Bjarne Johansen

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.

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.

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.

The Ser(186) phospho-acceptor site within ERK4 is essential for its ability to interact with and activate PRAK/MK5.

ERK (extracellular-signal-regulated kinase) 4 [MAPK (mitogen-activated protein kinase) 4] and ERK3 (MAPK6) are atypical MAPKs. One major difference between these proteins and the classical MAPKs is substitution of the conserved T-X-Y motif within the activation loop by a single phospho-acceptor site within an S-E-G motif. In the present study we report that Ser(186) of the S-E-G motif in ERK4 is phosphorylated in vivo. Kinase-dead ERK4 is also phosphorylated on Ser(186), indicating that an ERK4 kinase, rather than autophosphorylation, is responsible. Co-expression of MK5 [MAPK-activated protein kinase 5; also known as PRAK (p38-regulated/activated kinase)], a physiological target of ERK4, increases phosphorylation of Ser(186). This is not dependent on MK5 activity, but does require interaction between ERK4 and MK5 suggesting that MK5 binding either prevents ERK4 dephosphorylation or facilitates ERK4 kinase activity. ERK4 mutants in which Ser(186) is replaced with either an alanine residue or a phospho-mimetic residue (glutamate) are unable to activate MK5 and Ser(186) is also required for cytoplasmic anchoring of MK5. Both defects seem to reflect an impaired ability of the ERK4 mutants to interact with MK5. We find that there are at least two endogenous pools of wild-type ERK4. One form exhibits reduced mobility when analysed using SDS/PAGE. This is due to MK5-dependent phosphorylation and only this retarded ERK4 species is both phosphorylated on Ser(186) and co-immunoprecipitates with wild-type MK5. We conclude that binding between ERK4 and MK5 facilitates phosphorylation of Ser(186) and stabilization of the ERK4-MK5 complex. This results in phosphorylation and activation of MK5, which in turn phosphorylates ERK4 on sites other than Ser(186) resulting in the observed mobility shift.

Activation of the coactivator four-and-a-half-LIM-only protein FHL2 and the c-fos promoter through inhibition of protein phosphatase 2A

Previous studies have demonstrated that the serine/threonine protein phosphatase 2A (PP2A) can modulate the transcriptional activity of several sequence-specific DNA-binding proteins. However, less is known about the effect of PP2A on the activities of general transcription factors and transcriptional coregulators. Here we describe that the activity of a general coactivator, the four-and-a-half-LIM-only protein 2 (FHL2), is regulated in a PP2A-dependent manner. Specific inhibition of PP2A by simian virus 40 (SV40) small t-antigen (st-ag) stimulated the intrinsic transcriptional activity of FHL2 more than 10-fold, while a st-ag mutant unable to bind PP2A had no effect. Overexpression of the B56 subunits alpha, beta, and gamma1 of PP2A impaired the induction of FHL2 by st-ag. FHL2 functioned as a coactivator for CREB-mediated transcription, and inactivation of PP2A further increased FHL2-induced CREB-directed transcription. Overexpression of FHL2 readily enhanced the transcription of the luciferase reporter gene driven by the c-fos promoter, and inhibition of PP2A further stimulated FHL2-induced transactivation of this promoter. These results suggest that dephosphorylation of the general coactivator FHL2 may represent a novel mechanism by which PP2A modulates the transcription of FHL2-responsive genes.

Concerted expression of BK virus large T- and small t-antigens strongly enhances oestrogen receptor-mediated transcription.

Previous studies have shown that the human polyomavirus BK (BKV) genome contains an oestrogen response element (ERE). This isolated element binds its cognate receptor in vitro and can mediate 17beta-oestradiol-induced gene expression when linked to a heterologous promoter. The roles of the ERE- and the AP-1-binding sites in oestrogen receptor-directed transcription from the complete BKV promoter/enhancer (Dunlop strain) have been examined and the effects of the general co-activator CBP and large T- and small t-antigens on oestrogen receptor-mediated transcription have been investigated. A constitutive activated oestrogen receptor stimulated BKV promoter activity in HeLa cells. Mutations in either the ERE- or the AP-1-binding sites did not impair oestrogen receptor-induced activation of the BKV Dunlop promoter, while mutations in both binding motifs almost completely abolished oestrogen receptor-induced transcription. Simultaneous expression of large T- and small t-antigens strongly activated oestrogen receptor-mediated transcription. When expressed separately, only large T-antigen moderately stimulated oestrogen receptor-mediated transcription. The stimulatory effect of large T-antigen on the activity of the oestrogen receptor is probably indirect because no physical interaction between the two proteins was detected in a two-hybrid assay. Large T-antigen abrogated the synergistic effect on transcription between this nuclear receptor and the general co-activator CBP. The findings that the BKV early proteins amplify oestrogen receptor-mediated transcription may have important biological implications in individuals with raised oestrogen concentrations.

Simian Virus 40 Large T-antigen, but not Small T-antigen, Trans-activatesthe Human Cytomegalovirus Major Immediate Early Promoter

Cytomegalovirus infection is a major cause of morbidity in immunocompromised patients. The major immediate early promoter/enhancer (MIEP) of the human cytomegalovirus controls the expression of the immediate early genes 1 and 2 which play a central role both in primary and reactivated human cytomegalovirus (HCMV)-infections. Our previous studies have shown that co-infection of A549 cells with human cytomegalovirus and human polyomavirus BK resulted in enhanced expression of the immediate early genes 1 and 2 and that the early gene products of BK virus trans-activated the MIEP. However, neither the MIEP sequences required for mediating this trans-activation, nor the contribution of the individual BK virus early gene products were examined. The MIEP contains multiple binding sites for the transcription factors CREB, AP1, Sp1 and NFκB, which may mediate polyomavirus large T- or small t-antigens-induced promoter activation. Transient transfection studies in A549 cells demonstrated that SV40 large T-antigen, but not small t-antigen, trans-activated MIEP activity approximately 9-fold. Mutations in individual binding motifs in the context of the complete MIEP did not impair trans-activation by large T-antigen. The level of induction of a truncated MIEP consisting of a single set of CRE/AP1, NFκB, and Sp1 binding motifs by large T-antigen was reduced 2-fold compared to the full length MIEP. Extended truncations diminished trans-activation by large T-antigen. To determine the contribution of a single binding motif in the trans-activation by large T-antigen, a CRE/AP1, an NFκB, an Sp1, or a non-consensus Sp1-motif, respectively, was linked to the MIEP TATA-sequence respecting the natural spacing between the two transcription regulatory elements. Only the MIEP TATA-box with the correctly spaced non-consensus Sp1 binding site (GT-motif) was stimulated by large T-antigen. These results suggest that an isolated non-consensus Sp1-motif is important for trans-activation of the MIEP by large T-antigen, but that other cis-acting elements can compensate for this element in the context of the whole MIEP.