Vimentin is a widely expressed intermediate filament protein thought to be involved mainly in structural processes, such as wound healing. We now demonstrate that activated human macrophages secrete vimentin into the extracellular space. The maturation of blood-derived monocytes into macrophages involves several signalling pathways. We show that secretion of vimentin, which is phosphorylated at serine and threonine residues, is enhanced by the phosphatase inhibitor okadaic acid and blocked by the specific protein kinase C inhibitor GO6983. These findings are consistent with previous observations that phosphorylation of vimentin affects its intracellular localization and that vimentin is a substrate for protein kinase C (PKC). We also show that the anti-inflammatory cytokine interleukin-10 (IL-10), which inhibits PKC activity, blocks secretion of vimentin. In contrast, the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) can trigger secretion of vimentin. Finally, we found that extracellular vimentin is involved in bacterial killing and the generation of oxidative metabolites, two important functions of activated macrophages. These data establish that vimentin is secreted by macrophages in response to pro-inflammatory signalling pathways and is probably involved in immune function.

The natural product withaferin A (WFA) exhibits antitumor and antiangiogenesis activity in vivo, which results from this drug's potent growth inhibitory activities. Here, we show that WFA binds to the intermediate filament (IF) protein, vimentin, by covalently modifying its cysteine residue, which is present in the highly conserved alpha-helical coiled coil 2B domain. WFA induces vimentin filaments to aggregate in vitro, an activity manifested in vivo as punctate cytoplasmic aggregates that colocalize vimentin and F-actin. WFA's potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, we show that WFA-induced inhibition of capillary growth in a mouse model of corneal neovascularization is compromised in vimentin-deficient mice. These findings identify WFA as a chemical genetic probe of IF functions, and illuminate a potential molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases.

DEK is a nuclear phosphoprotein implicated in oncogenesis and autoimmunity and a major component of metazoan chromatin. The intracellular cues that control the binding of DEK to DNA and its pleiotropic functions in DNA-and RNA-dependent processes have remained mainly elusive so far. Our recent finding that the phosphorylation status of DEK is altered during death receptor-mediated apoptosis suggested a potential involvement of DEK in stress signaling. In this study, we show that in cells committed to die, a portion of the cellular DEK pool is extensively posttranslationally modified by phosphorylation and poly(ADP-ribosyl)ation. Through interference with DEK expression, we further show that DEK promotes the repair of DNA lesions and protects cells from genotoxic agents that typically trigger poly(ADP-ribose) polymerase activation. The posttranslational modification of DEK during apoptosis is accompanied by the removal of the protein from chromatin and its release into the extracellular space. Released modified DEK is recognized by autoantibodies present in the synovial fluids of patients affected by juvenile rheumatoid arthritis/juvenile idiopathic arthritis. These findings point to a crucial role of poly(ADP-ribosyl)ation in shaping DEK's autoantigenic properties and in its function as a promoter of cell survival.Human DEK is an abundant and highly conserved nuclear protein that has long been implicated in carcinogenesis and autoimmune disorders (for a review, see references 50 and 60). Originally isolated from a specific subtype of acute myeloid leukemia (55), the gene encoding DEK is expressed under the control of transcription factors E2F and YY1 (10, 49). High levels of DEK support cell immortalization and inhibit both senescence and apoptosis, as shown in cells infected with highrisk human papillomavirus E7 (1,62,63). DEK is also upregulated in a variety of aggressive human tumors, including retinoblastoma, colon and bladder cancer, and melanoma (e.g., see references 10, 19, 28, 30, and 37).In the nucleus, DEK is involved in a variety of DNA-and RNA-dependent processes, such as DNA replication (2), splice site recognition (51), and gene transcription. Here it can function as either an activator (9) or a repressor (16,20,45). The diversity of these effects is in line with DEK's described function as a possible regulator of chromatin architecture, which may affect genome activity at various levels in a highly contextdependent manner. In fact, DEK has been shown in vitro to be a modifier of DNA higher-order structure, acting in concert with topoisomerase I to introduce constrained positive supercoils in closed circular DNA plasmids and simian virus 40 (SV40) minichromosomes (24,25,58,59). Accordingly, DEK was shown to bind to DNA in a structure-specific rather than sequence-specific manner and to reduce the accessibility of chromatin to components of the replication machinery (2, 58). Beyond its effects on DNA topology, DEK can modulate the activity of other chromatin-associated proteins, such as P/CAF and p3...

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen Shigella flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.