Ole Morten Seternes

Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway

Extracellular signal‐regulated kinase 3 (ERK3) is an atypical mitogen‐activated protein kinase (MAPK), which is regulated by protein stability. However, its function is unknown and no physiological substrates for ERK3 have yet been identified. Here we demonstrate a specific interaction between ERK3 and MAPK‐activated protein kinase‐5 (MK5). Binding results in nuclear exclusion of both ERK3 and MK5 and is accompanied by ERK3‐dependent phosphorylation and activation of MK5 in vitro and in vivo. Endogenous MK5 activity is significantly reduced by siRNA‐mediated knockdown of ERK3 and also in fibroblasts derived from ERK3−/− mice. Furthermore, increased levels of ERK3 protein detected during nerve growth factor‐induced differentiation of PC12 cells are accompanied by an increase in MK5 activity. Conversely, MK5 depletion causes a dramatic reduction in endogenous ERK3 levels. Our data identify the first physiological protein substrate for ERK3 and suggest a functional link between these kinases in which MK5 is a downstream target of ERK3, while MK5 acts as a chaperone for ERK3. Our findings provide valuable tools to further dissect the regulation and biological roles of both ERK3 and MK5.

Noncoding control region of naturally occurring BK virus variants: sequence comparison and functional analysis.

The human polyomavirus BK (BKV) has a proven oncogenic potential, but its contribution to tumorigenesis under natural conditions remains undetermined. As for other primate polyomaviruses, the approximately 5.2 kbp double-stranded circular genome of BKV has three functional regions: the coding regions for the two early (T, t antigens) and four late (agno, capsid proteins; VP1-3) genes separated by a noncoding control region (NCCR). The NCCR contains the origin of replication as well as a promoter/enhancer with a mosaic ofcis-acting elements involved in the regulation of both early and late transcription. Since the original isolation of BKV in 1971, a number of other strains have been identified. Most strains reveal a strong sequence conservation in the protein coding regions of the genome, while the NCCR exhibits considerable variation between different BKV isolates. This variation is due to deletions, duplications, and rearrangements of a basic set of sequence blocks. Comparative studies have proven that the anatomy of the NCCR may determine the transcriptional activities governed by the promoter/enhancer, the host cell tropism and permissivity, as well as the oncogenic potential of a given BKV strain. In most cases, however, the NCCR sequence of new isolates was determined after the virus had been passaged several times in more or less arbitrarily chosen cell cultures, a process known to predispose for NCCR rearrangements. Following the development of the polymerase chain reaction (PCR), it has become feasible to obtain naturally occurring BKV NCCRs, and their sequences, in samples taken directly from infected human individuals. Hence, the biological significance of BKV NCCR variation may be studied without prior propagation of the virus in cell culture. Such variation has general interest, because the BKV NCCRs represent typical mammalian promoter/enhancers, with a large number of binding motifs for cellular transacting factors, which can be conveniently handled for experimental purposes. This communication reviews the naturally occurring BKV NCCR variants, isolated and sequenced directly from human samples, that have been reported so far. The sequences of the different NCCRs are compared and analyzed for the presence of proven and putative cellular transcription factor binding sites. Differences in biological properties between BKV variants are discussed in light of their aberrant NCCR anatomies and the potentially modifying influence of transacting factors.

Both Binding and Activation of p38 Mitogen-Activated Protein Kinase (MAPK) Play Essential Roles in Regulation of the Nucleocytoplasmic Distribution of MAPK-Activated Protein Kinase 5 by Cellular Stress

ABSTRACT The p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of cellular responses to environmental stress. Targets of p38 include transcription factors, components of the translational machinery, and downstream serine/threonine kinases, including MAPK-activated protein kinase 5 (MK5). Here we have used enhanced green fluorescent protein fusion proteins to analyze the subcellular localization of MK5. Although this protein is predominantly nuclear in unstimulated cells, MK5 shuttles between the nucleus and the cytoplasm. Furthermore, we have shown that the C-terminal domain of MK5 contains both a functional nuclear localization signal (NLS) and a leucine-rich nuclear export signal (NES), indicating that the subcellular distribution of this kinase reflects the relative activities of these two signals. In support of this, we have shown that stress-induced activation of the p38 MAPK stimulates the chromosomal region maintenance 1 protein-dependent nuclear export of MK5. This is regulated by both binding of p38 MAPK to MK5, which masks the functional NLS, and stress-induced phosphorylation of MK5 by p38 MAPK, which either activates or unmasks the NES. These properties may define the ability of MK5 to differentially phosphorylate both nuclear and cytoplasmic targets or alternatively reflect a mechanism whereby signals initiated by activation of MK5 in the nucleus may be transmitted to the cytoplasm.

Mechanisms of Transcriptional Regulation of Cellular Genes by SV40 Large T- and Small T-Antigens

During the past decade a number of virus-encoded transcriptional trans-activators that regulate the expression of viral genes have been reported. These trans-activators may also affect the expression or activity of several cellular genes or gene products to create an optimal cellular environment that favors viral replication. Among the better-studied viral trans-activating proteins are the Simian virus 40 large T- and small t-antigens. During the last few years, mechanisms by which these two viral proteins influence cellular gene expression start to emerge. They are grouped provisionally and reflect the methods used to determine the effects of large T-antigen. Large T-antigen may influence cellular gene expression by: i. altering mRNA levels of cellular transcription factors; ii. interacting with and regulating the DNA-binding or transcriptional activity of specific transcription factors; iii. functionally substitution of eukaryotic transcription factors; iv. direct binding to DNA; or v. regulating components of signaling transduction pathways. Small t-ag seems to exert its effect mainly through inhibiting a cellular phosphatase, protein phosphatase 2A, thereby modulating components of signal transduction pathways and preventing dephosphorylation of several transcription factors. However, small t-ag may also control cellular gene expression by regulating mRNA levels of transcription factors or by interacting with other transcription factors.

Regulation of MAPK-activated protein kinase 5 activity and subcellular localization by the atypical MAPK ERK4/MAPK4.

MAPK-activated protein kinase 5 (MK5) was recently identified as a physiological substrate of the atypical MAPK ERK3. Complex formation between ERK3 and MK5 results in phosphorylation and activation of MK5, concomitant stabilization of ERK3, and the nuclear exclusion of both proteins. However, ablation of ERK3 in HeLa cells using small interfering RNA or in fibroblasts derived from ERK3 null mice reduces the activity of endogenous MK5 by only 50%, suggesting additional mechanisms of MK5 regulation. Here we identify the ERK3-related kinase ERK4 as a bona fide interaction partner of MK5. Binding of ERK4 to MK5 is accompanied by phosphorylation and activation of MK5. Furthermore, complex formation also results in the relocalization of MK5 from nucleus to cytoplasm. However unlike ERK3, ERK4 is a stable protein, and its half-life is not modified by the presence or absence of MK5. Finally, although knock-down of ERK4 protein in HeLa cells reduces endogenous MK5 activity by ∼50%, a combination of small interfering RNAs targeting both ERK4 and ERK3 causes a further reduction in the MK5 activity by more than 80%. We conclude that MK5 activation is dependent on both ERK3 and ERK4 in these cells and that these atypical MAPKs are both physiological regulators of MK5 activity.

Synergistic increase in c-fos expression by simultaneous activation of the ras/raf/map kinase- and protein kinase A signaling pathways is mediated by the c-fos AP-1 and SRE sites.

Expression of the c-fos proto-oncogene is induced by numerous stimuli some of which are transmitted through the Ras/Raf/MAP kinase or the cAMP-dependent protein kinase (PKA) pathways. The effect of cell-specific interactions between these pathways on c-fos expression was investigated by exposing quiescent NIH3T3 cells to serum, forskolin, or a combination. Co-stimulation with serum and forskolin resulted in a more than additive increase in c-fos transcription. Synergistic increase in c-fos promoter activity was also observed in transient transfection studies after co-stimulation with serum plus forskolin or co-transfection with c-Raf and PKA expression plasmids. Analysis of the cAMP signaling pathway revealed that the synergy was neither due to an increase in PKA activity nor to Ser-133 phosphorylation/activation of CREB. The activation status of the MAP kinases ERK1 and ERK2 in co-treated cells was comparable to that in serum-treated cells. Co-stimulation with forskolin did not alter the phosphorylation state of Elk-1 compared to serum-induced phosphorylation of Elk-1. Deletion of c-fos promoter elements previously shown to be important for regulation of c-fos expression in response to mitogens indicates a role for SRE and FAP-1 elements.

Biochemical analysis of mouse FKBP60, a novel member of the FKPB family

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).

The human polyomavirus BK T antigen induces gene expression in human cytomegalovirus.

Co-infections or co-habitations of cells by two or more viruses may occur in the human organism. Human cytomegalovirus (HCMV) and the human polyomavirus BK (BKV) have common host cells and may both establish lifelong latency/persistence following primary infection. Both viruses may become reactivated by immunosuppression or other conditions which upset host-virus balance, and they encode gene products with the inherent potential of acting as heterologous transacting factors for expression of cellular or viral genes. It has been shown that HCMV induces gene expression and replication of primate polyomaviruses. We now demonstrate that BKV is able to enhance the expression of HCMV immediate early (IE1 and 2) as well as the early (E) protein pp65 during double infections in semi-permissive cells. By transfection experiments it was established that the phenomenon is due to heterologous transcriptional transactivation of the HCMV major IE promoter (MIEP) by the BKV large T antigen, without contribution from the small t antigen.

Docking of PRAK/MK5 to the Atypical MAPKs ERK3 and ERK4 Defines a Novel MAPK Interaction Motif

ERK3 and ERK4 are atypical MAPKs in which the canonical TXY motif within the activation loop of the classical MAPKs is replaced by SEG. Both ERK3 and ERK4 bind, translocate, and activate the MAPK-activated protein kinase (MK) 5. The classical MAPKs ERK1/2 and p38 interact with downstream MKs (RSK1–3 and MK2–3, respectively) through conserved clusters of acidic amino acids, which constitute the common docking (CD) domain. In contrast to the classical MAPKs, the interaction between ERK3/4 and MK5 is strictly dependent on phosphorylation of the SEG motif of these kinases. Here we report that the conserved CD domain is dispensable for the interaction of ERK3 and ERK4 with MK5. Using peptide overlay assays, we have defined a novel MK5 interaction motif (FRIEDE) within both ERK4 and ERK3 that is essential for binding to the C-terminal region of MK5. This motif is located within the L16 extension lying C-terminal to the CD domain in ERK3 and ERK4 and a single isoleucine to lysine substitution in FRIEDE totally abrogates binding, activation, and translocation of MK5 by both ERK3 and ERK4. These findings are the first to demonstrate binding of a physiological substrate via this region of the L16 loop in a MAPK. Furthermore, the link between activation loop phosphorylation and accessibility of the FRIEDE interaction motif suggests a switch mechanism for these atypical MAPKs in which the phosphorylation status of the activation loop regulates the ability of both ERK3 and ERK4 to bind to a downstream effector.

Does MK5 reconcile classical and atypical MAP kinases

MAP kinase-activated protein kinase 5 (MK5) was originally described as a protein kinase activated downstream of the p38 MAP kinase and is also named p38-regulated/activated protein kinase (PRAK). However, while MK5 is most similar in sequence to the two p38 regulated MAPKAP kinases MK2 and MK3, recent data has shown that in contrast to these enzymes MK5 is not activated in response to either cellular stress or pro-inflammatory cytokines. This lack of response to stimuli which cause robust activation of p38 MAP kinase in vivo is supported by data obtained using transgenic mice lacking MK5. Unlike animals lacking MK2 and MK3, MK5 null mice respond normally to endotoxic shock and display an unchanged pattern of cytokine expression in response to LPS. Clues as to the physiological function of MK5 have come from the recent observation that MK5 is uniquely regulated and activated following complex formation with the atypical MAP kinases ERK3 and ERK4. Thus, it is possible that MK5 is unique amongst the MAPKAP kinases in being regulated downstream of signaling pathways other than the classical MAP kinases p38 and ERK1/2.