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.
Keywordsmicrotubule-associated protein
extracellular signal–regulated kinase
cyclin-dependent kinase
Src homology 3 domain
PKA
NMR
growth factor receptor-bound protein 2 (GRB2)
AbbreviationsAKAPA-kinase anchoring protein
CDKcyclin-dependent kinase
ERK2extracellular signal–regulated kinase 2
GSKglycogen synthase kinase
MAPmicrotubule-associated protein
MARKMAP/microtubule affinity-regulating kinase
MSTmicroscale thermophoresis
MTBDmicrotubule-binding domain
VCRvariable central region
© 2022 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.
留言 (0)