Phosphorylation of vimentin head domain inhibits interaction with the carboxyl‐terminal end of α‐helical rod domain studied by surface plasmon resonance measurements

Abstract

The amino-terminal head domain of vimentin is the target site for several protein kinases and phosphorylation induces disassembly of the vimentin intermediate filaments in vivo and in vitro. To better understand molecular mechanisms involved in phosphorylation-dependent disassembly, we examined domain interactions involving the head domain and the effect of phosphorylation on the interaction, using surface plasmon resonance. We observed that the head domain binds to the carboxyl-terminal helix 2B in the rod domain, under physiological ionic strength. This interaction was interfered with by Akinase phosphorylation of the head domain. Deletion of the carboxyl-terminal 20 amino acids of helix 2B resulted in loss of the interaction. Furthermore, peptide representing the carboxylterminal 20 residues of helix 2B had a substantial affinity with the head domain but not with the phosphorylated one. These findings support the idea that the interaction between the head domain and the last 20 residues of helix 2B is essential for association of vimentin tetramers into the intermediate filaments and that the phosphorylation-dependent disassembly is the result of loss of the interaction. ß

Identification of Phosphorylation-Induced Changes in Vimentin Intermediate Filaments by Site-Directed Spin Labeling and Electron Paramagnetic Resonance

“…This implies interactions between the head domain and rod 2B, and evidence exists to support this (48, 49). Binding affinity measurements have placed this head domain/2B interaction in the far carboxy terminus of region 2B in residues 390-411 a highly conserved region spanning the Helix Termination Motif.…”

“…Binding affinity measurements have placed this head domain/2B interaction in the far carboxy terminus of region 2B in residues 390-411 a highly conserved region spanning the Helix Termination Motif. Surface plasmon resonance measurements showed that this interaction is substantially lowered, and possibly eliminated, when the vimentin head domain is phosphorylated in vitro with PKA (48). Additional studies identified the ability of arginine but not lysine to interfere with filament assembly and the identification of the di-arginine motif in the head as critical for IF assembly offers an explanation (49, 50).…”

Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression

“…To that end, several in vitro studies demonstrate the importance of the ordered assembly of filaments (29), and of intrinsic determinants within IF proteins, for their organization (4,30). Among these determinants are interactions of the head-and-rod domains, which can be modulated by phosphorylation as demonstrated by phosphorylation-mediated inhibition of purified vimentin head-domain interaction with helix 2B of the rod domain (31), supporting the hypothesis that keratin (and likely other IF proteins) filament organization and reorganization are hierarchical and involve multiple phosphorylation͞dephosphorylation events that may be coupled to associations͞dissociations with other IF-binding proteins.…”

Novel bacterial ADP-ribosylating toxins: structure and function

“…These residues are located in the head domain of vimentin, and modification of residues in the head domain prevents vimentin from forming higher order structures 148 . ADP-ribosylation likely blocks vimentin oligomerization into intermediate filaments, resulting in disruption of vimentin filaments and cytoskeletal collapse 147 .…”