH
- Protein Binding * MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- GRB2 Adaptor Protein * MeSH
- GRB2 protein, human MeSH Browser
- Microtubule-Associated Proteins * MeSH
- Proto-Oncogene Proteins c-fyn MeSH
Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal-regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies. Our comparison with the phosphorylation of MAP2c by a model cyclin-dependent kinase CDK2 and with phosphorylation of the MAP2c homolog Tau revealed differences in phosphorylation profiles that explain specificity of regulation of biological functions of MAP2c and Tau. To probe the molecular basis of the regulatory effect of ERK2, we investigated the interactions of phosphorylated and unphosphorylated MAP2c by NMR with single-residue resolution. As ERK2 phosphorylates mostly outside the regions binding microtubules, we studied the binding of proteins other than tubulin, namely regulatory subunit RIIα of cAMP-dependent PKA, adapter protein Grb2, Src homology domain 3 of tyrosine kinases Fyn and Abl, and ERK2 itself. We found ERK2 phosphorylation interfered mostly with binding to proline-rich regions of MAP2c. Furthermore, our NMR experiments in SH-SY5Y neuroblastoma cell lysates showed that the kinetics of dephosphorylation are compatible with in-cell NMR studies and that residues targeted by ERK2 and PKA are efficiently phosphorylated in the cell lysates. Taken together, our results provide a deeper characterization of MAP2c phosphorylation and its effects on interactions with other proteins.
- Keywords
- NMR, PKA, Src homology 3 domain, cyclin-dependent kinase, extracellular signal–regulated kinase, growth factor receptor-bound protein 2 (GRB2), microtubule-associated protein,
- MeSH
- Extracellular Signal-Regulated MAP Kinases * metabolism MeSH
- Phosphorylation MeSH
- Humans MeSH
- Microtubules metabolism MeSH
- Cell Line, Tumor MeSH
- Proline-Directed Protein Kinases * metabolism MeSH
- Microtubule-Associated Proteins * metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- CDK2 protein, human MeSH Browser
- Extracellular Signal-Regulated MAP Kinases * MeSH
- MAP2 protein, human MeSH Browser
- MAPK1 protein, human MeSH Browser
- Proline-Directed Protein Kinases * MeSH
- Microtubule-Associated Proteins * MeSH
Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.
The stability and dynamics of cytoskeleton in brain nerve cells are regulated by microtubule associated proteins (MAPs), tau and MAP2. Both proteins are intrinsically disordered and involved in multiple molecular interactions important for normal physiology and pathology of chronic neurodegenerative diseases. Nuclear magnetic resonance and cryo-electron microscopy recently revealed propensities of MAPs to form transient local structures and long-range contacts in the free state, and conformations adopted in complexes with microtubules and filamentous actin, as well as in pathological aggregates. In this paper, we compare the longest, 441-residue brain isoform of tau (tau40), and a 467-residue isoform of MAP2, known as MAP2c. For both molecules, we present transient structural motifs revealed by conformational analysis of experimental data obtained for free soluble forms of the proteins. We show that many of the short sequence motifs that exhibit transient structural features are linked to functional properties, manifested by specific interactions. The transient structural motifs can be therefore classified as molecular recognition elements of tau40 and MAP2c. Their interactions are further regulated by post-translational modifications, in particular phosphorylation. The structure-function analysis also explains differences between biological activities of tau40 and MAP2c.
- Keywords
- intrinsically disordered protein, microtubule associated protein, nuclear magnetic resonance, phosphorylation, tau,
- MeSH
- Humans MeSH
- Microtubules chemistry metabolism MeSH
- Microtubule-Associated Proteins chemistry metabolism MeSH
- tau Proteins chemistry metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- Microtubule-Associated Proteins MeSH
- tau Proteins MeSH
