Vimentin binding to phosphorylated Erk sterically hinders enzymatic dephosphorylation of the kinase.
Cleavage fragments of de novo synthesized vimentin were recently reported to interact with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling complex retrogradely transported on importins and dynein. Here we clarify the structural basis for this interaction, which explains how pErk is protected from dephosphorylation while bound to vimentin. Pull-down and ELISA experiments revealed robust calcium-dependent binding of pErk to the second coiled-coil domain of vimentin, with observed affinities of binding increasing from 180 nM at 0.1 microM calcium to 15 nM at 10 microM calcium. In contrast there was little or no binding of non-phosphorylated Erk to vimentin under these conditions. Geometric and electrostatic complementarity docking generated a number of solutions wherein vimentin binding to pErk occludes the lip containing the phosphorylated residues in the kinase. Binding competition experiments with Erk peptides confirmed a solution in which vimentin covers the phosphorylation lip in pErk, interacting with residues above and below the lip. The same peptides inhibited pErk binding to the dynein complex in sciatic nerve axoplasm, and interfered with protection from phosphatases by vimentin. Thus, a soluble intermediate filament fragment interacts with a signaling kinase and protects it from dephosphorylation by calcium-dependent steric hindrance.
Perlson
Eran
E
Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel.
Michaelevski
Izhak
I
Kowalsman
Noga
N
Ben-Yaakov
Keren
K
Shaked
Maya
M
Seger
Rony
R
Eisenstein
Miriam
M
Fainzilber
Mike
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008647:Mesocricetus; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014746:Vimentin / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014746:Vimentin; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium