The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain.

The protein kinase Bcr is a negative regulator of cell proliferation and oncogenic transformation. We identified Bcr as a ligand for the PDZ domain of the cell junction and Ras-interacting protein AF-6. The Bcr kinase phosphorylates AF-6, which subsequently allows efficient binding of Bcr to AF-6, showing that the Bcr kinase is a regulator of the PDZ domain-ligand interaction. Bcr and AF-6 colocalize in epithelial cells at the plasma membrane. In addition, Bcr, AF-6, and Ras form a trimeric complex. Bcr increases the affinity of AF-6 to Ras, and a mutant of AF-6 that lacks a specific phosphorylation site for Bcr shows a reduced binding to Ras. Wild-type Bcr, but not Bcr mutants defective in binding to AF-6, interferes with the Ras-dependent stimulation of the Raf/MEK/ERK pathway. Since AF-6 binds to Bcr via its PDZ domain and to Ras via its Ras-binding domain, we propose that AF-6 functions as a scaffold-like protein that links Bcr and Ras to cellular junctions. We suggest that this trimeric complex is involved in downregulation of Ras-mediated signaling at sites of cell-cell contact to maintain cells in a nonproliferating state.

Specific association of Mil/Raf proteins with a 34 kDa phosphoprotein.

Mil/Raf protein kinases are intermediates in signaling pathways leading to differentiation, mitogenesis and cellular transformation. To gain insight into the activity of Mil/Raf kinases at the molecular level we aimed to identify proteins specifically interacting with Mil/Raf proteins. A phosphoprotein of 34 kDa (pp34) was found to be associated with c-Raf as well as with viral and activated forms of Mil/Raf proteins in exponentially growing interphase cells. pp34 association was not detectable in mitotic cells. Serum stimulation or coexpression of activated Ras led to decreased electrophoretic mobility of pp34 complexed to Mil/Raf proteins while serum starvation rendered pp34 undetectable. Moreover, the association with pp34 became undetectable in parallel with the onset of morphological cellular transformation caused by overexpression of a constitutively activated mutant of c-Raf in an inducible expression system. Thus, the association of Mil/Raf proteins with pp34 is altered in the course of cell cycle progression, serum stimulation and cellular transformation. These events represent hallmarks of cellular Mil/Raf functions, rendering pp34 a candidate protein involved in Mil/Raf function

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.

Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1.

The serine/threonine protein kinase c-Raf-1 interacts with a number of cellular proteins including 14-3-3 isoforms which may be regulators or substrates of c-Raf-1 in signal transduction pathways. In vivo and in vitro binding analyses of c-Raf-1 and mutant proteins with 14-3-3 zeta indicate bivalent binding of 14-3-3 zeta to the amino terminus as well as to the carboxy terminus of c-Raf-1. Although 14-3-3 zeta and Ras use different binding regions on the amino terminal regulatory domain of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino terminus upon binding of activated Ras. In contrast, if c-Raf-1 full length is analysed instead of the separately expressed c-Raf-NT, binding of 14-3-3 zeta is only slightly effected by co-expression of activated Ras. This is explained by a second binding site of 14-3-3 zeta at the carboxy terminus of c-Raf-1. The mutant c-Raf-NT (S259A) cannot bind 14-3-3 zeta, suggesting a regulatory role of this in vivo phosphorylation site. However, c-Raf-NT phosphorylated or unphosphorylated at S259, is able to bind 14-3-3 zeta. Even though 14-3-3 zeta can be phosphorylated in vivo, only the unphosphorylated form binds to the amino terminus of c-Raf-1. The data presented indicate, that 14-3-3 zeta binds to c-Raf-1 in a bivalent fashion in unstimulated cells. 14-3-3 zeta is displaced from the amino terminus but not from the carboxy terminus of c-Raf-1 by binding of activated Ras to c-Raf-1.

Negative regulation of Raf activity by binding of 14-3-3 to the amino terminus of Raf in vivo.

In the developing eye of Drosophila the protein kinase D-Raf controls the specification of the R7 photoreceptor cells. We show that overexpression of wild-type D-Raf inhibits the formation of R7 cells in a dose-dependent manner. Conversely, overexpression of mutant D-Raf proteins in which the conserved S388 is replaced by A or by D promotes the formation of supernumerary R7 cells, indicating increased D-Raf activity in vivo. S388 in D-Raf corresponds to S259 in c-Raf; shown to be involved in binding of 14-3-3. We show that analogous substitutions of S259 in c-Raf prevent binding of 14-3-3 zeta to the amino terminus of c-Raf and cause a Ras-independent constitutively increased c-Raf kinase activity. Binding of 14-3-3 zeta to the second binding site at the carboxy terminal catalytic domain was unaffected by these mutations. These results suggest that the increased kinase activity of mutant D-Raf is caused by the selective loss of 14-3-3 binding to its amino terminus. Therefore, binding of 14-3-3 to the amino terminus of Raf appears to negatively regulate Raf kinase activity in vivo.

Retroviral gene transfer of dominant negative raf-1 mutants suppresses ha-ras-induced transformation and delays tumor formation.

Activating mutants of ras are among the most frequently found genetic alterations in human cancers. Therefore, Ras appears to be an attractive target for therapeutic intervention using gene transfer. The protein kinase Raf-1 acts as a direct downstream effector of Ras and is involved in Ras-induced cellular transformation. Using the NIH3T3 fibroblast-derived tumor cell line PEJ, which expresses oncogenic Ha-rasG12V, we analyzed whether dominant negative mutants of Raf-1 can inhibit Ras-mediated transformation. Retroviral gene transfer was used to stably transduce PEJ cells with three different dominant negative mutants of Raf-1. This resulted in reversion of the transformed phenotype in vitro as evidenced by an increase in contact inhibition and reduced anchorage-independent growth. However, tumor formation in nude mice was significantly delayed only by one of these mutants. Therefore, dominant negative mutants of the oncoprotein Myc, which is known to synergize with Raf-1 in tumor formation, were transduced into PEJ cells expressing a dominant negative Raf mutant. This leads to killing of the cells. These results indicate that although interference with Ras-induced transformation using dominant negative mutants of Raf is feasible and effective in vitro using retroviral vectors, an additional block (e.g., that of Myc) is necessary to kill PEJ cells. These results also indicate that interference with Ras-dependent signaling is not sufficient for inhibition of tumor formation of PEJ cells in vivo.

Signal transduction as target of gene therapy.

Cancer development involves multistep events. Therapeutic approaches can be targeted against any of these events individually or in combination. Pancreatic cancer can involve activation of Ki-Ras, overexpression of Myc or ErbB-2, and mutational inactivation of functional p53. Three approaches with nucleic acid-based therapies have been taken. A ribozyme specific for Ki-ras mRNA carrying the activating mutation in codon 12 (GGU to GUU) was designed and shown to be stimulated by interaction with an RNA-binding protein NCp7 of HIV-1. The same protein was targeted to cell-adhesion molecules, which allowed transfer of the ribozyme and an antisense oligodeoxynucleotide (ODN) into the cell. Furthermore, proliferation may be prevented by blocking signal transduction. A transdominant negative mutant of the signaling kinase c-Raf-1 efficiently blocked transmission. A retroviral vector will be used as carrier. Furthermore, the concept of using naked DNA injected intramuscularly as an immunogen against cancer is discussed.

CNK1 is a novel Akt interaction partner that promotes cell proliferation through the Akt-FoxO signalling axis.

The scaffold proteins connector enhancer of KSR (CNK) participate in Raf-, Rho- and NF-kappaB-dependent signalling and promote cell differentiation and invasion. In this study, we demonstrate that CNK1 downregulation inhibits, whereas CNK1 overexpression stimulates the proliferation of breast cancer cells and human embryonic kidney cells, respectively. This stimulatory effect depends on a functional phosphatidylinositol-3 kinase (PI3K) pathway because treatment of cells with the PI3K inhibitor, LY294002, abrogates CNK1-induced proliferation. CNK1 interacts with the PI3K effector Akt and knockdown of CNK1 decreases Akt activity in breast cancer cells. CNK1 controls Akt-dependent phosphorylation and transcriptional activity of FoxO, which is a negative regulator of proliferation. Consistent with this, CNK1-induced cell proliferation is blocked by FoxO overexpression. Moreover, CNK1 regulates anchorage-independent proliferation and focus formation of breast cancer cells. CNK1 is predominantly localized at the plasma membrane of breast cancer cells, whereas in non-transformed mammary epithelial cells, CNK1 is cytoplasmatic. Accordingly, CNK1 is found preferentially at the plasma membrane in carcinoma in situ and invasive breast cancer tumours compared with normal breast tissue sections. Analysis of multiple breast cancer samples reveals that CNK1-negative tumours show less Akt activity. Thus, CNK1 promotes oncogenic signalling through Akt in breast cancer cell lines and tumours.

Complementation analysis of mutants defective in different steps of HBV reverse transcription.

The HBV P gene encodes a multifunctional polyprotein which contains the major enzymatic activities required for hepadnaviral reverse transcription (protein primer, DNA polymerase, and RNase H). Mutant HBV genomes unable to synthesize fully functional P gene products were analysed for their potential to be rescued by a second mutant HBV genome that either contained a wild type P gene (intergenic complementation) or a mutation in a functionally different P gene domain (intragenic complementation). This analysis was carried out by cotransfecting two mutants at a time into HepG2 cells and assaying for the production of core particles containing DNA polymerase activity. The results obtained demonstrate the existence of intergenic, but not of intragenic complementation. This indicates that the primary P gene product is not processed before core assembly, and furthermore that there is a rather free mixing of all HBV gene products in the HBV infected cell which can lead to the efficient propagation of defective viral genomes.

Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity.

To correlate the hepatitis B virus P gene with the enzymatic activities predicted to participate in hepadnavirus reverse transcription, a series of P gene mutants containing missense mutations, in-phase insertions, and in-phase deletions was constructed by site-directed mutagenesis. These mutants were tested in the context of otherwise intact hepatitis B virus genomes for the ability to produce core particles containing the virus-associated polymerase activity. The results obtained suggest that the P protein consists of three functional domains and a nonessential spacer arranged in the following order: terminal protein, spacer, reverse transcriptase/DNA polymerase, and RNase H. The first two domains are separated by a spacer region which could be deleted to a large extent without significant loss of endogenous polymerase activity. In cotransfection experiments, all P gene mutants could be complemented in trans by constructs expressing the wild-type gene product but not by a second P gene mutant. This indicates that the multifunctional P gene is expressed as a single translational unit and independent of the core gene and furthermore that the gene product is freely diffusible and not processed before core assembly.

Direct interaction and N-terminal phosphorylation of c-Jun by c-Mil/Raf.

c-Mil is the avian homologue of the mammalian serine/threonine kinase c-Raf-1. c-Mil/Raf is a mediator of signal transduction leading to gene expression via the c-Jun DNA-binding site, AP-1. Here we show that c-Mil immunopurified from MC29-virus-transformed quail fibroblasts phosphorylates c-Jun in vitro near its N terminus (Ser-63 and -73). Furthermore, the viral oncogene product Gag-Mil of the avian wild-type retrovirus MH2 phosphorylates c-Jun in vitro. A contribution by other known kinases phosphorylating c-Jun, such as the mitogen-activated protein kinases (MAPKs) and the c-Jun N-terminal kinases, was excluded by control reactions. c-Raf-1 and c-Jun directly interact in vitro as shown by various immobilized glutathione S-transferase-Raf fusion proteins which specify the cysteine-rich region of c-Mil/Raf as the major N-terminal binding site. An additional minor binding site is located in the C-terminal region. The biological relevance of these results is demonstrated by coimmunoprecipitation of c-Jun and c-Mil from 32P-labeled MC29- and MH2-transformed fibroblasts as well as normal quail embryo fibroblasts, whereby c-Jun was identified by tryptic phosphopeptide analysis. The complexed c-Jun exhibits a decreased electrophoretic mobility corresponding to a more highly phosphorylated state. Cell fractionation analyses indicate that the c-Mil/c-Jun complex is located in the cytoplasm. The data demonstrate that c-Jun can be a direct target of the protein kinase c-Mil/Raf, suggesting an alternative pathway, which leads to c-Jun phosphorylation independent of the MAPKs and MAPK-related proteins.

A small peptide derived from the aminoterminus of c-Raf-1 inhibits c-Raf-1/Ras binding.

Various domains of the aminoterminal part of c-Raf-1 expressed as glutathione-S-transferase fusion proteins were analyzed for Ras binding. The binding site was localized at the aminoterminus outside of the cysteine-rich region. A single aminoacid exchange at aminoacid residue 89 (Arg89 to Leu) of c-Raf-1 inhibits binding. A small synthetic peptide corresponding to c-Raf-1 aminoacids 77 to 101 comprising Arg89 in a central position competes for Ras binding and thereby characterizes the relevant binding domain of Ras on c-Raf-1.

The aminoterminus of c-Raf-1 binds a protein kinase phosphorylating Ser259.

The kinase negative aminoterminal domain of c-Raf-1 expressed as glutathione S-transferase fusion protein was phosphorylated in vitro after treatment with lysates from A431 cells and subsequent in vitro protein kinase assay. This phosphorylation was independent of stimulation of the cells with EGF; it occurred exclusively on serine and was mapped to Ser259. The identical site of c-Raf-1 was phosphorylated in A431 cells by metabolic labelling in vivo. The kinase binding domain was mapped by various GST-Raf deletion mutants to c-Raf-1 aminoacid residues 181 to 255.

The duck hepatitis B virus DNA polymerase is tightly associated with the viral core structure and unable to switch to an exogenous template.

The duck hepatitis B virus (DHBV) has a DNA polymerase associated with it which uses the incomplete viral genome as endogenous template. A prerequisite for studying this polymerase is the availability of conditions to open viral cores without destroying their enzymatic activity. In this study, this was achieved by a brief treatment with low pH. DHBV DNA in low-pH-treated cores was susceptible to digestion with deoxyribonuclease I and restriction enzymes, and large restriction fragments diffused out of the viral cores. However, the DHBV polymerase remained tightly associated with its DNA template in the viral core structure and could still incorporate nucleotides into those DNA fragments which carried the DNA-bound protein and remained in the core. The DHBV polymerase could not switch to any of several exogenously supplied templates although these were most likely accessible to it. The manner in which this tight association of the DHBV polymerase with the core may occur, and the possible implications of this interaction during the DHBV replication cycle, is discussed.

The duck hepatitis B virus P-gene codes for protein strongly associated with the 5'-end of the viral DNA minus strand.

A number of antisera, elicited against different segments of the duck hepatitis B virus (DHBV) P-gene translation product, were used to immunoprecipitate the protein that is covalently bound to the 5'-end of the DHBV DNA minus strand. For monitoring purposes, a small DNA minus-strand fragment, carrying this protein, was radioactively labeled. All of the P-specific antisera specifically immunoprecipitated this DNA fragment demonstrating that the protein species attached to the immunoprecipitated DNA fragment were products of the DHBV P-gene. The electrophoretic behavior, in SDS gels, of the DNA minus-strand fragment-protein complex indicated that it was present mostly in the form of aggregates. However, a small fraction consisted of DNA minus-strand fragments carrying P-gene proteins, encoded solely within the 5'-region of the P-gene. This indicated that different P-gene proteins, presumably covalently bound at a common region and subsequently processed, were bound to the 5'-end of the DHBV DNA minus strand. The DHBV P-gene presumably codes for the virus-associated reverse transcriptase and DNA polymerase activities. Using the P-gene-specific antisera, it was not possible to detect putative P-gene-coded polymerase proteins in a free form, i.e., not bound to viral DNA. This may be due to insufficient sensitivity or to the polymerase protein(s) being heterogeneous and/or aggregated. In addition, it is possible that the genome-bound protein itself may have polymerase activity.

Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins.

The expression strategy of the duck hepatitis B virus (DHBV) P gene, which is assumed to encode the viral reverse transcriptase, was investigated by mutational analysis. This study showed that P gene expression starts in the region where the P gene overlaps the viral core gene. However, in contrast to retroviral reverse transcriptases, which are expressed via gag-pol fusion protein intermediates, the DHBV P gene product was found to be synthesized starting at a P gene ATG codon. The resulting protein can complement polymerase-negative mutants in trans and can reverse transcribe viral pregenomic RNA that does not encode an active polymerase. These findings raise the question of how reverse transcription of cellular RNAs can be avoided in infected cells.

Inhibition of Raf/MAPK signaling in Xenopus oocyte extracts by Raf-1-specific peptides.

Raf-1 is an upstream element of the mitogen-activated protein kinase (MAPK) pathway which leads to cell proliferation and differentiation. In this study Raf-1 derived peptides comprising the conserved amino acid residues Arg89 and Ser259, involved in binding of activated Ras and 14-3-3 proteins, respectively, were shown to interfere with MAPK activation in extracts from immature Xenopus oocytes. Lipids prepared from oocyte extracts can stimulate MAPK in a Ras- and protein kinase C-independent manner. This lipid-induced MAPK activation is blocked by a Raf-1 derived peptide comprising Ser259.

Mitotic Raf-1 is stimulated independently of Ras and is active in the cytoplasm.

Raf-1 is a Ser/Thr protein kinase that is involved in regulation of proliferation, differentiation, and apoptosis. Recently, we and others showed that Raf-1 is not only activated in mitogenic pathways leading to cell cycle entry but also during mitosis. Transient expression studies in COS cells now demonstrate that, in contrast to growth factor-dependent activation of Raf-1, mitotic activation of Raf-1 is Ras-independent. Dominant negative RasS17N does not interfere with mitotic activation of Raf-1, whereas epidermal growth factor-dependent stimulation of Raf-1 is inhibited. In addition, the Raf-1 mutant RafR89L, which cannot bind to activated Ras, is still stimulated in mitotic cells. Mitotic activation of Raf-1 seems to be partially dependent on tyrosine phosphorylation since the kinase activity of the Raf mutant RafYY340/341FF, which can no longer be activated by Src, is reduced in mitotic cells. Surprisingly, cell fractionation experiments showed that mitotic-activated Raf-1 is predominantly located in the cytoplasm in contrast to the mitogen-activated Raf-1 that is bound to the plasma membrane. In addition, mitotic activation of Raf-1 does not lead to stimulation of the mitogen-activated protein kinase kinase (MAPKK or MEK) and the extracellular signal-regulated protein kinase (ERK). These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway. These data demonstrate that in mitotic cells a Ras-independent mechanism results in a cytoplasmic active Raf-1 kinase which does not signal via the MEK/ERK pathway.

Phosphorylation of c-Raf-1 by protein kinase A interferes with activation.

c-Raf-1 is a serine/threonine-specific protein kinase which is regulated by phosphorylation. A putative c-AMP dependent protein kinase PKA phosphorylation site with the consensus sequence RRXS, Ser43, and a predominant phosphorylation site of c-Raf-1, Ser259, can be phosphorylated by PKA in vitro as shown by comparison of phosphopeptide maps of recombinant wild-type c-Raf-1 and the corresponding mutants. In vivo stimulation of the PKA pathway by treatment of A431 cells with Forskolin results in increase of phosphorylation in Ser43. Forskolin reduces the upshift of c-Raf-1 induced by EGF-treatment. It inhibits the EGF-activation of the c-Raf-1 protein kinase activity tested in vitro with a peptide substrate.

Host cell-virus cross talk: phosphorylation of a hepatitis B virus envelope protein mediates intracellular signaling.

Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S-->A) or mimicking (S-->D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.