Identification of phosphorylation-induced changes in vimentin intermediate filaments by site-directed spin labeling and electron paramagnetic resonance

Abstract

Phosphorylation drives the disassembly of the vimentin intermediate filament (IF) cytoskeleton at mitosis. Chromatographic analysis has suggested that phosphorylation produces a soluble vimentin tetramer, but little has been determined about the structural changes that are caused by phosphorylation or the structure of the resulting tetramer. In this study, site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) were used to examine the structural changes resulting from protein kinase A phosphorylation of vimentin IFs in vitro. EPR spectra suggest that the tetrameric species resulting from phosphorylation is the A11 configuration. EPR spectra also establish that the greatest degree of structural change was found in the linker 2 and the C-terminal half of the rod domain, despite the fact that most phosphorylation occurs in the N-terminal head domain. The phosphorylation-induced changes notably affected the proposed "trigger sequences" located in the linker 2 region, which have been hypothesized to mediate the induction of coiled-coil formation. These data are the first to document specific changes in IF structure resulting from a physiologic regulatory mechanism and provide further evidence, also generated by SDSL-EPR, that the linker regions play a key role in IF structure and regulation of assembly/disassembly.

Figure 1

A. Schematic depiction of the vimentin molecule with variable head/tail regions, and rod domain separated by linker regions. Cysteine mutants spin labeled for EPR are listed above the molecule; phosphorylation sites identified in this study are listed below. B. Schematic depiction of A₁₁ and A₂₂ alignments of the vimentin tetramer, and the relative proximity of positions 191 and 348.

Figure 2

A-D Electron microscope images of vimentin IFs spin labeled at position 309. A. No treatment. B. ATP only treatment. C. Kinase only treatment. D. Kinase and ATP treatment. E. Normalized EPR spectra of vimentin spin labeled at position 309 following the indicated treatments. The amplitude of each spectrum is normalized to the same number of spins. Arrows “a” and “b” indicate components of the spectra discussed in the text.

Figure 3

Normalized EPR spectra for vimentin mutants observed. For each indicated position, the black trace is the buffer control; the grey trace is the kinase +ATP experimental sample.

Figure 4

EPR spectra of frozen vimentin containing a spin-labeled side chain at position 348. The spectrum of phosphorylated vimentin is shown by the gray line, with control vimentin shown by the black trace. The extent of broadening is revealed by the qualitative parameter d₁/d, which is larger for labels experiencing greater dipolar interaction.