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I Sadowski

Publications and source records attributed to I Sadowski.

31 records · Page 2Linked to original sources
PubMed1991

GAL4 is phosphorylated as a consequence of transcriptional activation.

GAL4 protein isolated from yeast in which it is active is phosphorylated predominantly on two different serine residues. One of these was identified as Ser-837; substitution of this residue for alanine has no detectable effect on transcriptional activation by GAL4. Phosphorylation at Ser-837 requires that both the DNA binding and transcriptional activation functions be intact. We propose that some phosphorylations of GAL4, including that at Ser-837, occur concomitantly with activation of transcription.

Amino Acid Sequence↗
PubMed1990

Mutations that increase the activity of a transcriptional activator in yeast and mammalian cells.

Activating region I of GAL4 protein is a stretch of amino acids, positioned adjacent to the DNA-binding region, that activates transcription in yeast and, as we show here, in mammalian cells. Here we describe mutations located throughout a 65-amino acid region that increase the activation function of region I. Most of these mutations replace positively charged amino acids in the region with neutral ones, although we also describe substitutions at one position that do not alter the charge of the region. Mutations of region I that alter the activation function in yeast have similar effects on activation when assayed in mammalian cells. When individual mutations that raise the acidity of the activating region are recombined, the activities of the mutant proteins increase with increasing negative charge in both yeast and mammalian cells. These results extend and modify the correlation between acidity and activation and suggest that the requirements for a strong activating region are conserved in yeast and mammals.

Amino Acids↗
PubMed1989

The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function.

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

Amino Acid Sequence↗
PubMed1988

GAL4-VP16 is an unusually potent transcriptional activator.

Recent work has defined a class of transcriptional activators, members of which activate transcription in yeast, plant, insect and mammalian cells. These proteins contain two parts: one directs DNA binding and the other, called the activating region, presumably interacts with some component of the transcriptional machinery. Activating regions are typically acidic and require some poorly-understood aspect of structure, probably at least in part an alpha-helix. Here we describe a new member of this class, formed by fusing a DNA-binding fragment of the yeast activator GAL4 to a highly acidic portion of the herpes simplex virus protein VP16 (ref. 11; also called Vmw65). VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters. We show that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene. We suggest that the activating region of VP16 may be near-maximally potent and that it is not coincidental that such a strong activator is encoded by a virus.

Animals↗
PubMed1988

Mutational analysis of a phosphotransfer motif essential for v-fps tyrosine kinase activity.

The catalytic domains of protein-tyrosine kinases such as the P130gag-fps oncoprotein contain the sequence HRDLAARN, followed thirteen residues C-terminal by DFG (P130gag-fps residues 1041-1048 and 1061-1063). These residues define a structural motif conserved among eucaryotic protein kinases (-RD----N, DFG) and shared with several procaryotic 3'aminoglycoside phosphotransferases (H-D----N, D-G). Functional analysis of mutant v-fps proteins employing bacterial and mammalian expression systems indicated that this motif is critical for P130gag-fps kinase activity and oncogenicity. In particular, conservative substitutions of the two invariant aspartates (asp1043, asp1061) with glutamate or asparagine completely eliminated enzymatic activity, suggesting that these residues are essential for catalysis. In contrast, substitution of arg1042 with glutamate decreased but did not eliminate v-fps kinase activity in bacteria. The effects of these and other amino acid substitutions within the phosphotransfer motif and at the nearby autophosphorylation site (tyr1073) of P130gag-fps indicate that these conserved residues are intrinsically essential to the execution or regulation of catalytic activity, and suggest that tight spatial constraints operate within the active centre of the v-fps tyrosine kinase domain.

Amino Acid Sequence↗
PubMed1988

v-fps protein-tyrosine kinase coordinately enhances the malignancy and growth factor responsiveness of pre-neoplastic lung fibroblasts.

The v-fps oncoprotein was expressed in a pre-neoplastic, growth factor-dependent Chinese hamster lung fibroblast line (CCL39) to study its effect on growth controls and on the induction of malignancy. Two transfectants were characterized which expressed low (39FPS-8) or high (51FPS-6) levels of P130gag-f ps protein-tyrosine kinase activity. 39FPS-8 cells still arrested in quiescence when deprived of growth factors, but developed an increased sensitivity to the mitogenic actions of epidermal growth factor (20-fold) and alpha-thrombin (50-fold), although not to insulin. In contrast, 51FPS-6 cells completely escaped growth controls, divided in serum-free medium, and were insensitive to further growth factor stimulation. Both transfectants produced rapidly growing tumors in nude mice that formed pulmonary metastases from a subcutaneous site, unlike the parental cells which are non-metastatic. 51FPS-6 cells were comparatively more efficient than 39FPS-8 cells in colonizing the lungs after intravenous inoculation. The v-fps tyrosine kinase therefore induces a partial to complete relaxation of growth factor-mediated controls on the CCL39 cell cycle, with the extent of factor independence reflecting the amount of P130gag-f ps synthesized. This reduction in growth factor requirements correlates with the capacity of v-fps to confer the attributes of metastatic tumors upon preneoplastic CCL39 fibroblasts. We speculate that increased sensitivity to growth factor stimulation represents a common mechanism by which tumor cells acquire metastatic properties.

Animals↗
PubMed1987

A conserved domain regulates interactions of the v-fps protein-tyrosine kinase with the host cell.

All cytoplasmic protein-tyrosine kinases (PTKs) share a noncatalytic domain, termed SH2, which comprises approximately 100 residues located immediately N-terminal to the kinase domain. A linker in the AX9m mutant of Fujinami avian sarcoma virus (FSV) introduces a dipeptide insertion into the SH2 domain of the P130gag-fps PTK, which abolishes its ability to transform Rat-2 cells. However, at 36 degrees C AX9m FSV elicits focus formation and agar colony formation in infected chicken embryo fibroblasts (CEF) with single hit kinetics. At 41.5 degrees C AX9m FSV is nontransforming for CEF, and the mutant is therefore both host and temperature dependent for transforming activity. Both in vitro and in vivo, the specific kinase activity of AX9m FSV P130gag-fps, measured by autophosphorylation and phosphorylation of exogenous substrates, correlated with transforming activity. The consequences of the AX9m mutation for enzymatic function and transforming activity therefore depend on the cellular environment in which the altered v-fps protein is expressed. We conclude that the SH2 domain directs the interaction of the P130gag-fps catalytic domain with cellular proteins such as substrates for phosphorylation or regulators of kinase activity important for its transforming ability.

Animals↗
PubMed1987

Catalytic and non-catalytic domains of the Fujinami sarcoma virus P130gag-fps protein-tyrosine kinase distinguished by the expression of v-fps polypeptides in Escherichia coli.

While protein-tyrosine kinases share a region of sequence identity corresponding to their kinase domains, the specific elements essential for catalysis, substrate binding and substrate specificity are largely undefined. The P130gag-fps transforming protein of Fujinami avian sarcoma virus is a cytoplasmic tyrosine kinase with a complex structure that includes a C-terminal kinase domain. To identify the precise N-terminal border of the v-fps catalytic region and to assess its interactions with non-catalytic domains, C-terminal v-fps polypeptide fragments of decreasing size were expressed in E. coli as trpE-v-fps hybrid proteins. All such polypeptides containing 263 or more residues derived from the C-terminus of P130gag-fps (i.e. residues 920-1182) were enzymatically active as tyrosine kinases. They autophosphorylated at physiological sites in vivo and phosphorylated exogenous substrates such as enolase and poly(glu,tyr) at tyrosine in vitro. Deletion of a further five amino acids from P130gag-fps residues 920-925 abolished all enzymatic activity. This deletion coincides with the predicted N-terminus of the v-fps ATP-binding site at residue 922. These data indicate that the N-terminal border of the ATP-binding site defines the start of the minimal v-fps tyrosine kinase catalytic domain, and show that this minimal domain is competent to bind substrates. More N-terminal non-catalytic sequences appear to functionally interact with the catalytic domain.

Catalysis↗
PubMed1986

A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.

Proteins encoded by oncogenes such as v-fps/fes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-fps proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genomes and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-fps bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130gag-fps is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.

Animals↗
PubMed1984

RK bacterial test for independently measuring chemical toxicity and mutagenicity: short-term forward selection assay.

A short-term bacterial assay system for determining the mutagenic potential of environmental substances was developed and validated. Genotoxic activity was demonstrated for selected substances from 10 categories of chemical agents. The RK test results were obtained with one Escherichia coli assay strain that was transiently exposed to, and then removed from the test substance prior to the selection step for mutant cells. The RK test employs a hitherto unused short-term assay technique for selecting forward mutations in the wild-type selector strain cells. The cells of the selector strain are killed upon shifting to 42 degrees C as a consequence of thermal derepression and subsequent expression of the replication genes from an integrated 10-kilobase fragment of phage lambda. Cells that acquire mutations in the responsible killing genes are detected by their colony-forming ability at 42 degrees C. A substance is determined to be genotoxic if it is capable of increasing the forward mutation frequency for appearance of these mutant cells. Toxicity of the agent is independently evaluated by examining its effect on the viability of the selector strain at 30 degrees C, when the viral replication genes remain repressed. The flexible assay protocol enables determination of the effect of pH on mutagenic activity, the requirement for metabolic activation, and assays of nearly insoluble or highly toxic substances.

Biotransformation↗
PubMedDate not supplied

Purification of RBF-2, a transcription factor with specificity for the most conserved cis-element of naturally occurring HIV-1 LTRs.

The combination of high turnover and error-prone reverse transcription results in naturally occurring human immunodeficiency virus-1 long terminal repeats that differ considerably from the prototype sequence. Although no transcription-factor-binding site escapes mutation, the only mutated site that appears to be invariably compensated by co-occurrence of its duplication is the RBE III site, a target for the transcription factor RBF-2. In this work, we characterize RBF-2 further by biochemical purification. RBF-2 was purified by chromatography on heparin agarose and Mono-Q ion exchange chromatography, followed by affinity chromatography on mutant and wild-type RBE III oligonucleotide columns. The purified RBF-2 preparation contained 4 major and 1 minor polypeptides of 50, 100, 110, 120 and 125 kD, as detected by silver staining in SDS-PAGE gels. UV cross-linking revealed a specific 100-kD species, indicating that this protein likely represents the DNA-binding component of a complex. A second factor with DNA-binding specificity similar to that of RBF-2, called RBF-B, was also identified by heparin-agarose fractionation, which suggests that effects of the RBE III cis-element may be mediated by a combination of factors in vivo.

Anion Exchange Resins↗