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Andrew D Sharrocks

Publications and source records attributed to Andrew D Sharrocks.

At least 19 recordsLinked to original sources
PubMed2010

MAP Kinase: SUMO pathway interactions.

The convergence and coordinated cross talk of different signalling pathways forms a regulatory network which determines the biological outcome to environmental cues. The MAPK pathways are one of the important routes by which extracellular signals are transduced into intracellular responses. Through protein phosphorylation mechanisms, they can play a pivotal role in regulating other posttranslational modifications such as protein acetylation and ubiquitination. In addition, protein sumoylation has emerged as an important pathway which also functions through post-translational modification. The SUMO pathway modulates a diverse range of cellular processes including signal transduction, chromosome integrity, and transcription. Interestingly, recent studies have provided links between the SUMO and MAPK signalling pathways which converge to modulate transcription factor activity. This was first demonstrated by the observation that the activation of the ERK pathway caused de-sumoylation of the transcription factor, Elk-1. Furthermore, a growing number of links are now being made between the MAPK pathway and protein sumoylation. Given the nature of protein sumoylation in diverse biological functions, it is not surprising that the effect of MAPK pathways on sumoylation varies between different proteins. Here, we describe protocols that can be used in studying the cross talk between the MAPK and SUMO pathways, particularly at the level of gene regulation.

Amino Acid Sequence↗
PubMed2006

Polo kinase controls cell-cycle-dependent transcription by targeting a coactivator protein.

Polo kinases have crucial conserved functions in controlling the eukaryotic cell cycle through orchestrating several events during mitosis. An essential element of cell cycle control is exerted by altering the expression of key regulators. Here we show an important function for the polo kinase Cdc5p in controlling cell-cycle-dependent gene expression that is crucial for the execution of mitosis in the model eukaryote Saccharomyces cerevisiae. In particular, we find that Cdc5p is temporally recruited to promoters of the cell-cycle-regulated CLB2 gene cluster, where it targets the Mcm1p-Fkh2p-Ndd1p transcription factor complex, through direct phosphorylation of the coactivator protein Ndd1p. This phosphorylation event is required for the normal temporal expression of cell-cycle-regulated genes such as CLB2 and SWI5 in G2/M phases. Furthermore, severe defects in cell division occur in the absence of Cdc5p-mediated phosphorylation of Ndd1p. Thus, polo kinase is required for the production of key mitotic regulators, in addition to previously defined roles in controlling other mitotic events.

Cell Cycle↗
PubMed2006

An extended consensus motif enhances the specificity of substrate modification by SUMO.

Protein modification by SUMO conjugation is an important regulatory event. Sumoylation usually takes place on a lysine residue embedded in the core consensus motif psiKxE. However, this motif confers limited specificity on the sumoylation process. Here, we have probed the roles of clusters of acidic residues located downstream from the core SUMO modification sites in proteins such as the transcription factor Elk-1. We demonstrate that these are functionally important in SUMO-dependent transcriptional repression of Elk-1 transcriptional activity. Mechanistically, the acidic residues are important in enhancing the efficiency of Elk-1 sumoylation by Ubc9. Similar mechanisms operate in other transcription factors and phosphorylation sites can functionally substitute for acidic residues. Thus, an extended sumoylation motif, termed the NDSM (negatively charged amino acid-dependent sumoylation motif), helps define functional SUMO targets. We demonstrate that this extended motif can be used to correctly predict novel targets for SUMO modification.

Amino Acid Motifs↗
PubMed2006

Assembly of transcription factor IIB at a promoter in vivo requires contact with RNA polymerase II.

The general transcription factor TFIIB has a central role in the assembly of the preinitiation complex at the promoter, providing a platform for the entry of RNA polymerase II/TFIIF. We used an RNA interference (RNAi)-based system in which TFIIB expression is ablated in vivo and replaced with a TFIIB derivative that contains a silent mutation and is refractory to the RNAi. Using this approach, we found that transcriptionally defective TFIIB amino-terminal mutants showed distinct effects on the basis of their ability to compete with wild-type TFIIB in vivo. Moreover, analysis of the TFIIB mutant derivatives by chromatin immunoprecipitation showed that promoter occupancy by TFIIB is dependent on the association with RNA polymerase II. Together, our results support a mode of preinitiation complex assembly in which TFIIB/RNA polymerase II recruitment to the promoter occurs in vivo.

Cell Line↗
PubMed2006

Cell cycle: sustained ERK signalling represses the inhibitors.

The duration of ERK pathway signalling determines the proliferative response of cells. A recent study has shown that cell cycle re-entry requires the downregulation of an anti-proliferative gene cluster following sustained activation of the ERK pathway.

Cell Cycle↗
PubMed2006

PIASxalpha differentially regulates the amplitudes of transcriptional responses following activation of the ERK and p38 MAPK pathways.

Activation of the MAP kinase pathways leads to changes in gene expression profiles through direct targeting of transcription factors and their coregulators. Here we identify PIASxalpha as a key regulator that determines the differential response of the transcription factor Elk-1 to the ERK and the stress-activated p38 MAP kinase pathways. While PIASxalpha functions as a coactivator to facilitate SUMO and HDAC-2 removal from Elk-1 in response to ERK pathway activation, PIASxalpha acts in the opposite manner to inhibit HDAC-2 and SUMO loss following stress-activated MAP kinase pathway signaling. Thus, PIASxalpha either enhances or dampens down the activation of Elk-1 target genes, depending on the pathway activated. p38 MAP kinase-mediated PIASxalpha phosphorylation allows it to switch between these two alternative modes of operation. Thus, PIASxalpha acts as a key signal integrator that permits different responses from the same transcription factor, depending on the signaling pathway that is activated.

Anisomycin↗
PubMed2006

Convergence of the SUMO and MAPK pathways on the ETS-domain transcription factor Elk-1.

The ETS-domain transcription factor Elk-1 is regulated by phosphorylation in response to activation of the MAPK (mitogen-activated protein kinase) pathways. This phosphorylation triggers a series of molecular events that convert Elk-1 from a transcriptionally silent state into a highly active state and then back to a basal level. At the same time, activation of the ERK (extracellular-signal-regulated kinase) MAPK pathway leads to loss of modification of Elk-1 by SUMO (small ubiquitin-related modifier). As SUMO imparts repressive properties on Elk-1, ERK-mediated SUMO loss leads to de-repression at the same time as the ERK pathway promotes activation of Elk-1. Thus a two-step mechanism is employed to convert Elk-1 into its fully activated state. Here, the molecular events underlying these changes in Elk-1 status, and the role of PIASxalpha [protein inhibitor of activated STAT (signal transducer and activator of transcription) xalpha] as a co-activator that facilitates this process, are discussed.

Animals↗
PubMed2006

The LIM domain protein LPP is a coactivator for the ETS domain transcription factor PEA3.

PEA3 is a member of a subfamily of ETS domain transcription factors which is regulated by a number of signaling cascades, including the mitogen-activated protein (MAP) kinase pathways. PEA3 activates gene expression and is thought to play an important role in promoting tumor metastasis and also in neuronal development. Here, we have identified the LIM domain protein LPP as a novel coregulatory binding partner for PEA3. LPP has intrinsic transactivation capacity, forms a complex with PEA3, and is found associated with PEA3-regulated promoters. By manipulating LPP levels, we show that it acts to upregulate the transactivation capacity of PEA3. LPP can also functionally interact in a similar manner with the related family member ER81. Thus, we have uncovered a novel nuclear function for the LIM domain protein LPP as a transcriptional coactivator. As LPP continually shuttles between the cell periphery and the nucleus, it represents a potential novel link between cell surface events and changes in gene expression.

Base Sequence↗
PubMed2005

PIASx acts as an Elk-1 coactivator by facilitating derepression.

The ETS-domain transcription factor Elk-1 is a MAP kinase-inducible transcriptional activator protein. However, in the basal state, its activity is repressed by SUMO-dependent histone deacetylase (HDAC) recruitment. Relief of this repression accompanies the activation process. Here, we demonstrate that PIASx(alpha) acts to facilitate this derepression process. Members of the PIAS family of proteins can act as E3 enzymes that enhance the sumoylation status of a variety of substrates. However, PIASx-mediated coactivation of Elk-1 occurs in an E3 activity-independent manner. PIASx(alpha) binds to Elk-1 in vivo and enhances its transcriptional activity. The coactivating properties of PIASx(alpha) require Elk-1 to be modified with SUMO and the integrity of the SUMO binding motif in PIASx(alpha). PIASx(alpha) activates Elk-1 through alterations in the HAT/HDAC activities associated with Elk-1. In particular, PIASx(alpha) facilitates the loss of the repressive HDAC-2 from sumoylated Elk-1, a key event in the activation of Elk-1 in response to signalling through the ERK MAP kinase pathway. Our data therefore reveal a novel coactivator function for PIASx(alpha) through reversing SUMO-mediated repression of transcription factor activity.

Acetylation↗
PubMed2004

The ETS domain transcription factor Elk-1 regulates the expression of its partner protein, SRF.

The ternary complex factors (TCF) are a subfamily of ETS domain transcription factors that bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). Here, we have identified the SRF gene as a target for the TCFs, thereby providing a positive feedback loop whereby TCF activation leads to the enhancement of the expression of its partner protein SRF. The binding of the TCF Elk-1 to the SRF promoter and subsequent regulation of SRF expression occurs in a ternary complex-dependent manner. Our data therefore reveal that SRF is an important target for the ERK and Rho signaling pathways that converge on a ternary TCF-SRF complex at the SRE on the SRF promoter.

Base Sequence↗
PubMed2004

ERK5 is targeted to myocyte enhancer factor 2A (MEF2A) through a MAPK docking motif.

One critical component in determining the specificity, and efficiency of MAPK (mitogen-activated protein kinase) substrate phophorylation is the presence of distinct docking domains in the substrate proteins. Docking domains have been shown to be important for the activities of members of the ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 subfamilies of MAPKs towards their substrates. Here, we demonstrate that docking domains also play an important role in ERK5-mediated substrate phosphorylation. The presence of a docking domain promotes both phosphorylation of myocyte enhancer factor, MEF2A, in vitro and its activation in vivo by ERK5. Mutational analysis of the MEF2A docking domain demonstrates that the specificity determinants for ERK5 are similar to those observed with members of the p38 subfamily. A docking domain recognized by ERK5 can direct ERK5 to activate heterologous substrates. Deletion analysis demonstrates that as with other MAPKs, it is the catalytic domain of ERK5 that recognizes the docking domain. Our data therefore extend previous observations on other MAPKs and demonstrate that the requirement for specific docking domains in promoting MAPK action towards substrates is a general property of MAPKs.

Amino Acid Motifs↗
PubMed2004

SUMO promotes HDAC-mediated transcriptional repression.

Recently, SUMO modification has been shown to impart repressive properties on several transcriptional regulatory proteins. Indeed, the ETS domain transcription factor Elk-1 is modified by SUMO, and this modification is reversed by ERK MAP kinase pathway activation. This causes a switch from a repressive to activated state. However, the mechanism(s) of SUMO-mediated transcriptional repression is unclear. Here, we have investigated how sumoylation of Elk-1 leads to transcriptional repression. We demonstrate that sumoylation of Elk-1 results in the recruitment of histone deacetylase activity to promoters. In particular, our data point to a key role for HDAC-2. This recruitment leads to decreased histone acetylation and hence transcriptional repression at Elk-1 target genes. Thus, our data demonstrate an important integration point for two protein-modifying pathways in the cell, the SUMO and deacetylation pathways, that combine to promote transcriptional repression.

Blotting, Western↗
PubMed2004

Regulation of cell cycle-specific gene expression through cyclin-dependent kinase-mediated phosphorylation of the forkhead transcription factor Fkh2p.

The forkhead transcription factor Fkh2p acts in a DNA-bound complex with Mcm1p and the coactivator Ndd1p to regulate cell cycle-dependent expression of the CLB2 gene cluster in Saccharomyces cerevisiae. Here, we demonstrate that Fkh2p is a target of cyclin-dependent protein kinases and that phosphorylation of Fkh2p promotes interactions between Fkh2p and the coactivator Ndd1p. These phosphorylation-dependent changes in the Fkh2p-Ndd1p complex play an important role in the cell cycle-regulated expression of the CLB2 cluster. Our data therefore identify an important regulatory target for cyclin-dependent kinases in the cell cycle and further our molecular understanding of the key cell cycle regulatory transcription factor Fkh2p.

Binding Sites↗
PubMed2004

Ternary complex factor-serum response factor complex-regulated gene activity is required for cellular proliferation and inhibition of apoptotic cell death.

Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCF-mediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.

Alleles↗
PubMed2003

Transcriptional regulation by the MAP kinase signaling cascades.

A major function of the mitogen-activated protein kinase (MAPK) pathways is to control eukaryotic gene expression programmes in response to extracellular signals. MAPKs directly control gene expression by phosphorylating transcription factors. However, it is becoming clear that transcriptional regulation in response to MAPK signaling is more complex. MAPKs can also target coactivators and corepressors and affect nucleosomal structure by inducing histone modifications. Furthermore, multiple inputs into individual promoters can be elicited by MAPKs by targeting different components of the same coregulatory complex or by triggering different events on the same transcription factor. "Postgenomic approaches" are beginning to impact on our understanding of these gene regulatory networks. In this review, we summarise the current knowledge of MAPK-mediated gene regulation, and focus on how complexities in signaling outcomes are achieved and how this relates to physiological processes.

Animals↗
PubMed2003

Cell cycle-regulated transcription through the FHA domain of Fkh2p and the coactivator Ndd1p.

Recent studies in Saccharomyces cerevisiae by using global approaches have significantly enhanced our knowledge of the components involved in the transcriptional regulation of the cell cycle. The Mcm1p-Fkh2p complex, in combination with the coactivator Ndd1p, plays an important role in the cell cycle-dependent expression of the CLB2 gene cluster during the G2 and M phases ([4-7]; see [8-10]for reviews). Fkh2p is phosphorylated in a cell cycle-dependent manner, and peak phosphorylation occurs coincidentally with maximal expression of Mcm1p-Fkh2p-dependent gene expression. However, the mechanism by which this complex is activated in a cell cycle-dependent manner is unknown. Here, we demonstrate that the forkhead-associated (FHA) domain of Fkh2p directs cell cycle-regulated transcription and that the activity of this domain is dependent on the coactivator Ndd1p. Ndd1p was found to be phosphorylated in a cell cycle-dependent manner by Cdc28p-Clb2p, and, importantly, this phosphorylation event promotes interactions between Ndd1p and the FHA domain of Fkh2p. Furthermore, mutation of the FHA domain blocks these phosphorylation-dependent interactions and abolishes transcriptional activity. Our data therefore link the transcriptional activity of the FHA domain with cell cycle-dependent phosphorylation of the coactivator Ndd1p and reveal a mechanism that permits precise temporal activation of the Mcm1p-Fkh2p complex.

Blotting, Western↗
PubMed2003

Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

Amino Acid Sequence↗