Harald Mischak

MECHANISM OF INHIBITION OF RAF-1 BY PROTEIN KINASE A

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.

Negative regulation of Raf-1 by phosphorylation of serine 621.

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation.

The Raf-1 kinase plays a key role in relaying proliferation signals elicited by mitogens or oncogenes. Raf-1 is regulated by complex and incompletely understood mechanisms including phosphorylation. A number of studies have indicated that phosphorylation of serines 259 and 621 can inhibit the Raf-1 kinase. We show that both serines are hypophosphorylated during early mitogenic stimulation and that hypophosphorylation correlates with peak Raf-1 activation. Concentrations of okadaic acid that selectively inhibit protein phosphatase 2A (PP2A) induce phosphorylation of these residues and prevent maximal activation of the Raf-1 kinase. This effect is mediated via phosphorylation of serine 259. The PP2A core heterodimer forms complexes with Raf-1 in vivo and in vitro. These data identify PP2A as a positive regulator of Raf-1 activation and are the first indication that PP2A may support the activation of an associated kinase.

Cell type-specific activation of mitogen-activated protein kinases by CpG-DNA controls interleukin-12 release from antigen-presenting cells.

Activation of antigen‐presenting cells (APCs) by invariant constituents of pathogens such as lipopolysaccharide (LPS) or bacterial DNA (CpG‐DNA) initiates immune responses. We have analyzed the mitogen‐activated protein kinase (MAPK) pathways triggered by CpG‐DNA and their significance for cytokine production in two subsets of APCs, i.e. macrophages and dendritic cells (DCs). We found that CpG‐DNA induced extracellular signal‐regulated kinase (ERK) activity in macrophages in a classic MEK‐dependent way. This pathway up‐regulated tumor necrosis factor production but down‐regulated interleukin (IL)‐12 production. However, in DCs, which produce large amounts of IL‐12, CpG‐DNA and LPS failed to induce ERK activity. Consistent with a specific negative regulatory role for ERK in macrophages, chemical activation of this pathway in DCs suppressed CpG‐DNA‐induced IL‐12 production. Overall, these results imply that differential activation of MAP kinase pathways is a basic mechanism by which distinct subsets of innate immune cells regulate their effector functions.

Structural Mechanism for Lipid Activation of the Rac-Specific GAP, β2-Chimaerin

The lipid second messenger diacylglycerol acts by binding to the C1 domains of target proteins, which translocate to cell membranes and are allosterically activated. Here we report the crystal structure at 3.2 A resolution of one such protein, beta2-chimaerin, a GTPase-activating protein for the small GTPase Rac, in its inactive conformation. The structure shows that in the inactive state, the N terminus of beta2-chimaerin protrudes into the active site of the RacGAP domain, sterically blocking Rac binding. The diacylglycerol and phospholipid membrane binding site on the C1 domain is buried by contacts with the four different regions of beta2-chimaerin: the N terminus, SH2 domain, RacGAP domain, and the linker between the SH2 and C1 domains. Phospholipid binding to the C1 domain triggers the cooperative dissociation of these interactions, allowing the N terminus to move out of the active site and thereby activating the enzyme.

The molecular make-up of a tumour: proteomics in cancer research

The enormous progress in proteomics, enabled by recent advances in MS (mass spectrometry), has brought protein analysis back into the limelight of cancer research, reviving old areas as well as opening new fields of study. In this review, we discuss the basic features of proteomic technologies, including the basics of MS, and we consider the main current applications and challenges of proteomics in cancer research, including (i) protein expression profiling of tumours, tumour fluids and tumour cells; (ii) protein microarrays; (iii) mapping of cancer signalling pathways; (iv) pharmacoproteomics; (v) biomarkers for diagnosis, staging and monitoring of the disease and therapeutic response; and (vi) the immune response to cancer. All these applications continue to benefit from further technological advances, such as the development of quantitative proteomics methods, high-resolution, high-speed and high-sensitivity MS, functional protein assays, and advanced bioinformatics for data handling and interpretation. A major challenge will be the integration of proteomics with genomics and metabolomics data and their functional interpretation in conjunction with clinical results and epidemiology.

INHIBITION OF THE RAF-1 KINASE BY CYCLIC AMP AGONISTS CAUSES APOPTOSIS OF V-ABL-TRANSFORMED CELLS

Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as chronic myeloid leukemias.

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)

Induction of Apoptosis by Protein Kinase Cδ Is Independent of Its Kinase Activity

Protein kinase C, a multigene family of phospholipid-dependent and diacylglycerol-activated Ser/Thr protein kinases, is a key component in many signal transduction pathways. The kinase activity was thought to be essential for a plethora of biological processes attributed to these enzymes. Here we show that at least one protein kinase C function, the induction of apoptosis by protein kinase Cδ, is independent of the kinase activity. Stimulation of green fluorescent protein-protein kinase Cδ fusion protein with phorbol ester or diacylglycerol led to its redistribution within seconds after the stimulus. Membrane blebbing, an early hallmark of apoptosis, was visible as early as 20 min after stimulation, and nuclear condensation was visible after 3–5 h. Apoptosis could be inhibited by expression of Bcl-2 but not by specific protein kinase C inhibitors. In addition, a kinase-negative mutant of protein kinase Cδ also induced apoptosis to the same extent as the wild type enzyme. Apoptosis was confined to the protein kinase Cδ-overexpressing cells. Stimulation of overexpressed protein kinase Cε did not result in increased apoptosis. Our results indicate that distinct protein kinase C isozymes induce apoptosis in vascular smooth muscle cells. More importantly, they show that some protein kinase C effector functions are independent of the catalytic activity.

Unique expression pattern of protein kinase C‐θ: high mRNA levels in normal mouse testes and in T‐lymphocytic cells and neoplasms

A 2.2‐kb cDNA that contains the entire coding region of mouse protein kinase C‐θ (PKC‐θ) was cloned from skeletal muscle mRNA using reverse transcription and the polymerase chain reaction (PCR). This clone was used as a probe to study the expression of this PKC isoform in normal and transformed hemopoietic cells and other normal tissues. By far the highest steady‐state level of PKC‐0 MRNA was found as a 2.8‐kb transcript on a Northern blot of poly(A)+ RNA from testes. High levels were also found in skeletal muscle, spleen, T lymphomas and purified normal T lymphocytes, but these tissues and cells expressed two transcripts, 3.3 kb and 3.8 kb. Lower levels of similar size transcripts were found in normal brain, B lymphocytes and B‐lymphocytic tumors and cell lines.