Josip Lovrić

Protein kinase C-epsilon associates with the Raf-1 kinase and induces the production of growth factors that stimulate Raf-1 activity.

Several observations indicate that the Raf-1 kinase is a downstream effector of protein kinase C-ε (PKCε). We recently have shown that Raf-1 is constitutively activated in PKCε transformed Rat6 fibroblasts, and transformation can be reverted by expression of a dominant negative Raf-1, but not a dominant negative Ras mutant (Cacace et al., 1996). Cai et al. (1997) demonstrated that PKCε induced proliferation of NIH3T3 cells is independent of Ras or Src, but depends on Raf-1. These authors further suggested that PKCε activates Raf-1 by direct phosphorylation. Here we have investigated the functional interaction between PKCε and Raf-1. PKCε, but not PKCα, was found to bind to the Raf-1 kinase domain. The association appeared to be direct, as it could be reconstituted in vitro with purified proteins. Raf-1 and PKCε could be co-precipitated from Sf-9 insect cells and PKCε transformed NIH313 cells (NIH/ε). The association was negatively regulated by ATP in vitro and by TPA treatment in NIH/ε cells, but not in Sf-9 insect cells. Raf-1 was constitutively activated in NIH/ε cells. However, using coexpression experiments in Sf-9 cells and transiently transfected A293 cells we did not obtain any evidence for a direct activation of Raf-1 by PKCε. PKCε did not induce translocation of Raf-1 to the membrane. Furthermore, PKCε did not activate Raf-1 nor enhance the kinase activity of Raf-1 that had been pre-activated by coexpression of Ras or the Lck tyrosine kinase. In contrast, conditioned media from PKCε transformed cells induced a robust activation of Raf-1. This activation could be partially reproduced by recombinant TGFβ, a growth factors secreted by PKCε transformed Rat6 cells. In conclusion, our results suggest that PKCε stimulates Raf-1 indirectly by inducing the production of autocrine growth factors.

Lamin B1 maintains the functional plasticity of nucleoli.

The dynamic ability of genomes to interact with discrete nuclear compartments appears to be essential for chromatin function. However, the extent to which structural nuclear proteins contribute to this level of organization is largely unresolved. To test the links between structure and function, we evaluated how nuclear lamins contribute to the organization of a major functional compartment, the nucleolus. HeLa cells with compromised expression of the genes encoding lamins were analyzed using high-resolution imaging and pull-down assays. When lamin B1 expression was depleted, inhibition of RNA synthesis correlated with complex structural changes within the nucleolar active centers until, eventually, the nucleoli were dispersed completely. With normal lamin expression, the nucleoli were highly plastic, with dramatic and freely reversible structural changes correlating with the demand for ribosome biogenesis. Preservation of the nucleolar compartment throughout these structural transitions is shown to be linked to lamin B1 expression, with the lamin B1 protein interacting with the major nucleolar protein nucleophosmin/B23.

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 ki‐nases. 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 poly‐merization 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.—Janosch, P., Kieser, A., Eulitz, M., Lovric, J., Sauer, G., Reichert, M., Gounari, F., Büscher, D., Baccarini, M., Mischak, H., Kolch, W. The Raf‐1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments. FASEB J. 14, 2008–2021 (2000)

Identification of Epstein-Barr Virus (EBV) Nuclear Antigen 2 (EBNA2) Target Proteins by Proteome Analysis: Activation of EBNA2 in Conditionally Immortalized B Cells Reflects Early Events after Infection of Primary B Cells by EBV

ABSTRACT The Epstein-Barr virus (EBV) is a ubiquitous B-lymphotropic herpesvirus associated with several malignant tumors, e.g., Burkitts lymphoma and Hodgkins disease, and is able to efficiently immortalize primary B lymphocytes in vitro. The growth program of infected B cells is initiated and maintained by the viral transcription factor EBV nuclear antigen 2 (EBNA2), which regulates viral and cellular genes, including the proto-oncogene c-myc. In our study, patterns of protein expression in B cells with and without EBNA2 were analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. For this purpose, we used a conditional immortalization system for EBV, a B cell line (EREB2-5) that expresses an estrogen receptor-EBNA2 fusion protein. In order to discriminate downstream targets of c-Myc from c-Myc-independent EBNA2 targets, we used an EREB2-5-derived cell line, P493-6, in which c-Myc is expressed under the control of a tetracycline-regulated promoter. Of 20 identified EBNA2 target proteins, 11 were c-Myc dependent and therefore most probably associated with proliferation, and one of these proteins was a posttranslationally modified protein, i.e., hypusinylated eIF5a. Finally, to estimate the relevance of EBNA2 targets during early EBV infection, we analyzed the proteomes of primary B cells before and after infection with EBV. The protein expression pattern induced upon EBV infection was similar to that following EBNA2 activation. These findings underscore the value of EREB2-5 cells as an appropriate model system for the analysis of early events in the process of EBV-mediated B-cell immortalization.

Changed Genome Heterochromatinization Upon Prolonged Activation of the Raf/ERK Signaling Pathway

The Raf/ERK (Extracellular Signal Regulated Kinase) signal transduction pathway controls numerous cellular processes, including growth, differentiation, cellular transformation and senescence. ERK activation is thought to involve complex spatial and temporal regulation, to achieve a high degree of specificity, though precisely how this is achieved remains to be confirmed. We report here that prolonged activation of a conditional form of c-Raf-1 (BXB-ER) leads to profound changes in the level and distribution of a heterochromatic histone mark. In mouse fibroblasts, the heterochromatic trimethylation of lysine 9 in histone H3 (H3K9Me3) is normally confined to pericentromeric regions. However, following ERK activation a genome-wide redistribution of H3K9Me3 correlates with loss of the histone modification from chromocentres and the appearance of numerous punctuate sites throughout the interphase nucleus. These epigenetic changes during interphase correlate with altered chromosome structure during mitosis, where robust H3K9Me3 signals appear within telomeric heterochromatin. This pattern of heterochromatinization is distinct from previously described oncogene induced senescence associated heterochromatin foci (SAHF), which are excluded from telomeres. The H3K9Me3 histone mark is known to bind the major heterochromatin protein HP1 and we show that the alterations in the distribution of this histone epistate correlate with redistribution of HP1β throughout the nucleus. Interestingly while ERK activation is fully reversible, the observed chromatin changes induced by epigenetic modifications are not reversible once established. We describe for the first time a link from prolonged ERK activation to stable changes in genome organization through redistribution of heterochromatic domains involving the telomeres. These epigenetic changes provide a possible mechanism through which prolonged activation of Raf/ERK can lead to growth arrest or the induction of differentiation, senescence and cancer.

Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics

The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-l-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles.

MEK1 mediates a positive feedback on Raf-1 activity independently of Ras and Src

Growth factor stimulated receptor tyrosine kinases activate a protein kinase cascade via the serine/threonine protein kinase Raf-1. Direct upstream activators of Raf-1 are Ras and Src. This study shows that MEK1, the direct downstream effector of Raf-1, can also stimulate Raf-1 kinase activity by a positive feedback loop. Activated MEK1 mediates hyperphosphorylation of the amino terminal regulatory as well as of the carboxy terminal catalytic domain of Raf-1. The hyperphosphorylation of Raf-1 correlates with a change in the tryptic phosphopeptide pattern only at the carboxy terminus of Raf-1 and an increase in Raf-1 kinase activity. MEK1-mediated Raf-1 activation is inhibited by co-expression of the MAPK specific phosphatase MKP-1 indicating that the MEK1 effect is exerted through a MAPK dependent pathway. Stimulation of Raf-1 activity by MEK1 is independent of Ras, Src and tyrosine phosphorylation of Raf-1. MEK1 can however synergize with Ras and leads to further increase of the Raf-1 kinase activity. Thus, MEK1 can mediate activation of Raf-1 by a novel positive feedback mechanism which allows fast signal amplification and could prolong activation of Raf-1.

Identification of the smooth muscle-specific protein, sm22, as a novel protein kinase C substrate using two-dimensional gel electrophoresis and mass spectrometry

We report a novel method to identify protein kinase C (PKC) substrates. Tissue lysates were fractionated by ion exchange chromatography and used as substrates in in vitro kinase reactions. The phosphorylated proteins were separated using two‐dimensional gel electrophoresis. Spots that contained isolated phosphoproteins were excised and digested with trypsin. The tryptic peptides were analyzed using mass spectrometry. While several of the proteins identified using this technique represent known PKC substrates, we identified a new PKC substrate in the initial screen. This protein, sm22, is expressed in smooth muscle cells and served well as a substrate for PKC in vitro. Sm22 is predominantly associated with the actin cytoskeleton. Upon activation of PKC in vivo, sm22 dissociates from the actin cytoskeleton and is distributed diffusely in the cytoplasm. Our data strongly suggest that phosphorylation by PKC controls the intracellular localization of sm22. This demonstrates that our approach, using a complex mixture of proteins as in vitro kinase substrates and subsequently identifying the newly phosphorylated proteins by mass spectrometry, is a powerful method to identify new kinase substrates.

Reduced Expression of Lamin A/C Results in Modified Cell Signaling and Metabolism Coupled with Changes in Expression of Structural Proteins

Nuclear lamins are intermediate filament proteins that define the shape and stability of nuclei in mammalian cells. In addition to this dominant structural role, recent studies have suggested that the lamin proteins also regulate fundamental aspects of nuclear function. In order to understand different roles played by lamin proteins, we used RNA interference to generate a series of HeLa cell lines to study loss-of-function phenotypes associated with depletion of lamin protein expression. In this study, we used genome-wide proteomic approaches to monitor global changes in protein expression in cells with <10% of normal lamin A/C expression. Of approximately 2000 protein spots analyzed by two-dimensional electrophoresis, only 38 showed significantly altered expression in lamin A/C depleted cells. Of these, 4 protein spots were up-regulated, and 34 were down-regulated. Significant changes were seen to involve the general reduction in expression of cytoskeletal proteins, consistent with altered functionality of the structural cellular networks. At the same time, alterations in expression of proteins involved in cellular metabolism correlated with altered patterns of metabolic activity. In order to link these two features, we used antibody microarrays to perform a focused analysis of expression of cell cycle regulatory proteins. This confirmed a general reduction in expression of proteins regulating cell cycle progression and alteration in signaling pathways that regulate the metabolic activity of cells. The cross-talk between signal transduction and the cytoskeleton emphasizes how structural and kinase-based networks are integrated in mammalian cells to fine-tune metabolic responses.