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B Schaffhausen

Publications and source records attributed to B Schaffhausen.

At least 19 recordsLinked to original sources
PubMed1996

NMR analysis of interactions of a phosphatidylinositol 3'-kinase SH2 domain with phosphotyrosine peptides reveals interdependence of major binding sites.

The interactions of the N-terminal src homology (SH2) domain (N-SH2) of the 85 kDa subunit of phosphatidylinositol 3'-kinase (PI-3K) with phosphotyrosine (ptyr) and a series of ptyr-containing peptides have been examined by NMR spectroscopy. HSQC (heteronuclear single-quantum coherence) NMR spectra of 15N-labeled SH2 were used to evaluate its interactions with ptyr-containing ligands. The ability of ligands to cause chemical shift changes was compared to their potency as competitors in in vitro binding experiments using polyoma virus middle T antigen (MT). The results suggest the interdependence of SH2 binding elements. Chemical shifts of residues involved in the ptyr binding were altered by variations of the sequence of the bound peptide, suggesting that the ptyr fit can be adjusted by the peptide sequence. Perturbations of chemical shifts of residues coordinating the methionine three residues C-terminal to the ptyr (the +3 residue) were affected by substitution in the binding peptide at +1 and vice versa. Such results show synergistic interplay between regions of the SH2 binding residues C-terminal to the ptyr.

Amino Acid Sequence↗
PubMed1995

SH2 domain structure and function.

An emerging theme in both the biology of signal transduction and the biochemistry of proteins has been the modular function of small protein domains. In some cases these can directly regulate catalytic activity. In others, they serve to interconnect important regulatory proteins. SH2 (src homology 2) domains represent some of the best studied models. Originally identified on the basis of homology in src and fps [1], SH2s are elements that ordinarily respond to tyrosine phosphorylation by binding the phosphorylated sequence. As such, they are key elements in tyrosine kinase regulation of cellular processes. Because SH2 interactions result from phosphorylation, such elements provide a regulatable circuitry along which signals can be transmitted in a timely manner. Because the regulation is based on a common mechanism, signal generators can target several different proteins coordinately. The PDGF receptor (PDGFr), for example, may interact with as many as ten different elements [2,3]. There are a number of excellent reviews on SH2 domains available [4-11]. This discussion will try to show how genetic, biochemical and biophysical results can be integrated in a satisfying way.

Amino Acid Sequence↗
PubMed1995

Multiple change in E2F function and regulation occur upon muscle differentiation.

We have examined regulation of the E2F transcription factor during differentiation of muscle cells. E2F regulates many genes involved in growth control and is also the target of regulation by diverse cellular signals, including the RB family of growth suppressors (e.g., the retinoblastoma protein [RB], p107, and p130). The following aspects of E2F function and regulation during muscle differentiation were investigated: (i) protein-protein interactions, (ii) protein levels, (iii) phosphorylation of the E2F protein, and (iv) transcriptional activity. A distinct E2F complex was present in differentiated cells but not in undifferentiated cells. The p130 protein was a prominent component of the E2F complex associated with differentiation. In contrast, in undifferentiated cells, the p107 protein was the prominent component in one of three E2F complexes. In addition, use of a differentiation-defective muscle line provided genetic and biochemical evidence that quiescence and differentiation are separable events. Exclusive formation of the E2F-p130 complex did not occur in this differentiation-defective line; however, E2F complexes diagnostic of quiescence were readily apparent. Thus, sole formation of the E2F-p130 complex is a necessary event in terminal differentiation. Other changes in E2F function and regulation upon differentiation include decreased phosphorylation and increased repression by E2F. These observations suggest that the regulation of E2F function during terminal differentiation may proceed through differential interaction within the RB family and/or phosphorylation.

Carrier Proteins↗
PubMed1994

Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

Antigens, Polyomavirus Transforming↗
PubMed1994

Retinoblastoma antioncogene is involved in the inhibition of myogenesis by polyomavirus large T antigen.

The expression of polyomavirus large T antigen in stably transfected C2 myoblast cells inhibits terminal differentiation without inducing a transformed phenotype. In the present work, we report on the lifting of this inhibition by a mutation that prevents polyomavirus large T antigen from binding to the product of the retinoblastoma susceptibility gene (p105 RB). In contrast with cells containing wild-type large T, those with the Rb binding site mutant large T showed the same up-regulation of myosine heavy chain and myogenin mRNA expression as control cells. Furthermore, we correlate the cell cycle alteration induced by polyomavirus large T antigen expression with the inability of the cells to undergo terminal differentiation.

Animals↗
PubMed1993

Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit.

Tyrosine-phosphorylated peptides based on the regions of polyoma virus middle t antigen and the platelet-derived growth factor receptor that bind phosphoinositide 3-kinase are shown to activate this enzyme 2-3-fold in vitro. The concentrations of the peptides required to activate the enzyme are at least 10-1000-fold higher than the dissociation constants of these peptides for the individual SH2 domains of the 85-kDa subunit (KD < 100 nM). Doubly phosphorylated peptides are more effective than singly phosphorylated peptides. The results suggest that a fraction of the cellular phosphoinositide 3-kinase has SH2 domains with relatively low affinity for phosphopeptides and that binding of phosphopeptides to these enzymes causes activation. Thus, SH2 domains may be involved not only in recruiting the enzyme but also in regulating activity.

Amino Acid Sequence↗
PubMed1993

A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.

Animals↗
PubMed1992

IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains.

3T3 Cells↗
PubMed1991

The T/t common region of simian virus 40 large T antigen contains a distinct transformation-governing sequence.

Simian virus 40 large T antigen (T) can transform cultured cells, but the mechanisms by which it functions are not entirely understood. Several lines of evidence have suggested that the amino-terminal approximately 130 residues of T may be sufficient to confer the transforming capability. Oligonucleotide-directed mutagenesis was used to generate a series of deletion and substitution mutants within the amino-terminal 82 residues of T, the segment which is shared with simian virus 40 small t antigen (t). Results of stability and transformation assays of these mutants strongly suggest that the 1-to-82 region of T contains sequences which govern T transforming activity and affect in vivo stability. Instability and a defect in transforming activity could be separated from one another genetically. Thus, the 1-to-82 region appears to contain a specific region that contributes to the transforming function of the protein. This segment operates by means other than the simple binding of pRb and/or p107.

Animals↗
PubMed1989

Mutations in polyomavirus middle T antigen affecting tumorigenesis.

P155 is a polyomavirus mlt mutant with normal transforming ability but impaired tumorigenic potential. The mutation, a 12-bp deletion (nucleotides 1348-1359), removes amino acids 372 to 375 from middle T and affects its ability to function in tumorigenesis (C. Gelinas, S. Masse, and M. Bastin, 1984, J. Virol. 51, 242-246). We used deletion loop mutagenesis to introduce point mutations within the wild-type sequence spanned by the P155 deletion. A mutant phenotype resembling that of P155 could be produced by as little as one alanine to valine substitution at residue 373. The mutants were impaired in their ability to induce tumors in rats but they could still transform established cell lines or primary fibroblasts in culture. To define the biochemical defect, we examined the mutant middle T antigen both for association with pp60c-src, the cellular src gene product, as well as its pattern of phosphorylation. No obvious differences explaining the phenotype were observed. The mutant middle T associated with, and activated pp60c-src, but exhibited a slightly altered pattern of phosphorylation, presumably because of additional sites on the middle T protein.

Animals↗
PubMed1989

Mechanisms of transformation by polyoma virus middle T antigen.

This review addresses a fundamental question of polyoma virus biology: What is the molecular mechanism by which the polyoma virus middle T antigen (MTAg) transforms cells in culture? Since MTAg has no known intrinsic biochemical activity, it is believed to act by modulating the properties of the host cell's proteins (see review by Courtneidge [26]). Experiments to date have largely focused on the interaction of MTAg with the cellular tyrosine kinase, pp60c-src. However, recent data from a number of laboratories have demonstrated the importance of other MTAg-associating cellular proteins in MTAg-mediated transformation, including pp62c-yes and a phosphatidylinositol kinase. In this review, we will summarize what is presently known about the proteins interacting with MTAg. The extent to which the currently known details of the biochemistry of MTAg and its associated proteins can explain the transforming properties of the various mutant alleles of MTAg will be assessed.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

Cellular proteins that associate with the middle and small T antigens of polyomavirus.

We have used two-dimensional gel electrophoresis to analyze in more detail the cellular proteins which associate with the middle and small tumor antigens (MT and ST, respectively) of polyomavirus. Proteins with molecular masses of 27, 29, 36, 51, 61, 63, and 85 kilodaltons (kDa) that specifically coimmunoprecipitated with MT were identified on these gels. The 36-, 51-, 61-, 63-, and 85-kDa proteins are probably the same as the proteins of similar sizes previously reported by a number of groups, whereas the 27- and 29-kDa proteins represent proteins that are heretofore undescribed. The 27- and 29-kDa proteins were abundant cellular proteins, whereas the others were minor cellular constituents. The association of each of these proteins with MT was sensitive to one or more mutations in MT that rendered it transformation defective. The association of the 85-kDa protein was the most sensitive indicator of the transformation competence of MT mutants. In addition, the 85-kDa protein was the only associated protein whose association with MT changed consistently in parallel with MT-associated phosphatidylinositol kinase activity. Furthermore, the fraction of the 85-kDa protein which was found associated with the MT complex contained 15 to 20% of its phosphate content on tyrosine. The 36- and 63-kDa proteins complexed with both polyomavirus MT and ST and comigrated on two-dimensional gels with two simian virus 40 ST-associated proteins originally described by Rundell and coworkers (K. Rundell, E. O. Major, and M. Lampert, J. Virol. 37:1090-1093, 1981). None of the other MT-associated proteins associated significantly with ST.

Antigens, Polyomavirus Transforming↗
PubMed1987

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity.

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Phosphorylation of polyomavirus large T antigen: effects of viral mutations and cell growth state.

Phosphorylation is responsible for the shift in electrophoretic mobility of polyomavirus large T antigen observed in pulse-chase or continuous-labeling experiments. Phosphorylated forms migrated more slowly than newly synthesized [35S]methionine large T antigen, and alkaline phosphatase treatment reversed the mobility shift. Analysis of phosphopeptides with Staphylococcus aureus V8 protease showed that large T antigen forms of intermediate mobility were enriched in peptides 1 to 4, 8, and 9, while the slower migrating species had all nine phosphopeptides, including peptides 5 and 7. The phosphorylations represented by phosphopeptides 5 and 7 were of particular interest. These phosphopeptides were entirely lacking in large T antigen from tsa mutants such as ts616 labeled at the nonpermissive temperature. Also, the phosphorylation of peptides 5 and 7 depends on the growth state of the cell. Early in infection of quiescent cells intermediate mobility forms of large T antigen with little or no phosphorylation, particularly of peptides 5 and 7, were seen, whereas peptides 5 and 7 were well represented at the same time in patterns from growing cells. Later in infection of growth-arrested cells, these phosphorylations were observed, suggesting that infection stimulates the relevant kinase. Because large T antigen of hrt mutants, which lack middle and small T antigens, showed phosphorylation of peptides 5 and 7, large T antigen was apparently responsible for the stimulation. Because some differences in the distribution of phosphopeptides were noted between hrt mutants and the wild type, middle T antigen, small T antigen, or both may play a modulating role in large T antigen phosphorylation.

Alkaline Phosphatase↗
PubMed1987

Localization of the phosphorylations of polyomavirus large T antigen.

Polyomavirus large T antigen is phosphorylated on both serine and threonine residues at a ratio of approximately 6 to 1. This phosphorylation could be resolved into a series of nine Staphylococcus aureus V8 phosphopeptides. All of these were found in an N-terminal chymotryptic fragment with a molecular weight of 57,000. A C-terminal formic acid fragment of 50,000-molecular-weight lacked phosphate. Therefore, unlike simian virus 40 large T antigen, polyomavirus large T antigen has no significant C-terminal phosphorylation. Limited V8 and hydroxylamine cleavage showed that the phosphorylations can be localized to two different portions of the molecule. A significant fraction of the phosphate was localized in the N-terminal portion of the molecule before residue 183. Within this region V8 peptides 4, 8, and 9 represented phosphorylations that were more proximal, while peptides 1, 2, and 3 included more distal phosphorylations. None of these phosphorylations appeared analogous to those of simian virus 40 large T antigen. V8 phosphopeptides 5 and 7 were more distal and could be distinguished in biological experiments from the N-terminal phosphorylations. Formic acid mapping suggested that much, if not all, of this phosphorylation is located between residues 257 and 285.

Animals↗