scholarly journals Phosphorylation of the Human Cytomegalovirus 86-Kilodalton Immediate-Early Protein IE2

1998 ◽  
Vol 72 (7) ◽  
pp. 5481-5492 ◽  
Author(s):  
Noam Y. Harel ◽  
James C. Alwine
Keyword(s):  
Map Kinase ◽  
Human Cytomegalovirus ◽  
Transcriptional Activation ◽  
Immediate Early ◽  
Wild Type ◽  
Independent Manner ◽  
Early Protein ◽  
Mitogen Activated Protein

ABSTRACT We have investigated the phosphorylation state of the human cytomegalovirus 86-kDa immediate-early (IE) protein IEP86 from transfected and infected cells. We show that multiple domains of IEP86 are phosphorylated by cellular kinases, both in vitro and in vivo. Our data suggest that serum-inducible kinases play a significant role in cell-mediated IE protein phosphorylation and that a member of the mitogen-activated protein (MAP) kinase (MAPK) family, extracellular regulated kinase 2 (ERK2), phosphorylates several domains of IEP86 in vitro. Alanine substitution mutagenesis was performed on specific serines or threonines (T27, S144, T233/S234, and T555) found in consensus MAP kinase motifs. Analysis of these mutations showed that T27 and T233/S234 are the major sites for serum-inducible kinases and are the major ERK2 sites in vitro. S144 appeared to be phosphorylated in a serum-independent manner in vitro. All of the mutations except T555 eliminated specific phosphorylation in vivo. In transient transfection analyses, IEP86 isoforms containing mutations in S144 and, especially, T233/S234displayed increased transcriptional activation relative to the wild type, suggesting that phosphorylation at these sites in wild-type IEP86 may result in reduction of its transcriptional activation ability.

scholarly journals In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins.

1993 ◽  
Vol 13 (2) ◽  
pp. 1238-1250 ◽  
Author(s):  
K M Klucher ◽  
M Sommer ◽  
J T Kadonaga ◽  
D H Spector
Keyword(s):  
Human Cytomegalovirus ◽  
Hela Cells ◽  
Transcriptional Activation ◽  
Histone H1 ◽  
Early Gene ◽  
Immediate Early ◽  
Early Promoter ◽  
Drosophila Embryos

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The results of the in vitro experiments correlated well with those obtained in vivo. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein. Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter. However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold. The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Krüppel promoter in either the presence or absence of histone H1. These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.

scholarly journals The carboxyl terminus of the human cytomegalovirus UL37 immediate-early glycoprotein is conserved in primary strains and is important for transactivation

2001 ◽  
Vol 82 (7) ◽  
pp. 1569-1579 ◽  
Author(s):  
Wail A. Hayajneh ◽  
Despina G. Contopoulos-Ioannidis ◽  
Marci M. Lesperance ◽  
Ana M. Venegas ◽  
Anamaris M. Colberg-Poley
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Immediate Early ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Reading Frame ◽  
Cytosolic Domains ◽  
Carboxyl Terminal

The human cytomegalovirus (HCMV) UL37 exon 3 (UL37x3) open reading frame (ORF) encodes the carboxyl termini of two immediate-early glycoproteins (gpUL37 and gpUL37M). UL37x3 homologous sequences are not required for mouse cytomegalovirus (MCMV) growth in vitro; yet, they are important for MCMV growth and pathogenesis in vivo. Similarly, UL37x3 sequences are dispensable for HCMV growth in culture, but their requirement for HCMV growth in vivo is not known. To determine this requirement, we directly sequenced the complete UL37x3 gene in multiple HCMV primary strains. A total of 63 of the 310 amino acids in the UL37x3 ORF differ non-conservatively in one or more HCMV primary strains. The HCMV UL37x3 genetic diversity is non-random: the N-glycosylation (46/186 aa) and basic (9/15 aa) domains have the highest proportion of non-conservative variant amino acids. Nonetheless, most (15/17 signals) of the N-glycosylation signals are retained in all HCMV primary strains. Moreover, new N-glycosylation signals are encoded by 5/20 primary strains. In sharp contrast, the UL37x3 transmembrane (TM) ORF completely lacks diversity in all 20 HCMV sequenced primary strains, and only 1 of 28 cytosolic tail residues differs non-conservatively. To test the functional significance of the conserved carboxyl terminus, gpUL37 mutants lacking the TM and/or cytosolic tail were tested for transactivating activity. The gpUL37 carboxyl-terminal mutants are partially defective in hsp70 promoter transactivation even though they trafficked similarly to the wild-type protein into the endoplasmic reticulum and to mitochondria. From these results, we conclude that N-glycosylated gpUL37, particularly its TM and cytosolic domains, is important for HCMV growth in humans.

In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins

1993 ◽  
Vol 13 (2) ◽  
pp. 1238-1250
Author(s):  
K M Klucher ◽  
M Sommer ◽  
J T Kadonaga ◽  
D H Spector
Keyword(s):  
Human Cytomegalovirus ◽  
Hela Cells ◽  
Transcriptional Activation ◽  
Histone H1 ◽  
Early Gene ◽  
Immediate Early ◽  
Early Promoter ◽  
Drosophila Embryos

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The results of the in vitro experiments correlated well with those obtained in vivo. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein. Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter. However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold. The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Krüppel promoter in either the presence or absence of histone H1. These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.

scholarly journals Targeting of p38 Mitogen-Activated Protein Kinases to MEF2 Transcription Factors

1999 ◽  
Vol 19 (6) ◽  
pp. 4028-4038 ◽  
Author(s):  
Shen-Hsi Yang ◽  
Alex Galanis ◽  
Andrew D. Sharrocks
Keyword(s):  
Transcription Factors ◽  
Transcriptional Activation ◽  
Cellular Response ◽  
Map Kinases ◽  
Biological Response ◽  
Extracellular Signals ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases which phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. In this study, we demonstrate that among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38α and p38β2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to our understanding of signalling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38).

scholarly journals Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fosPromoter

2000 ◽  
Vol 20 (4) ◽  
pp. 1140-1148 ◽  
Author(s):  
Dae-Won Kim ◽  
Brent H. Cochran
Keyword(s):  
Map Kinase ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Binding Motif ◽  
Dependent Manner ◽  
Consensus Map ◽  
Interaction Domain ◽  
Extracellular Signal

ABSTRACT We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fospromoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.

scholarly journals GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

2001 ◽  
Vol 21 (24) ◽  
pp. 8565-8574 ◽  
Author(s):  
Anthony J. Greenberg ◽  
Paul Schedl
Keyword(s):  
Fusion Protein ◽  
Transcriptional Activation ◽  
Functional Difference ◽  
Wild Type ◽  
Gaga Factor ◽  
Phenotypic Analysis ◽  
Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides

1994 ◽  
Vol 14 (5) ◽  
pp. 3484-3493
Author(s):  
T J Wu ◽  
G Monokian ◽  
D F Mark ◽  
C R Wobbe
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transcriptional Activation ◽  
Deletion Mutant ◽  
Full Length ◽  
Early Gene ◽  
Immediate Early ◽  
Simplex Virus

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

scholarly journals MDM2 Suppresses p73 Function without Promoting p73 Degradation

1999 ◽  
Vol 19 (5) ◽  
pp. 3257-3266 ◽  
Author(s):  
Xiaoya Zeng ◽  
Lihong Chen ◽  
Christine A. Jost ◽  
Ruth Maya ◽  
David Keller ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Feedback Loop ◽  
Human Lung ◽  
Activation Function ◽  
Wild Type ◽  
Mdm2 Protein ◽  
Induced Apoptosis ◽  
P53 Inactivation

ABSTRACT The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73α and p73β stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.

Role of IRS-1-GRB-2 complexes in insulin signaling

1994 ◽  
Vol 14 (6) ◽  
pp. 3577-3587
Author(s):  
M G Myers ◽  
L M Wang ◽  
X J Sun ◽  
Y Zhang ◽  
L Yenush ◽  
...  
Keyword(s):  
Map Kinase ◽  
Insulin Receptor ◽  
Insulin Receptors ◽  
Growth Factor Signaling ◽  
Insulin Stimulation ◽  
Mitogen Activated Protein ◽  
D Cells ◽  
Stimulation Of

GRB-2 is a small SH2- and SH3 domain-containing adapter protein that associates with the mammalian SOS homolog to regulate p21ras during growth factor signaling. During insulin stimulation, GRB-2 binds to the phosphorylated Y895VNI motif of IRS-1. Substitution of Tyr-895 with phenylalanine (IRS-1F-895) prevented the IRS-1-GRB-2 association in vivo and in vitro. The myeloid progenitor cell line, 32-D, is insensitive to insulin because it contains few insulin receptors and no IRS-1. Coexpression of IRS-1 or IRS-1F-895 with the insulin receptor was required for insulin-stimulated mitogenesis in 32-D cells, while expression of the insulin receptor alone was sufficient to mediate insulin-stimulated tyrosine phosphorylation of Shc and activation of p21ras and mitogen-activated protein (MAP) kinase. The Shc-GRB-2 complex formed during insulin stimulation is a possible mediator of p21ras and MAP kinase activation in IRS-1-deficient 32-D cells. Interestingly, IRS-1, but not IRS-1F-895, enhanced the stimulation of MAP kinase by insulin in 32-D cells expressing insulin receptors. Thus, IRS-1 contributes to the stimulation of MAP kinase by insulin, probably through formation of the IRS-1-GRB-2 complex at Tyr-895. Our results suggest that the Shc-GRB-2 complex and the activation of p21ras-dependent signaling pathways, including MAP kinase, are insufficient for insulin-stimulated mitogenesis and that the essential function(s) of IRS-1 in proliferative signaling is largely unrelated to IRS-1-GRB-2 complex formation.

scholarly journals Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

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