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W Krek

Publications and source records attributed to W Krek.

At least 19 recordsLinked to original sources
PubMed1999

The von Hippel-Lindau tumor suppressor protein is a component of an E3 ubiquitin-protein ligase activity.

pVHL, the product of the VHL tumor suppressor gene, plays an important role in the regulation of cell growth and differentiation of human kidney cells, and inactivation of the VHL gene is the most frequent genetic event in human kidney cancer. The biochemical function of pVHL is unknown. Here we report that pVHL exists in vivo in a complex that displays ubiquitination-promoting activity in conjunction with the universally required components E1, E2, and ubiquitin. pVHL-associated ubiquitination activity requires, at a minimum, pVHL to bind elongin C and Cul-2, relatives of core components of SCF (Skp1-Cdc53/Cul-1-F-box protein) E3 ligase complexes. Notably, certain tumor-derived mutants of pVHL demonstrate loss of associated ubiquitination promoting activity. These results identify pVHL as a component of a potential SCF-like E3 ubiquitin-protein ligase complex and suggest a direct link between pVHL tumor suppressor and the process of ubiquitination.

Amino Acid Sequence↗
PubMed1999

Association of human SCF(SKP2) subunit p19(SKP1) with interphase centrosomes and mitotic spindle poles.

In Saccharomyces cerevisiae, the initiation of DNA replication and mitotic progression requires SKP1p function. SKP1p is an essential subunit of a newly identified class of E3 ubiquitin protein ligases, the SCF complexes, that catalyze ubiquitin-mediated proteolysis of key cell-cycle-regulatory proteins at distinct times in the cell cycle. SKP1p is also required for proper kinetochore assembly. Little is known about the corresponding human homolog, p19(SKP1), except that it is expressed throughout the cell cycle and that it too is a component of an S-phase-regulating SCF-E3 ligase complex. Here we show by immunofluorescence microscopy that p19(SKP1) localizes to the centrosomes. Centrosome association occurs throughout the mammalian cell cycle, including all stages of mitosis. These findings suggest that p19(SKP1) is a novel component of the centrosome and the mitotic spindle, which, in turn, implies a physiological role of this protein in the regulation of one or more aspects of the centrosome cycle.

Animals↗
PubMed1999

Increased levels of E2F-1-dependent DNA binding activity after UV- or gamma-irradiation.

In mammalian cells, DNA damage induces robust changes in gene expression and these changes contribute to the proper execution of cellular responses to DNA damage, including DNA repair, cell cycle arrest and apoptosis. The transcription factor E2F-1 has been suggested to play a key role in the regulation of cell cycle-dependent gene expression and apoptosis. These activities depend on the ability of E2F-1 to form functionally active DNA binding complexes. Here we describe an assay that allows one to measure E2F-1 DNA binding activity in naive cells. We find that DNA damage, generated by UV- or gamma-irradiation, prompts increased production of E2F-1 DNA binding activity, which, at least in part, originates from alterations in E2F-1 protein levels. These findings represent an indication for a role of the transcription factor E2F-1 in the DNA damage response pathway.

Amino Acid Sequence↗
PubMed1999

E2F-1 has properties of a radiosensitizer and its regulation by cyclin A kinase is required for cell survival of fibrosarcoma cells lacking p53.

Negative regulation of E2F-1 DNA binding function by cyclin A kinase represents part of an S-phase checkpoint control system that, when activated, leads to apoptosis. In this study, we examined the cellular sensitivity and resistance of isogenic mouse fibrosarcoma cell lines, differing primarily in their p53 status, to ectopic expression of wild-type (wt) E2F-1 and cyclin A kinase binding-defective mutants of it. We found that E2F-1 (wt) potently affected the survival of p53+/+ tumor cells but not that of p53-/- cells. In contrast, expression of cyclin A kinase binding-defective E2F-1 species interfered with cell survival of fibrosarcoma cells irrespective of their p53 status. Finally, expression of E2F-1 (wt) in p53-/- fibrosarcoma cells enhanced the cytotoxic effect of ionizing radiation in vitro and in vivo in a mouse tumor model. These results suggest that E2F-1-dependent activation of an S-phase checkpoint is p53 independent and that E2F-1 possesses radiosensitizing properties in the absence of p53.

Animals↗
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↗