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B Lüscher

Publications and source records attributed to B Lüscher.

At least 73 records · Page 4Linked to original sources
PubMed1994

Expression of mad, mxi1, max and c-myc during induced differentiation of hematopoietic cells: opposite regulation of mad and c-myc.

The Myc proto-oncoprotein family is considered to play an important role in the control of cell growth and differentiation. It appears that the interaction of Myc with its heterodimeric partner Max is essential for Myc function. Recently two other partners of Max, called Mad and Mxi1, have been identified. In an effort to gain insight into the network of these four proteins we have started to analyse the expression of the c-myc, max, mad and mxi1 genes at the mRNA level during hematopoietic cell growth and differentiation. In the human myeloid cell lines U-937, HL-60 and ML-1 c-myc expression was down-regulated as shown previously after induction of differentiation, whereas the expression of max was only slightly affected. In contrast to these two genes the expression of mad was induced upon differentiation in the three cell lines by TPA, retinoic acid, vitamin D3, dimethyl sulfoxide, and interferon-gamma and remained elevated for at least 3 days. A kinetic analysis showed that the induction of mad in U-937 in response to TPA was rapid (15 min) and at least in part transcriptional, reminiscent of immediate early genes. The expression of mxi1 was induced in U-937 by some inducers but not in HL-60 or ML-1. Its induction occurred slowly, peaking around 48 h. These analysis thus suggest that the expression of mad and c-myc is inversely regulated during induced hematopoietic differentiation.

Cell Differentiation↗
PubMed1993

Casein kinase II in signal transduction and cell cycle regulation.

Casein kinase II is a protein serine/threonine kinase that is ubiquitously distributed in eukaryotes. Molecular cloning studies and protein sequence analysis of purified proteins have demonstrated the existence of two related, but distinct, isoenzymic forms of its catalytic subunit in mammals and birds. At present, the precise role of the individual casein kinase II isoforms in biological responses is poorly understood. However, a great deal of evidence indicates that casein kinase II is an important component of signalling pathways that control the growth and division of cells. In particular, casein kinase II is known to phosphorylate, and in several cases, regulate the activity of a variety of regulatory nuclear proteins including nuclear oncoproteins, transcription factors, and enzymes involved in other aspects of DNA metabolism. In this review, we will summarize evidence relating to the involvement of casein kinase II in signal transduction events that are relevant to cell proliferation.

Amino Acid Sequence↗
PubMed1993

Phosphorylation sites mapping in the N-terminal domain of c-myc modulate its transforming potential.

The nuclear proto-oncoprotein c-Myc is involved in the regulation of cell growth and differentiation. c-Myc is phosphorylated at multiple sites in vivo, two of which we have identified near the amino terminus. In chicken Thr-61/Ser-65 are phosphorylated, as are the comparable positions, Thr-58/Ser-62 in human c-Myc. These residues are located within a domain that is implicated in transactivation and is important for the transforming potential of the protein. Furthermore, these phosphorylation sites or nearby amino acids are frequently mutated in v-myc and in several c-myc genes from Burkitt's lymphoma cells. In vitro these two phosphorylation sites can be modified by glycogen synthase kinase 3 and mitogen activated protein kinase. To address their biological importance we mutated these amino terminal phosphorylation sites separately and together. Stably transfected Rat1A cells expressing the mutated proteins have an increased growth potential in soft agar compared to wt-c-myc transfectants. These altered transformation characteristics indicate that Myc function may be negatively regulated by the amino terminal phosphorylation.

Amino Acid Sequence↗
PubMed1993

Identification of casein kinase II phosphorylation sites in Max: effects on DNA-binding kinetics of Max homo- and Myc/Max heterodimers.

Myc proteins have been implicated in the regulation of cell growth and differentiation. The identification of Max, a basic region/helix-loop-helix/leucine zipper protein, as a partner for Myc has provided insights into Myc's molecular function as a transcription factor. Recent evidence indicates that the relative abundance of Myc and Max is important to determine the level of specific gene transcription. In this report we have identified two major in vivo phosphorylation sites in Max (Ser-2 and -11) which can be modified in vitro by casein kinase II (CKII). Phosphorylation of these sites modulates DNA-binding by increasing both the on- and off-rates of Max homo- as well as Myc/Max heterodimers. In addition, our data indicate that the steady state binding of the shorter version of Max (p21) to DNA was similar yet its rate of dissociation faster than that of longer version of Max (p22). These data argue that different Max complexes have different kinetic properties and that these can be modified by CKII phosphorylation. We propose this as an important biological mechanism by which different dimeric complexes can exchange with varying efficiencies on DNA, thereby responding to changes in cell growth conditions.

Amino Acid Sequence↗
PubMed1993

[Risk factors in papillary thyroid cancer].

270 patients with papillary thyroid carcinoma were retrospectively analyzed. The mean age at initial diagnosis was 43.5 years. 74% of the patients were females. 15 patients (6%) had residual tumor after initial surgery and 61 patients (24%) showed recurrent disease. The recurrence was discovered clinically, radiologically and by increasing thyroglobulin levels in 72%, 23% and 5% of the patients, respectively. Recurrent disease was located in the tumor bed, locally and metastatic and only metastatic in 61%, 23% and 16% of the patients, respectively. After a mean follow-up of 9.4 years (range 3-20 years) the overall mortality was 13%. 6% of the patients died from tumor, 3% with tumor and 4% without tumor. 46% of patients with residual tumor died from tumor progression, in contrast to 18% of patients with early recurrence (< 5 years) and 6% of patients with late recurrence (> 5 years). We conclude that papillary thyroid carcinoma has a low mortality (6%) even in an endemic area. Patients with residual tumor after initial surgery and patients with early recurrence have an increased cancer mortality.

Adult↗
PubMed1992

Phosphorylation of casein kinase II by p34cdc2 in vitro and at mitosis.

In human epidermal carcinoma A431 cells, the beta subunit of casein kinase II is phosphorylated at an autophosphorylation site and at serine 209 which can be phosphorylated in vitro by p34cdc2 (Litchfield, D. W., Lozeman, F. J., Cicirelli, M. F., Harrylock, M., Ericsson, L. H., Piening, C. J., and Krebs, E. G. (1991) J. Biol. Chem. 266, 20380-20389). Given the importance of p34cdc2 in the regulation of cell cycle events, we were interested in examining the phosphorylation of casein kinase II during different stages of the cell cycle. In this study it is demonstrated that the extent of phosphorylation of serine 209 in the beta subunit is significantly increased relative to phosphorylation of the autophosphorylation site when chicken bursal lymphoma BK3A cells are arrested at mitosis by nocodazole treatment. This result suggests that serine 209 is a likely physiological target for p34cdc2. In addition, the alpha subunit of casein kinase II also undergoes dramatic phosphorylation with an associated alteration in its electrophoretic mobility when BK3A cells or human Jurkat cells are arrested with nocodazole. Phosphopeptide mapping studies indicate that p34cdc2 can phosphorylate in vitro the same peptides on the alpha subunit that are phosphorylated in cells arrested at mitosis. These phosphorylation sites were localized to serine and threonine residues in the carboxyl-terminal domain of alpha. Taken together, the results of this study indicate that casein kinase II is a probable physiological substrate for p34cdc2 and suggest that its functional properties could be affected in a cell cycle-dependent manner.

Animals↗
PubMed1992

Mitosis-specific phosphorylation of the nuclear oncoproteins Myc and Myb.

The c-myc and c-myb proto-oncogenes encode phosphorylated nuclear DNA binding proteins that are likely to be involved in transcriptional regulation. Here we demonstrate that both Myc and Myb proteins are hyperphosphorylated during mitosis. In the case of Myb, hyperphosphorylation is accompanied by the appearance of three M phase-specific tryptic phosphopeptides. At least one of these phosphopeptides corresponds to a phosphopeptide generated after phosphorylation of Myb in vitro by p34cdc2 kinase. By contrast, the mitotic hyperphosphorylation of Myc does not correlate with the appearance of unique phosphopeptides, suggesting that M phase and interphase sites may be clustered within the same peptides. In addition Myc does not appear to be a target for p34cdc2 phosphorylation. The hyperphosphorylated forms of Myc and Myb from mitotic cells are functionally distinct from the corresponding interphase proteins in that the former have reduced ability to bind nonspecificially to double-stranded DNA cellulose. Furthermore, mitotic Myb binds poorly to oligodeoxynucleotides containing an Myb response element. We surmise that the decreased DNA binding capacity of hyperphosphorylated Myb and Myc during M phase may function to release these proteins from chromatin during chromosome condensation.

Animals↗
PubMed1992

Myc and Max associate in vivo.

Max is a helix-loop-helix zipper protein that associates in vitro with Myc family proteins to form a sequence-specific DNA-binding complex. We show here, by means of a coimmunoprecipitation assay with anti-Myc and anti-Max antibodies, that Myc and Max are associated in vivo and essentially all of the newly synthesized Myc can be detected in a complex with Max. This complex possesses specific DNA-binding activity for CACGTG-containing oligonucleotides. Although Max itself is a highly stable protein, Myc is rapidly degraded during or after its association with Max. In vivo Max is shown to be a nuclear protein phosphorylated by casein kinase II, and alternatively spliced forms of Max are expressed in cells. Furthermore, the levels of Max expression are equivalent in quiescent, mitogen-stimulated, and cycling cells. We conclude that the highly regulated rate of Myc biosynthesis is likely to be a limiting step in the formation of Myc:Max complexes.

Basic-Leucine Zipper Transcription Factors↗
PubMed1991

Negative charge at the casein kinase II phosphorylation site is important for transformation but not for Rb protein binding by the E7 protein of human papillomavirus type 16.

The human papillomavirus E7 protein is phosphorylated at the two serines in positions 31/32, which are part of a consensus sequence for casein kinase II (CKII). In this study, we have investigated the effect of CKII phosphorylation site mutations, all of which lead to unphosphorylated E7 proteins. The replacement of the two serines by uncharged alanine residues drastically reduced the ability of E7 to cotransform primary cells with ras, whereas negatively charged aspartic acid at the same positions produced only a slight effect. This difference was not reflected in the p105Rb binding or the E2 promoter transactivation capability of these two mutants. Mutations that changed the CKII consensus without altering the serine residues also resulted in a loss of phosphorylation and transformation. This indicated that negative charge at positions 31/32 provided either by phosphorylation or by a negatively charged amino acid is necessary for efficient transformation without significantly affecting p105Rb binding or transactivation.

Adenoviridae↗
PubMed1991

A role for the p34cdc2 kinase and phosphatases in the regulation of phosphorylation and disassembly of lamin B2 during the cell cycle.

While the p34cdc2 kinase is considered to be a critical regulator of mitosis, its function has not yet been directly linked to one of the key events during the onset of mitosis: nuclear envelope breakdown. Here we show that a major structural protein of the nuclear envelope, lamin B2, is phosphorylated by p34cdc2. Results from two-dimensional phosphopeptide mapping experiments demonstrate that the p34cdc2-specific phosphopeptides represent both mitotic and interphase specific phosphorylations of lamin B2 and include the major interphase phosphorylation site. In mitotic cells we detected two distinct forms of lamin B2 which differ in electrophoretic mobility and in degree of phosphorylation. The phosphorylation pattern of lamin B2 generated in vitro by p34cdc2 was more closely related to the less phosphorylated mitotic lamin B2, suggesting that another kinase(s) in addition to p34cdc2 is involved in generating the mitotic phosphorylation pattern. In addition, we show that treatment of interphase cells with okadaic acid, a potent phosphatase inhibitor, leads to the acquisition of mitosis-specific phosphopeptides and can reversibly increase the detergent-solubility of lamin B2. However, the M-phase-like phosphorylation of lamin B2 in itself is not sufficient to induce its disassembly from the nuclear lamina suggesting that an additional event(s) besides phosphorylation is required.

Animals↗
PubMed1990

Myb DNA binding inhibited by phosphorylation at a site deleted during oncogenic activation.

The c-Myb nuclear oncoprotein is phosphorylated in vitro and in vivo at an N-terminal site near its DNA-binding domain by casein kinase II (CK-II) or a CK-II-like activity. This in vitro phosphorylation reversibly inhibits the sequence-specific binding of c-Myb to DNA. The site of this phosphorylation is deleted in nearly all oncogenically activated Myb proteins, resulting in DNA-binding that is independent of CK-II. Because CK-II activity is modulated by growth factors, loss of the site could uncouple c-Myb from its normal physiological regulator.

Amino Acid Sequence↗
PubMed1990

Mutational analysis of the carboxy-terminal casein kinase II phosphorylation site in human c-myc.

Myc proteins are phosphorylated within two critical regions by casein kinase II (CKII): the central acidic domain and a carboxy-terminal region bordering the basic region-helix-loop-helix segment. In order to test whether the carboxy-terminal phosphorylation site was functionally important we introduced three types of mutations into this region. Two of the mutations would be expected to prevent phosphorylation and minimize negative charge while the third introduced a permanent negative charge. The Myc CKII site mutants were cloned into a retroviral vector and were shown to be efficiently expressed in several different cell types. In one mutant we directly demonstrated loss of the phosphorylation site. When the Myc mutants were used in a cooperative transformation assay of Rat-1 cells with the bcr-abl oncogene we were unable to detect a significant difference in transformation efficiency between wild-type Myc and any of the mutants. While the CKII site is non-functional in this assay, the high levels of Myc produced may have overridden potential CKII regulation.

Amino Acid Sequence↗
PubMed1990

Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity.

Enhancer binding protein AP-4 is a transcription factor that activates both viral and cellular genes by binding to the symmetrical DNA sequence, CAGCTG. Here, we report the molecular cloning and characterization of human AP-4 cDNAs. The deduced amino acid sequence reveals that AP-4 is a helix-loop-helix (HLH) protein. Like other members of this family, the AP-4 HLH motif and the adjacent basic domain are necessary and sufficient to confer site-specific DNA binding. However, unlike other HLH proteins, AP-4 also contains two additional protein dimerization motifs consisting of leucine repeat elements LR1 and LR2. The analysis of various deletion and point mutants for their ability to dimerize in the presence or absence of DNA reveals several unusual features. Although the HLH basic region is sufficient for DNA recognition and binding, dimer formation between different truncated versions of AP-4 in solution requires an intact LR1 or LR2 domain. AP-4 is unable to form heterodimers with other helix-loop-helix family members such as the immunoglobulin enhancer binding factor, E12. In contrast, an AP-4 derivative, delta C222, which lacks LR1 and LR2 but retains an intact HLH, can form heterodimers with E12. Moreover, AP-4 molecules containing LR2 or LR1 are unable to form mixed dimers with carboxy-terminally truncated AP-4 molecules such as delta C222, but retain the ability to form complexes with longer versions of AP-4 that contain LR1 and/or LR2. Our findings strongly suggest that AP-4 contains multiple protein-protein interfaces that function to promote homodimer formation and restrict heterocomplexes. These findings provide a mechanism by which different members of the helix-loop-helix family of transcription factors can form functional dimers in a specific fashion with their appropriate partners to control transcriptional networks during cellular differentiation.

Amino Acid Sequence↗
PubMed1989

Activation of human B cells. Comparison of the signal transduced by IL-4 to four different competence signals.

The effects of the cytokine IL-4 on resting and activated human B cells were compared with the effects of known "competence" signals able to drive resting B cells into the cell cycle, including anti-Ig, PMA, anti-CD20, and a recently described competence signal, anti-Bgp95. In proliferation assays, IL-4 was costimulatory with anti-Ig and anti-Bgp95 but not with anti-CD20 or PMA. IL-4 alone triggered increases in expression of class II DR/DQ and CD40, but it did not trigger increases in intracellular free calcium [Ca2+]i in resting B cells or induce resting B cells to leave G0 and enter the G1 phase of the cell cycle. Although IL-4 has some characteristics of competence signals, it was most effective if added to B cells up to 12 h after anti-Ig or anti-Bgp95 rather than before, and thus, in this respect, works more like a progression signal. Like IL-4, all four competence signals for B cells triggered increases in class II and CD40, but only IL-4 consistently induced increases in CD23 surface levels. IL-4 was costimulatory only with anti-Ig and anti-Bgp95, each of which can trigger increases in [Ca2+]i and new protein synthesis of the proto-oncogene c-myc, and can increase attachment of protein kinase C to the plasma membrane. IL-4 was not costimulatory with signals that 1) did not affect [Ca2+]i yet induced c-myc protein synthesis (anti-CD20), 2) only stimulated the translocation of protein kinase C (PMA), or 3) only stimulated increases in [Ca2+]i (calcium ionophore). These results suggest that resting human B cells require at least two intracytoplasmic signals before IL-4 can effectively promote B cell proliferation.

Adjuvants, Immunologic↗