Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments

Abstract

Intermediate filaments (IFs) continuously exchange between a small, depolymerized fraction of IF protein and fully polymerized IFs. To elucidate the possible role of phosphorylation in regulating this equilibrium, we disrupted the exchange of phosphate groups by specific inhibition of dephosphorylation and by specific phosphorylation and site-directed mutagenesis of two of the major in vivo phosphorylation sites determined in this study. Inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases in BHK-21 fibroblasts with calyculin-A, induced rapid vimentin phosphorylation in concert with disassembly of the IF polymers into soluble tetrameric vimentin oligomers. This oligomeric composition corresponded to the oligopeptides released by cAMP-dependent kinase (PKA) following in vitro phosphorylation. Characterization of the 32P-labeled vimentin phosphopeptides, demonstrated Ser-4, Ser-6, Ser-7, Ser-8, Ser-9, Ser-38, Ser-41, Ser-71, Ser-72, Ser-418, Ser-429, Thr-456, and Ser-457 as significant in vivo phosphorylation sites. A number of the interphase-specific high turnover sites were shown to be in vitro phosphorylation sites for PKA and protein kinase C (PKC). The effect of presence or absence of phosphate groups on individual subunits was followed in vivo by microinjecting PKA-phosphorylated (primarily S38 and S72) and mutant vimentin (S38:A, S72:A), respectively. The PKA-phosphorylated vimentin showed a clearly decelerated filament formation in vivo, whereas obstruction of phosphorylation at these sites by site-directed mutagenesis had no significant effect on the incorporation rates of subunits into assembled polymers. Taken together, our results suggest that elevated phosphorylation regulates IF assembly in vivo by changing the equilibrium constant of subunit exchange towards a higher off-rate.

Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

“…These phosphorylation events are thought to mainly involve the vimentin N-terminal serine residues [15,26,40] and, in agreement, our data showed a major effect of PI3Kg signaling on Ser6 phosphorylation. Although multiple protein kinases are known to phosphorylate this residue, a critical role is played by PKC isoforms [15,26]: for example, phosphorylation of Ser6 on vimentin by PKCe enhances fiber disassembly and cell migration [19]. This PKC activation might potentially be controlled by GPCR-induced PI3K signaling, since the PI3K inhibitor LY294002 blocks bradykinin-induced translocation of PKCd and PKCe [41].…”

“…These changes, causing extension or shortening of fibers, can be triggered by specific phosphorylation events, particularly on serine residues within the vimentin N-terminal non-helical domain [10,13,14]. Consistently, treatment with protein phosphatase inhibitors, such as calyculin-A [15] or okadaic acid [16], results in a dose-and time-dependent increase in vimentin phosphorylation, causing rapid disassembly of pre-existing filaments, delay of fiber assembly and increased levels of soluble vimentin tetramers [15,17,18]. These phosphorylation events are triggered by a wide range of protein kinases, such as PKA and PKC [15,19] [25] and involve a large number of serine residues (e.g.…”

“…These phosphorylation events are triggered by a wide range of protein kinases, such as PKA and PKC [15,19] [25] and involve a large number of serine residues (e.g. Ser 4,[6][7][8][9]25,33,38,41, 50, 55, 65, 71, 72, 82) with a complex pattern of overlapping specificities [15,26,27].Although vimentin appears involved in multiple cellular processes, mice lacking this protein are viable and do not show overt phenotypes [28]. Nonetheless, fibroblasts from vimentindeficient mice show mechanical instability and impaired ability to migrate and to contract a 3-D collagen network [29], thus providing a genetic evidence for the involvement of vimentin in cell plasticity and motility [30,31].…”

Intermediate filaments: versatile building blocks of cell structure

“…At higher protein concentrations under more physiological conditions, the rate of elongation is faster (H. Herrmann, personal communication). Assembly in vivo is most likely to be much faster, since IF protein concentration is higher (~2-3% of total fibroblast protein, unpublished observations) and physiological conditions are optimal (e.g., the extent of phosphorylation [15,16]). Once longer IF are formed, there is a "radial compaction" phase in which molecular re-arrangements result in a decrease in filament diameter from ~17 nm to the mature dimension of 10 nm [17].…”

Withaferin A inhibits breast cancer invasion and metastasis at sub‐cytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation

“…Future studies evaluating the extent and duration of WFA retention in tumor tissue will fully characterize the disposition and pharmacodynamic properties of WFA in preventing metastatic growth. Vimentin phosphorylation regulates vimentin dynamics [42][43][44][45] and ser56 phosphorylation is associated with cell motility signaling pathways. 40,46 Specifically, vimentin ser56 phosphorylation inversely regulates PAK activation and is critical for vimentin filament spatial rearrangement elicited by agonists.…”