Role of stress fibers in the association of intermediate filaments with microtubules in fibroblast cells.

The association between intermediate filaments (IF) and microtubules (MT) has been demonstrated by several experiments using MT inhibitors and by microinjecting specific antibodies. The actin cytoskeleton has recently been assigned a role in this process of drug induced IF collapse. However, this was not found to be true in large cells with irregular morphology. For instance, in early passage diploid fibroblasts of human origin and in armadillo cell lines, where the cells are large, irregular in shape and exhibit prominent stress fibers ( SF ), depolymerization of MT with nocodazole did not lead to collapse of IF . Instead, the IF formed bundles of coils that seemed to associate with the SF . Disintegration of the SF with cytochalasin B led to the collapse of the IF . It appears that the actin organization in such large cells with extensive SF , is not as contractile as in typical spindle shaped fibroblasts which have relatively less stable actin organization. The stable SF may actually prevent IF collapse.     

Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.     

Protein kinase C signaling and oxidative stress

Oxidative stress is involved in the pathogenesis of various degenerative diseases including cancer. It is now recognized that low levels of oxidants can modify cell-signaling proteins and that these modifications have functional consequences. Identifying the target proteins for redox modification is key to understanding how oxidants mediate pathological processes such as tumor promotion. These proteins are also likely to be important targets for chemopreventive antioxidants, which are known to block signaling induced by oxidants and to induce their own actions. Various antioxidant preventive agents also inhibit PKC-dependent cellular responses. Therefore, PKC is a logical candidate for redox modification by oxidants and antioxidants that may in part determine their cancer-promoting and anticancer activities, respectively. PKCs contain unique structural features that are susceptible to oxidative modification. The N-terminal regulatory domain contains zinc-binding, cysteine-rich motifs that are readily oxidized by peroxide. When oxidized, the autoinhibitory function of the regulatory domain is compromised and, consequently, cellular PKC activity is stimulated. The C-terminal catalytic domain contains several reactive cysteines that are targets for various chemopreventive antioxidants such as selenocompounds, polyphenolic agents such as curcumin, and vitamin E analogues. Modification of these cysteines decreases cellular PKC activity. Thus the two domains of PKC respond differently to two different type of agents: oxidants selectively react with the regulatory domain, stimulate cellular PKC, and signal for tumor promotion and cell growth. In contrast, antioxidant chemopreventive agents react with the catalytic domain, inhibit cellular PKC activity, and thus interfere with the action of tumor promoters.     

Protein kinase C zeta associates with death inducing signaling complex and regulates Fas ligand-induced apoptosis

Previous studies have shown that Protein kinase C (PKC) stimulation may interfere with Fas signaling pathway and Fas ligand (FasL)-induced apoptosis. In this study, we investigated in Jurkat cells, a FasL-sensitive human T-cell model, whether PKC(zeta) targets apical events of Fas signaling. We describe for the first time that in Jurkat cells, both PKC(zeta) and Prostate apoptosis response-4 (Par-4), one of the major endogenous PKC(zeta) regulators, are components of the death inducing signaling complex (DISC). Using PKC(zeta) overexpressing cells or si-RNA depletion, we demonstrate that PKC(zeta) interferes neither with Fas expression nor Fas clustering in raft microdomains, but negatively regulates FasL-induced apoptosis by interfering with DISC formation and subsequent caspase-8 processing.     

Protein kinase C as a stress sensor

While there are many reviews which examine the group of proteins known as protein kinase C (PKC), the focus of this article is to examine the cellular roles of two PKCs that are important for stress responses in neurological tissues (PKC gamma and epsilon) and in cardiac tissues (PKC epsilon). These two kinases, in particular, seem to have overlapping functions and interact with an identical target, connexin 43 (Cx43), a gap junction protein which is central to proper control of signals in both tissues. While PKC gamma and PKC epsilon both help protect neural tissue from ischemia, PKC epsilon is the primary PKC isoform responsible for responding to decreased oxygen, or ischemia, in the heart. Both do this through Cx43. It is clear that both PKC gamma and PKC epsilon are necessary for protection from ischemia. However, the importance of these kinases has been inferred from preconditioning experiments which demonstrate that brief periods of hypoxia protect neurological and cardiac tissues from future insults, and that this depends on the activation, translocation, or ability for PKC gamma and/or PKC epsilon to interact with distinct cellular targets, especially Cx43. This review summarizes the recent findings which define the roles of PKC gamma and PKC epsilon in cardiac and neurological functions and their relationships to ischemia/reperfusion injury. In addition, a biochemical comparison of PKC gamma and PKC epsilon and a proposed argument for why both forms are present in neurological tissue while only PKC epsilon is present in heart, are discussed. Finally, the biochemistry of PKCs and future directions for the field are discussed, in light of this new information.     

The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments.

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.     

Protein kinase C delta.

The protein kinase C (PKC) family consists of 11 isoenzymes that, due to structural and enzymatic differences, can be subdivided into three groups: The Ca(2+)-dependent, diacylglycerol (DAG)-activated cPKCs (conventional PKCs: alpha, beta 1, beta 2, gamma); the Ca(2+)-independent, DAG-activated nPKCs (novel PKCs: delta, epsilon, eta, theta, mu), and the Ca(2+)-dependent, DAG non-responsive aPKCs (atypical PKCs: zeta, lambda/iota). PKC mu is a novel PKC, but with some special structural and enzymatic properties.     

Engagement of vimentin intermediate filaments in hypotonic stress

Intermediate filaments (IFs) play a key role in the control of cell structure and morphology, cell mechano-responses, migration, proliferation, and apoptosis. However, the mechanisms regulating IFs organization in motile adhesive cells under certain physical/pathological conditions remain to be fully understood. In this study, we found hypo-osmotic–induced stress results in a dramatic but reversible rearrangement of the IF network. Vimentin and nestin IFs are partially depolymerized as they are redistributed throughout the cell cytoplasm after hypo-osmotic shock. This spreading of the IFs requires an intact microtubule network and the motor protein associated transportation. Both nocodazole treatment and depletion of kinesin-1 (KIF5B) block the hypo-osmotic shock–induced rearrangement of IFs showing that the dynamic behavior of IFs largely depends on microtubules and kinesin-dependent transport. Moreover, we show that cell survival rates are dramatically decreased in response to hypo-osmotic shock, which was more severe by vimentin IFs depletion, indicating its contribution to osmotic endurance. Collectively, these results reveal a critical role of vimentin IFs under hypotonic stress and provide evidence that IFs are important for the defense mechanisms during the osmotic challenge.     

Vimentin intermediate filaments protect mitochondria from oxidative stress

The role of intermediate filaments (IFs) in eukaryotic cells is still unclear. The disturbance of mitochondria distribution and function, in particular the enhanced production of reactive oxygen species (ROS) and decreased membrane potential, is observed in cells devoid of IFs. The aim of this work was to study the dependence of mitochondria sensitivity to oxidative stress on the presence of vimentin IFs. It was found that mitochondria are less sensitive to ROS in cells containing vimentin than in cells devoid of vimentin. Besides, mitochondrial membrane potential was demonstrated to increase upon regeneration of vimentin IFs in the cells. Substitution of Pro-57 by Arg in N-terminal part of the vimentin molecule abandoned its protective ability and the effect on mitochondrial membrane potential.     

Bruton's tyrosine kinase (Btk) associates with protein kinase C mu

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.     

Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV

Type I and III interferons induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (“biomolecular condensates”). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.     

Protein kinase C isotypes in C. elegans

The protein kinase C (PKC) family, consisting of multiple isotypes, plays a major role in cellular signaling. In the nematode Caenorhabditis elegans, four pkc genes, tpa-1, pkc-1, pkc-2 and pkc-3, have been identified and investigated. Molecular analysis of tpa-1, pkc-1, and pkc-2 has shown that each gene encodes multiple PKC isoforms with different expression patterns. One of the tpa-1 isoforms, which is expressed in vulval cells, is found to play a role in nicotine-induced adaptation. The expression of pkc-1 seems to be specific to neurons, while that of pkc-2 is detected in several types of cells including neurons and muscle cells. An aPKC member encoded by pkc-3 has been shown to play an essential role in establishing the polarity of the zygote. Recent studies have revealed that the mechanism of polarity establishment mediated by aPKC is evolutionarily conserved in diverse organisms from nematodes to mammals. C. elegans provides an excellent model system for molecular dissection of the cellular signaling pathways involving PKC.     

Association of the beta isoform of protein kinase C with vimentin filaments.

Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305-312, 1986; Nature 334:661-665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621-623, 1983). The beta isoform of PKC is translocated and degraded much more rapidly than the alpha isoform in phorbolester-stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238-4243, 1989). We report here immunofluorescence evidence that the distributions of PKC alpha and beta are strikingly different in antigen-activated RBL cells. PKC beta associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC alpha concentrates in regions of the cell periphery. This distribution of PKC beta is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti-tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC beta and to the intermediate filament protein vimentin are almost identical, indicating that PKC beta associates with vimentin filaments. These bundles of 100 A filaments may provide docking sites for interactions of PKC beta with its substrates and thus confer specificity to the actions of this isoform.     

Casein kinase 2 associates with and phosphorylates dishevelled.

The dishevelled (dsh) gene of Drosophila melanogaster encodes a phosphoprotein whose phosphorylation state is elevated by Wingless stimulation, suggesting that the phosphorylation of Dsh and the kinase(s) responsible for this phosphorylation are integral parts of the Wg signaling pathway. We found that immunoprecipitated Dsh protein from embryos and from cells in tissue culture is associated with a kinase activity that phosphorylates Dsh in vitro. Purification and peptide sequencing of a 38 kDa protein co-purifying with this kinase activity showed it to be identical to Drosophila Casein Kinase 2 (CK2). Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by CK2 in vitro and in vivo, suggesting that CK2 is at least one of the kinases that phosphorylates Dsh. Overexpression of Dfz2, a Wingless receptor, also stimulated phosphorylation of Dsh, Dsh-associated kinase activity, and association of CK2 with Dsh, thus suggesting a role for CK2 in the transduction of the Wg signal.     

Diacylglycerol kinase δ associates with receptor for activated C kinase 1, RACK1

The δ-isozyme (type II) of diacylglycerol kinase (DGK) is known to positively regulate growth factor receptor signaling. DGKδ, which is distributed to clathrin-coated vesicles, interacts with DGKδ itself, protein kinase C and AP2α. To search for additional DGKδ-interacting proteins, we screened a yeast two-hybrid cDNA library from HepG2 cells using aa 896–1097 of DGKδ as a bait. We identified aa 184–317 (WD40 repeats 5–7) of receptor for activated C kinase 1 (RACK1), which interacts with various important signaling molecules, as a novel binding partner of DGKδ. Co-immunoprecipitation analysis, using COS-7 cells co-expressing RACK1 and DGKδ, revealed that RACK1 selectively interacted with DGKδ, but not with type I DGKs, in mammalian cells. The interaction was dynamically regulated by phorbol ester. Intriguingly, DGKδ appeared to recruit RACK1 to clathrin-coated vesicles and co-localized with RACK1. These results suggest that DGKδ serves as an adaptor protein to regulate the localization of the versatile scaffold protein, RACK1.     

Polycystin-1 interacts with intermediate filaments

Polycystin-1, the protein defective in a majority of patients with autosomal dominant polycystic kidney disease, is a ubiquitously expressed multi-span transmembrane protein of unknown function. Subcellular localization studies found this protein to be a component of various cell junctional complexes and to be associated with the cytoskeleton, but the specificity and nature of such associations are not known. To identify proteins that interact with the polycystin-1 C-tail (P1CT), this segment was used as bait in a yeast two-hybrid screening of a kidney epithelial cell library. The intermediate filament (IF) protein vimentin was identified as a strong polycystin-1-interacting partner. Cytokeratins K8 and K18 and desmin were also found to interact with P1CT. These interactions were mediated by coiled-coil motifs in polycystin-1 and IF proteins. Vimentin, cytokeratins K8 and K18, and desmin also bound directly to P1CT in GST pull-down and in in vitro filament assembly assays. Two observations confirmed these interactions in vivo: (i) a cell membrane-anchored form of recombinant P1CT decorated the IF network and was found to associate with the cytoskeleton in detergent-solubilized cells and (ii) endogenous polycystin-1 distributed with IF at desmosomal junctions. Polycystin-1 may utilize this association for structural, storage, or signaling functions.