Regulatory mechanisms and functions of intermediate filaments: a study using site- and phosphorylation state-specific antibodies

Abstract

Intermediate filaments (IF) form the structural framework of the cytoskeleton. Although histopathological detection of IF proteins is utilized for examining cancer specimens as reliable markers, the molecular mechanisms by which IF are involved in the biology of cancer cells are still unclear. We found that site-specific phosphorylation of IF proteins induces the disassembly of filament structures. To further dissect the in vivo spatiotemporal dynamics of IF phosphorylation, we developed site- and phosphorylation state-specific antibodies. Using these antibodies, we detected kinase activities that specifically phosphorylate type III IF, including vimentin, glial fibrillary acidic protein and desmin, during mitosis. Cdk1 phosphorylates vimentin-Ser55 from prometaphase to metaphase, leading to the recruitment of Polo-like kinase 1 (Plk1) to vimentin. Upon binding to Phospho-Ser55 of vimentin, Plk1 is activated, and then phosphorylates vimentin-Ser82. During cytokinesis, Rho-kinase and Aurora-B specifically phosphorylate IF at the cleavage furrow. IF phosphorylation by Cdk1, Plk1, Rho-kinase and Aurora-B plays an important role in the local IF breakdown, and is essential for the efficient segregation of IF networks into daughter cells. As another part of our research on IF, we have set out to find the binding partners with simple epithelial keratin 8/18. We identified tumor necrosis factor receptor type 1-associated death domain protein (TRADD) as a keratin 18-binding protein. Together with data from other laboratories, it is proposed that simple epithelial keratins may play a role in modulating the response to some apoptotic signals. Elucidation of the precise molecular functions of IF is expected to improve our understanding of tumor development, invasion and metastasis.

Figure 1

Regulation of intermediate filament (IF) organization by phosphorylation. The site‐specific phosphorylation in the amino‐terminal head domain of IF proteins induces the disassembly of IF. The balance between IF phosphorylation by protein kinases and dephosphorylation by protein phosphatases controls the continuous exchange of IF subunits between a soluble pool and polymerized IF.

Figure 2

Phosphorylation sites in the mouse vimentin head domain. Multiple serine residues are phosphorylated by cAMP‐dependent protein kinase (A kinase), protein kinase C (C kinase), Ca²⁺/calmodulin‐dependent protein kinase II (CaM kinase II), p21‐activated kinase (PAK), Cdk1/Cdc2 kinase, Rho‐kinase, Aurora‐B and Polo‐like kinase 1 (Plk1). Yellow circles indicate the important in vivo phosphorylation sites during mitosis.

Figure 3

Cooperative phosphorylation of vimentin by mitotic kinases. Cdk1 phosphorylates vimentin‐Ser55 from prometaphase to metaphase. Polo‐like kinase 1 (Plk1) binds to vimentin‐Ser55 phosphorylated by Cdk1, leading to Plk1 activation and phosphorylation at Ser82 by Plk1. During cytokinesis, Ser71 and Ser72 are specifically phosphorylated by Rho‐kinase and Aurora‐B, respectively. These phosphorylations play an important role in vimentin filament segregation.

Figure 4

A potential route to generate aneuploid cells. During mitosis, the phosphorylation of intermediate filaments (IF) by mitotic kinases is impaired, leading to the generation of tetraploid cells. If these cells lose the tetraploidy checkpoint, they will be transformed into aneuploid cells.