Biomedical subjects

Mona Johannessen

Publications and source records attributed to Mona Johannessen.

8 recordsLinked to original sources

PubMed2007

Multisite phosphorylation of the cAMP response element-binding protein (CREB) by a diversity of protein kinases.

The prevailing view of stimulus-induced activation of the transcription factor cAMP response element-binding protein (CREB) presumes phosphorylation at serine-133. Although, phosphorylation of this residue seems to be necessary, it is not sufficient to trigger CREB-driven transcription, indicating that other phosphoserine-133-independent mechanisms are required for full activation of CREB. One of these additional regulatory mechanisms influencing the transcriptional state of CREB may involve multiple phosphorylation events on other phosphoacceptor sites in the protein. This review focuses on the phosphorylation modifications of CREB by distinct protein kinases and discusses the possible implications for the function of CREB.

Animals↗

PubMed2005

Sequence- and position-dependent tagging protects extracellular-regulated kinase 3 protein from 26S proteasome-mediated degradation.

Extracellular-regulated kinase 3, an atypical member of the mitogen-activated protein kinase subfamily of extracellular-regulated kinases, was originally identified in 1991. Little is known about the biochemical properties, regulation, and biological functions of this protein kinase, partially due to the unstable nature of endogenous and low ectopical expression level of the protein. Here, we report that a single C-terminal c-myc tag increases the half-life of ectopic expressed tagged extracellular-regulated kinase 3 approximately four times compared to the reported 30 min half-life time for the endogenous protein and ectopic expressed extracellular-regulated kinase 3 deprived of its c-myc tag. These findings indicate that this C-terminal tag stabilizes the extracellular-regulated kinase 3. The stabilizing effect of the C-terminal c-myc tag is observed in all cell types tested, but is position- and tag sequence-dependent as neither N-terminal c-myc tag nor C-terminal HA tag stabilize the protein. The c-myc tag on extracellular-regulated kinase 3 did not interfere with its kinase activity, nor did it abrogate its ability to interacts with its bona fide substrate mitogen-activated protein kinase-activated protein kinase 5, indicating that tagging did not alter the known biological properties of the protein. Stabilization of the tagged extracellular-regulated kinase 3 protein probably results from reduced ubiquitination. In conclusion, position and sequence specific tagging should provide an easy and useful tool to generate a more stable protein that can functionally substitute the endogenous unstable protein. A stabilized variant may facilitate studies on the biological role of the protein.

Amino Acid Sequence↗

PubMed2005

The cAMP signalling pathway activates CREB through PKA, p38 and MSK1 in NIH 3T3 cells.

Cyclic adenosine 3',5'-monophosphate (cAMP) was originally shown to induce gene transcription through activation of cAMP-dependent protein kinase (PKA), and subsequent phosphorylation of the transcription factor cAMP response element-binding protein, CREB, at serine-133. However, elevated cAMP levels may activate multiple signalling pathways with protein kinases that can phosphorylate CREB at serine-133. We analysed the pathways involved in CREB phosphorylation and activation in NIH 3T3 cells exposed to the cAMP elevating agent forskolin. PKA represented the predominant pathway during the burst phase, while the mitogen-activated protein kinase p38 pathway became activated in a PKA-dependent fashion in forskolin treated cells. The phosphorylation kinetics of p38 was delayed compared to PKA activation. Activated p38 stimulated CREB-mediated transcription and potentiated the transcriptional strength of CREB provoked by forskolin. The p38-mediated activation of CREB was inhibited by dominant negative mutants of MSK-1 and by the PKA/MSK-1 inhibitor H89, but not by dominant negative mutants of MSK-2/RSK-B and MAPKAPK2. Our results suggest that forskolin-induced CREB phosphorylation and activation in NIH 3T3 cells is mediated directly by PKA and by a time-delayed PKA-dependent p38/MSK-1 pathway. This bifurcation and time-dependent regulation of the cAMP-responsive signalling pathways may enable the cell to endure and/or enforce a cellular response provoked by a cAMP-elevating stimulus.

Animals↗

PubMed2004

Synergistic activation of CREB-mediated transcription by forskolin and phorbol ester requires PKC and depends on the glutamine-rich Q2 transactivation domain.

Recruitment of a RNA polymerase II complex by the glutamine-rich Q2 domain of cAMP response element-binding protein (CREB) allows basal transcriptional activity, while recruitment of CBP/p300 through signal-induced phosphorylation of the kinase-inducible domain at serine-133 enhances CREB-dependent transcription. Here we demonstrate that co-administration of forskolin and phorbol ester TPA to NIH3T3 cells provoked a dose-dependent increase in phosphoserine-133. CREB- and Q2-dependent transcription, as well as transcription by other glutamine-rich transcription factors, but not by transcription factors lacking glutamine-rich regions, augmented synergistically in the presence of both stimuli. Synergistic activation was abograted by specific inhibition of protein kinase C (PKC), but not of PKA. Co-stimulation increased the basal activity of a minimal, CREB-independent promoter. Therefore, Q2, which directly interacts with the RNA polymerase II initiation complex, may transmit the increased basal promoter activity provoked by these stimuli to CREB, thereby contributing to synergistic activation of CREB-mediated transcription. This synergism may have important implications on glutamine-rich transcription factor-target genes.

Animals↗

PubMed2004

What turns CREB on?

The transactivation domain of the cAMP response element-binding protein (CREB) consists of two major domains. The glutamine-rich Q2 domain, which interacts with the general transcription factor TAFII130/135, is sufficient for the recruitment of a functional RNA polymerase II complex and allows basal transcriptional activity. The kinase-inducible domain, however, mediates signal-induced activation of CREB-mediated transcription. It is generally believed that recruitment of the coactivators CREB-binding protein (CBP) and p300 after signal-induced phosphorylation of this domain at serine-133 strongly enhances CREB-dependent transcription. Transcriptional activity of CREB can also be potentiated by phosphoserine-133-independent mechanisms, and not all stimuli that provoke phosphorylation of serine-133 stimulate CREB-dependent transcription. This review presents an overview of the diversity of stimuli that induce CREB phosphorylation at Ser-133, focuses on phosphoserine-133-dependent and -independent mechanisms that affect CREB-mediated transcription, and discusses different models that may explain the discrepancy between CREB Ser-133 phosphorylation and activation of CREB-mediated transcription.

Amino Acid Sequence↗

PubMed2003

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.

3T3 Cells↗

PubMed2003

A role of the TATA box and the general co-activator hTAF(II)130/135 in promoter-specific trans-activation by simian virus 40 small t antigen.

The small t antigen (st-ag) of simian virus 40 can exert pleiotropic effects on biological processes such as DNA replication, cell cycle progression and gene expression. One possible mode of achieving these effects is through stimulation of NFkappaB-responsive genes encoding growth factors, cytokines, transcription factors and cell cycle regulatory proteins. Indeed, a previous study has shown that st-ag enhanced NFkappaB-mediated transcription. This study demonstrates that promoters possessing a consensus TATA box (i.e. TATAAAAG) in the context of either NFkappaB- or Sp1-binding sites are trans-activated by st-ag. Overexpressing the general transcription factor hTAF(II)130/135, but not hTAF(II)28 or hTAF(II)80, stimulated the activity of promoters in a consensus TATA box-dependent mode. Converting the consensus TATA motif into a non-consensus TATA box strongly impaired activation by st-ag and hTAF(II)130/135. Conversely, mutating a non-consensus TATA motif into the consensus TATA box rendered the mutated promoter inducible by st-ag and hTAF(II)130/135. Mutation of the TATA box had no effect on TNFalpha- or RelA/p65-mediated induction of NFkappaB-responsive promoters, indicating a specific st-ag effect on hTAF(II)130/135. St-ag stimulated the intrinsic transcriptional activity of hTAF(II)130/135. Substitutions in the conserved HPDKGG motif in the N-terminal region or a mutation that impaired the interaction with protein phosphatase 2A abrogated the ability of st-ag to activate hTAF(II)130/135-mediated transcription. These results indicate that trans-activation of promoters by st-ag may depend on a consensus TATA motif and suggest that such promoters recruit the general transcription factor hTAF(II)130/135.

3T3 Cells↗

PubMed2002

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.

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.

3T3 Cells↗