Ichiro Izawa

PKCε‐mediated phosphorylation of vimentin controls integrin recycling and motility

PKCε controls the transport of endocytosed β1‐integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCε target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCε dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCε/integrin positive vesicles. Finally, introduction of ectopic wild‐type vimentin is shown to promote cell motility in a PKCε‐dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCε null cells. Our results indicate that PKC‐mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.

Autophosphorylation of a newly identified site of Aurora-B is indispensable for cytokinesis.

Mitotic kinases regulate cell division and its checkpoints, errors of which can lead to aneuploidy or genetic instability. One of these is Aurora-B, a key kinase that is required for chromosome alignment at the metaphase plate and for cytokinesis in mammalian cells. We report here that human Aurora-B is phosphorylated at Thr-232 through interaction with the inner centromere protein (INCENP) in vivo. The phosphorylation of Thr-232 occurs by means of an autophosphorylation mechanism, which is indispensable for the Aurora-B kinase activity. The activation of Aurora-B spatio-temporally correlated with the site-specific phosphorylation of its physiological substrates, histone H3 and vimentin. Overexpression of the TA mutant of Aurora-B, in which Thr-232 was changed into alanine, frequently induced multinuclearity in cells. These results indicate that the phosphorylation of Thr-232 is an essential regulatory mechanism for Aurora-B activation.

Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)–induced cell death. We have now identified human TNF receptor type 1 (TNFR1)–associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1–270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

Filament Formation of MSF-A, a Mammalian Septin, in Human Mammary Epithelial Cells Depends on Interactions with Microtubules

Septins are a family of conserved proteins implicated in a variety of cellular functions such as cytokinesis and vesicle trafficking, but their properties and modes of action are largely unknown. Here we now report findings of immunocytochemical and biochemical characterization of a mammalian septin, MSF-A. Using an antibody specific for MSF subfamily proteins, MSF-A was found to be expressed predominantly in mammary human mammary epithelial cells (HMEC). MSF-A was associated with microtubules in interphase HMEC cells as it localized with the mitotic spindle and the bundle of microtubule at midzone during mitosis. Biochemical analysis revealed direct binding of MSF-A with polymerized tubulin through its central region containing guanine nucleotide-interactive motifs. GTPase activity, however, was not required for the association. Conditions that disrupt the microtubule network also disrupted the MSF-A-containing filament structure, resulting in a punctate cytoplasmic pattern. Depletion of MSF-A using small interfering RNAs caused incomplete cell division and resulted in the accumulation of binucleated cells. Unlike Nedd5, an MSF mutant deficient in GTPase activity forms filament indistinguishable from that of the wild type in COS cells. These results strongly suggest that septin filaments may interact not only with actin filaments but also with microtubule networks and that GTPase activity of MSF-A is not indispensable to incorporation of MSF-A into septin filaments.

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

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. (Cancer Sci 2006; 97: 167–174)

Densin-180 Interacts with δ-Catenin/Neural Plakophilin-related Armadillo Repeat Protein at Synapses

Densin-180, a protein purified from the postsynaptic density fraction of the rat forebrain, is the founding member of a newly described family of proteins termed the LAP (leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains) family that plays essential roles in establishment of cell polarity. To identify Densin-180-binding proteins, we screened a yeast two-hybrid library using the carboxyl-terminal fragment of Densin-180 containing PDZ domain as bait, and we isolated δ-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) as a Densin-180-interacting protein. δ-catenin/NPRAP, a member of the armadillo repeat family, is a nervous system-specific adherens junction protein originally discovered as an interactor with presenilin-1, a protein involved in Alzheimers disease. Densin-180 PDZ domain binds the COOH terminus of δ-catenin/NPRAP containing the PDZ domain-binding sequence. Endogenous Densin-180 was co-immunoprecipitated with δ-catenin/NPRAP and N-cadherin. Although Densin-180 was reported to be a transmembrane protein, Densin-180 was not accessible to surface biotinylation in dissociated hippocampal neurons; hence Densin-180 may be a cytosolic protein. Densin-180 co-localized with δ-catenin/NPRAP at synapses in dissociated hippocampal neurons. We propose that Densin-180 is associated in vivo with δ-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with the δ-catenin/NPRAP-N-cadherin complex.

Identification of Mrj, a DnaJ/Hsp40 Family Protein, as a Keratin 8/18 Filament Regulatory Protein

To elucidate the function of keratins 8 and 18 (K8/18), major components of the intermediate filaments of simple epithelia, we searched for K8/18-binding proteins by screening a yeast two-hybrid library. We report here that human Mrj, a DnaJ/Hsp40 family protein, directly binds to K18. Among the interactions between DnaJ/Hsp40 family proteins and various intermediate filament proteins that we tested using two-hybrid methods, Mrj specifically interacted with K18. Immunostaining with anti-Mrj antibody showed that Mrj colocalized with K8/18 filaments in HeLa cells. Mrj was immunoprecipitated not only with K18, but also with the stress-induced and constitutively expressed heat shock protein Hsp/c70. Mrj bound to K18 through its C terminus and interacted with Hsp/c70 via its N terminus, which contains the J domain. Microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, without effects on the organization of actin filaments and microtubules. Taken together, these results suggest that Mrj may play an important role in the regulation of K8/18 filament organization as a K18-specific co-chaperone working together with Hsp/c70.

A Novel Protein, Densin, Expressed by Glomerular Podocytes

With the recent molecular findings, the podocyte is emerging as a key cell type involved in glomerular damage, but protein complexes involved remain poorly understood. To systematically search for additional podocyte molecules interacting with nephrin, a key structural molecule of the interpodocyte filtration slit, precipitation of glomerular lysates was set out with anti-nephrin antibodies to identify members of the nephrin-associated protein complex. Proteins of the precipitate were subsequently identified with MALDI-TOF mass analysis. One of the proteins thus obtained showed identity with densin, a protein originally purified from rat forebrain postsynaptic density fraction and so far shown to be highly brain-specific. The expression of densin appeared distinctly in the glomerulus and cultured podocytes by RT-PCR. Immunoblotting studies revealed a specific band of 185 kD in brain and cultured podocytes; in human glomerulus, densin appeared as a 210-kD band. By immunocytochemistry, densin localizes in glomeruli in a podocyte-like pattern. Electron microscopic studies revealed densin localization in the slit diaphragm area. Due to its known involvement in the synaptic organization, maintenance of cell shape and polarity in nerve cells, together with its demonstrated interactions with alpha-actinin-4, densin may share the same functions in podocytes by associating with the nephrin interacting protein complex at the slit diaphragm.

Trichoplein and Aurora A block aberrant primary cilia assembly in proliferating cells

The trichoplein–AurA pathway must suppress primary cilia assembly in order for cells to exit G1.

Aurora‐B and Rho‐kinase/ROCK, the two cleavage furrow kinases, independently regulate the progression of cytokinesis: possible existence of a novel cleavage furrow kinase phosphorylates ezrin/radixin/moesin (ERM)

Cytokinesis is regulated by several protein kinases, such as Aurora‐B and Rho‐kinase/ROCK. We have indicated that these two kinases are the cleavage furrow (CF) kinases that accumulate at the cleavage furrow and phosphorylate several intermediate filament (IF) proteins into two daughter cells. It has been reported that Aurora‐B phosphorylates MgcRacGAP to functionally convert to a RhoGAP during cytokinesis. Therefore, we investigated here the relationship between Aurora‐B and Rho‐kinase/ROCK in cytokinesis, by using small interfering RNA (siRNA) technique. Aurora‐B depletion did not alter the cleavage furrow‐specific localization of Rho‐kinase/ROCK and vice versa. Treatment of Aurora‐B or Rho‐kinase/ROCK siRNA increased multinucleate cells, and the effect of double depletion was additive. Aurora‐B depletion induced the reduction of cleavage furrow‐specific phosphorylation of vimentin at Ser72 but not vimentin at Ser71, myosin light chain (MLC) at Ser19, and myosin binding subunit of myosin phosphatase (MBS) at Ser852. In contrast, Rho‐kinase/ROCK depletion led to the reduction of cleavage furrow‐specific phosphorylation of MLC at Ser19, MBS at Ser852, and vimentin at Ser71 but not vimentin at Ser72. Cleavage furrow‐specific ezrin/radixin/moesin (ERM) phosphorylation was not altered in the Aurora‐B‐ and/or Rho‐kinase/ROCK‐depleted cells. In addition, C3 or toxin B treatment did not abolish ERM phosphorylation at the cleavage furrow in cells attaining cytokinesis. These results suggest that Aurora‐B and Rho‐kinase/ROCK regulate the progression of cytokinesis without communicating to each other, and there may exist a novel protein kinase which phosphorylates ERM at the cleavage furrow.