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W Krek

Publications and source records attributed to W Krek.

At least 19 recordsLinked to original sources
PubMed1998

Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway.

In normal and transformed cells, the F-box protein p45(SKP2) is required for S phase and forms stable complexes with p19(SKP1) and cyclin A-cyclin-dependent kinase (CDK)2. Here we identify human CUL-1, a member of the cullin family, and the ubiquitin-conjugating enzyme CDC34 as additional partners of p45(SKP2) in vivo. CUL-1 also associates with cyclin A and p19(SKP1) in vivo and, with p45(SKP2), they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box protein, a member of the cullin family and a homolog of p19(SKP1)) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45(SKP2)-CUL-1-p19(SKP1) complex may be a human representative of an SCF-type E3 ubiquitin protein ligase. We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiprotein complex formation involving p45(SKP2)-CUL-1 and p19(SKP1) is governed, in part, by periodic, S phase-specific accumulation of the p45(SKP2) subunit and by the p45(SKP2)-bound cyclin A-CDK2. The dependency of p45(SKP2)-p19(SKP1) complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway.

Amino Acid Sequence↗
PubMed1998

Proteolysis and the G1-S transition: the SCF connection.

Temporal control of ubiquitin-proteasome mediated protein degradation is critical for normal G1 and S phase progression. Recent work has shown that central to the temporal control mechanism is a relationship between newly identified E3 ubiquitin protein ligases, designated SCFs (Skp1-cullin-F-box protein ligase complexes), which confer substrate specificity on ubiquitination reactions and the activities of protein kinases that phosphorylate substrates destined for destruction at specific sites, thereby converting them into preferred targets for ubiquitin modification catalyzed by SCFs. The constituents of SCFs are members of evolutionary conserved protein families. SCF-based ubiquitination pathways may play a key role in diverse biological processes, such as cell proliferation, differentiation and development.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1996

CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F.

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B, and H1 is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified. Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp91-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.

Base Sequence↗
PubMed1995

Cyclin A-kinase regulation of E2F-1 DNA binding function underlies suppression of an S phase checkpoint.

Commitment of mammalian cells to enter S phase enables the transcription factor E2F-1 to activate certain genes whose products mediate cell cycle advance. In S phase, E2F-1 forms stable complexes with cyclin A-kinase, which in turn eliminates E2F-1DNA binding function. Here, we show that suppression of E2F-1 DNA-binding activity by cyclin A-kinase is linked to orderly S phase progression. Disruption of this linkage resulted in S phase delay/arrest followed by regrowth or apoptosis, depending upon whether the DNA-bound E2F-1 could transactivate. Hence, the unscheduled presence of E2F-1 on specific DNA sequences during S phase can activate a specific S phase checkpoint, thereby linking transcription, DNA replication, and cell cycle control.

3T3 Cells↗
PubMed1995

Multiple members of the E2F transcription factor family are the products of oncogenes.

The retinoblastoma gene product (pRB) is a known tumor suppressor, capable of arresting growth in mid-to-late G1. Part of its growth suppression action arises from interaction(s) with one or more members of the E2F family of transcription factors. These proteins most likely contribute to progression from G0 to S phase in mammalian cells, and pRB binding most likely inhibits aspects of their suspected growth-promoting function. Given their growth-stimulating potential, we asked whether one or more E2F alleles can function as oncogenes. Uncloned pools of NIH 3T3 cells producing the pRB binding target E2F-1, E2F-2, or E2F-3 grew in semisolid medium. In addition, they grew to much higher saturation density than controls. From the study of cells producing selected E2F-1 mutant species, it appears that E2F DNA-binding function contributes to, and pRB/E2F binding suppresses, soft-agar growth. Thus, three E2F family members can act as oncogene products, suggesting that part of the normal role of pRB is to down-modulate this potential activity.

3T3 Cells↗
PubMed1994

Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase.

Cyclin A-kinase, an enzyme required for coordinating S phase progression, forms stable in vivo complexes with E2F-1, a growth-promoting transcription factor, which binds to the retinoblastoma gene product and is involved in the timely activation of genes whose products contribute to G1 exit and S phase traversal. Complex formation results in a negative biochemical effect of cyclin A-kinase: the shut-off of E2F-1-dependent DNA binding function in S/G2. Thus, specific and timely cell cycle-dependent interactions of E2F-1 with proteins that inhibit its function (i.e., RB during G1 and cyclin A-kinase during S/G2) may contribute to the periodicity of expression of certain E2F-1-responsive genes at the G1/S transition.

3T3 Cells↗
PubMed1993

Binding to DNA and the retinoblastoma gene product promoted by complex formation of different E2F family members.

The E2F family of transcription factors functions in the control of the mammalian cell cycle. Here it is shown that two family members, E2F-1 and DP-1, form specific heterodimers in vivo, a process that enhances DNA binding, transactivation, and the binding of the retinoblastoma gene product. These results suggest that heterodimerization regulates E2F function and contributes to cell cycle control.

Base Sequence↗
PubMed1992

Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties.

An expression vector was modified to permit the rapid synthesis of purified, 32P-labeled, glutathione S-transferase (GST)-retinoblastoma (RB) fusion proteins. The products were used to screen lambda gt11 expression libraries, from which we cloned a cDNA encoding a polypeptide (RBAP-1) capable of binding directly to a putative functional domain (the pocket) of the retinoblastoma gene product (RB). The RB "pocket" is known to bind, directly or indirectly, to the cellular transcription factor, E2F, implicated in cell growth control. We have found that RBAP-1 copurifies with E2F, interacts specifically with the adenovirus E4 ORF 6/7 protein, binds specifically and directly to a known E2F DNA recognition sequence, and contains a functional tranasactivation domain. Therefore, RBAP-1 is a species of E2F and can bind specifically to the RB pocket.

Amino Acid Sequence↗
PubMed1992

Regulation of p34cdc2 protein kinase activity by phosphorylation and cyclin binding.

Activation of the protein kinase p34cdc2 is required for entry into meiotic or mitotic M phase in all eukaryotic cells. One important mechanism regulating the activity of p34cdc2 during the cell cycle is based on phosphorylation/dephosphorylation. Avian p34cdc2 is phosphorylated on threonine 14 (Thr14), tyrosine 15 (Tyr15), threonine 161 (Thr161) and serine 277 (Ser277). Dephosphorylation of both Thr14 and Tyr15 is required for activation of p34cdc2 at the G2/M transition, indicating that phosphorylation of these residues negatively regulates p34cdc2 activity. Conversely, phosphorylation of Thr161 is required for kinase activity. Whether modification of this residue is due to intramolecular autophosphorylation or to the action of an as yet unidentified kinase remains unresolved. Likewise, the role of phosphorylation of p34cdc2 on Ser277 during G1 phase of the cell cycle remains to be determined. The function of p34cdc2 is regulated also by cell cycle-dependent complex formation with cyclin proteins. We found that chicken cyclin B2 undergoes a striking redistribution from the cytoplasm to the nucleus just prior to the onset of mitosis. Expression of a non-destructible cyclin B2 mutant causes HeLa cells to arrest in mitosis. Frequently, arrested cells displayed multiple mitotic spindles.

Amino Acid Sequence↗
PubMed1992

Casein kinase II is a predominantly nuclear enzyme.

Casein kinase II (CK II) has been implicated in regulating multiple processes related to cell growth, proliferation, and differentiation. To better understand the function(s) and regulation of this ubiquitous kinase, it is important to know its subcellular distribution. However, this issue has been the subject of contradictory reports. In this study, we have used indirect immunofluorescence microscopy and cell fractionation to study the subcellular distribution of all three subunits of chicken CK II, alpha, alpha', and beta. We examined primary chick embryo fibroblasts, virally transformed chicken hepatoma cells, as well as HeLa cells transiently transfected with cDNAs encoding chicken CK II subunits. We found that each of the three CK II subunits was located predominantly in the cell nucleus, irrespective of the cell type analyzed or the procedure used for cell fixation. No major differences were detected in the subcellular distributions of individual CK II subunits, and no evidence was obtained for subunit redistributions during interphase of the cell cycle. During mitosis, the bulk of the enzyme was dispersed throughout the cell, though a fraction of all three subunits was associated with the mitotic spindle. Biochemical studies based on mechanical enucleation of chicken cells confirmed the predominantly nuclear location of all three CK II subunits. Finally, immunoblotting experiments were carried out to study the expression of CK II subunits. A survey of different adult chicken tissues revealed substantial tissue-specific differences in the levels of CK II protein, but no evidence was obtained for pronounced tissue specificity in the expression of individual CK II subunits. These results strongly suggest that CK II functions primarily in regulating nuclear activities, and that the two catalytic subunits, alpha and alpha', may carry out overlapping functions.

Animals↗
PubMed1992

Vertebrate p34cdc2 phosphorylation site mutants: effects upon cell cycle progression in the fission yeast Schizosaccharomyces pombe.

We have used the fission yeast Schizosaccharomyces pombe to analyse the effects of in vitro mutagenesis of the four known phosphorylation sites in the chicken p34(cdc2) protein, Thr 14, Tyr 15, Thr 161 and Ser 277, upon cell cycle progression. We have studied both the effect of overexpression of mutant proteins in a cdc2+ background and assayed their ability to rescue null and temperature-sensitive alleles of cdc2. Mutations of Thr 14 and Tyr 15 within the ATP binding domain of p34(cdc2) that mimic constitutive phosphorylation cause dominant negative cell cycle arrest when overexpressed. In contrast, some substitutions that simulate permanent dephosphorylation of the corresponding sites advance dephosphorylation of the corresponding sites advance mitosis. These data confirm the model that p34(cdc2) function is negatively regulated by phosphorylation of residues in the ATP binding site. Mutagenesis of the conserved residue Thr 161 functionally inactivates p34(cdc2), and our data suggest that both phosphorylation and dephosphorylation events at Thr 161 are required for progression through the cell cycle. Mutations at the fourth site of phosphorylation. Ser 277, lead to cold-sensitive cell cycle arrest, in minimal but not rich growth medium, suggesting that this site is involved in monitoring the nutritional status of the cell.

Amino Acid Sequence↗
PubMed1992

Cell cycle regulation of vertebrate p34cdc2 activity: identification of Thr161 as an essential in vivo phosphorylation site.

The protein kinase p34cdc2 is a key regulator of the cell cycle in all eukaryotes. Its activity is controlled by cell cycle-dependent interactions with other proteins, notably cyclins, and by changes in its phosphorylation state. Two inhibitory phosphorylation sites in chicken p34cdc2 have previously been mapped to threonine 14 and tyrosine 15. Here we describe the identification of threonine 161 as an additional in vivo phosphorylation site in vertebrate p34cdc2. Phosphorylation of this site is cell cycle dependent and likely to be required for p34cdc2 activity.

Amino Acid Sequence↗
PubMed1991

Casein kinase II. cDNA sequences, developmental expression, and tissue distribution of mRNAs for alpha, alpha', and beta subunits of the chicken enzyme.

We report the complete primary structures, as deduced from cloned cDNAs, of two catalytic (alpha and alpha') and one beta subunit of chicken casein kinase II. The alpha and alpha' subunits are closely related to each other but differ in their carboxyl termini; moreover, minor differences are distributed throughout the entire lengths of the two proteins, indicating that they are derived from separate genes. Comparison of the chicken protein sequences with those of their mammalian homologs reveals a high degree of evolutionary conservation of all three subunits. Northern analyses were carried out to study the expression of casein kinase II subunits during chicken embryonic development as well as in adult tissues. A single transcript migrating at 1 kilobase was detected when using a beta-specific probe, but multiple transcripts ranging in size from 1.45 to 2.8 kilobase were revealed by alpha- and alpha'-specific probes. Expression of mRNA for all three subunits was high in early embryos but decreased substantially during embryonic development. A comparative analysis of casein kinase II expression in different adult tissues revealed two major findings. First, relative levels of expression of the two catalytic subunits displayed a pronounced tissue specificity, consistent with the possibility that alpha and alpha' subunits may carry out specialized functions. Second, no correlation was observed between levels of transcripts for the catalytic subunits and those of mRNA for the beta subunit.

Amino Acid Sequence↗
PubMed1991

Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates.

In vertebrates, entry into mitosis is accompanied by dephosphorylation of p34cdc2 kinase on threonine 14 (Thr14) and tyrosine 15 (Tyr15). To examine the role of these residues in controlling p34cdc2 kinase activation, and hence the onset of mitosis, we replaced Thr14 and/or Tyr15 by non-phosphorylatable residues and transfected wild-type and mutant chicken p34cdc2 cDNAs into HeLa cells. While expression of wild-type p34cdc2 did not interfere with normal cell cycle progression, p34cdc2 carrying mutations at both Thr14 and Tyr15 displayed increased histone H1 kinase activity and rapidly induced premature mitotic events, including chromosome condensation and lamina disassembly. No phenotype was observed in response to mutation of only Thr14, and although single-site mutation at Tyr15 did induce premature mitotic events, effects were partial and their onset was delayed. These results identify both Thr14 and Tyr15 as sites of negative regulation of vertebrate p34cdc2 kinase, and they suggest that dephosphorylation of p34cdc2 represents the rate-limiting step controlling entry of vertebrate cells into mitosis.

Amino Acid Sequence↗
PubMed1991

Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites.

The cdc2 kinase is a key regulator of the eukaryotic cell cycle. The activity of its catalytic subunit, p34cdc2, is controlled by cell cycle dependent interactions with other proteins as well as by phosphorylation--dephosphorylation reactions. In this paper, we examine the phosphorylation state of chicken p34cdc2 at various stages of the cell cycle. By peptide mapping, we detect four major phosphopeptides in chicken p34cdc2; three phosphorylation sites are identified as threonine (Thr) 14, tyrosine (Tyr) 15 and serine (Ser) 277. Analysis of synchronized cells demonstrates that phosphorylation of all four sites is cell cycle regulated. Thr 14 and Tyr 15 are phosphorylated maximally during G2 phase but dephosphorylated abruptly at the G2/M transition, concomitant with activation of p34cdc2 kinase. This result suggests that phosphorylation of Thr 14 and/or Tyr 15 inhibits p34cdc2 kinase activity, in line with the location of these residues within the putative ATP binding site of the kinase. During M phase, p34cdc2 is also phosphorylated, but phosphorylation occurs on a threonine residue distinct from Thr 14. Finally, phosphorylation of Ser 277 peaks during G1 phase and drops markedly as cells progress through S phase, raising the possibility that this modification may contribute to control the proposed G1/S function of the vertebrate p34cdc2 kinase.

Amino Acid Sequence↗
PubMed1990

Structure and developmental expression of chicken nucleolin and NO38: coordinate expression of two abundant non-ribosomal nucleolar proteins.

We report the complete primary structures of two major chicken non-ribosomal nucleolar proteins known as nucleolin/C23 and NO38/B23, respectively. By comparison with homologous proteins from other species, this sequence information contributes to the identification of evolutionarily conserved motifs that may be relevant to the function and subcellular distribution of the two proteins. Using cDNA probes and monoclonal antibodies, we have also studied the expression of nucleolin and NO38 in the course of chicken embryogenesis. In all tissues examined, Northern analyses revealed single hybridization signals for nucleolin (at 3.0 kb) and NO38 (at 1.6 kb), and no evidence was obtained for multiple protein products. In total embryos between days 3 and 11 after egg laying, nucleolin and NO38 mRNA and protein levels decreased in parallel (2-5-fold), suggesting transcriptional down-regulation of expression. Coordinate expression of nucleolin and NO38 was observed also when examining individual tissues at various stages of development. Interestingly, however, there was no consistent correlation between relative mRNA and protein levels. In particular, several adult tissues contained exceedingly low levels of either nucleolin or NO38, despite the presence of large amounts of corresponding mRNAs. From these results we conclude, first, that the expression of nucleolin and NO38 is controlled coordinately, and, second, that regulation is likely to involve both transcriptional and posttranscriptional mechanisms.

Amino Acid Sequence↗