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B M Sefton

Publications and source records attributed to B M Sefton.

At least 55 records · Page 3Linked to original sources
PubMed1989

The lck tyrosine protein kinase interacts with the cytoplasmic tail of the CD4 glycoprotein through its unique amino-terminal domain.

The CD4 lymphocyte surface glycoprotein and the lck tyrosine protein kinase p56lck are found as a complex in T lymphocytes. We have defined the domains in both proteins that are responsible for this interaction by coexpressing hybrid and deleted forms of the two proteins in HeLa cells. We have found that the unique 32 amino-terminal residues of p56lck and the 38 carboxy-terminal residues of CD4 that comprise the cytoplasmic domain are both necessary and sufficient by themselves for the interaction of the two proteins. The interaction appears to be independent of other T cell-specific proteins and probably occurs before CD4 reaches the cell surface. Our findings suggest that the specialized amino-terminal domains of other members of the src family of intracellular tyrosine kinases may also mediate transmembrane signaling via coupling to the cytoplasmic domains of specific transmembrane proteins.

Amino Acid Sequence↗
PubMed1989

Activators of protein kinase C induce dissociation of CD4, but not CD8, from p56lck.

The CD4 and CD8 T cell receptor accessory molecules can both be isolated from T lymphocytes in association with p56lck, a membrane-associated, cytoplasmic tyrosine protein kinase that is expressed exclusively in lymphoid cells. The enzymatic activity of p56lck may therefore be regulated by CD4 and CD8 and be important in antigen-induced T cell activation. Exposure of human T cells and some mouse T cells to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, caused the dissociation of p56lck and CD4. Activation of protein kinase C may therefore interrupt regulation of p56lck by CD4 and alter the ability of p56lck to interact with polypeptide substrates. In contrast, exposure of cells to TPA did not cause dissociation of p56lck and CD8. Regulation of p56lck by CD4 may therefore differ from regulation by CD8.

Animals↗
PubMed1989

Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins.

Immobilon, a membrane of polyvinylidene difluoride to which gel-fractionated proteins can be transferred electrophoretically, was found to be an excellent matrix for the analysis of the phosphoamino acid content of phosphoproteins. Hydrolysis of 32P-labeled proteins bound to Immobilon with 5.7 N HCl resulted in the release of 90% of the 32P in the form of Pi, phosphoamino acids, and phosphopeptides. Two-dimensional electrophoretic analysis of the released phosphoamino acids yielded undistorted patterns. Because direct hydrolysis of proteins transferred to Immobilon eliminated the need for both preparative extraction of proteins from a gel and recovery by precipitation, analysis was rapid and yields of phosphoamino acids were extremely consistent. The yield of phosphoamino acids from proteins bound to Immobilon, unlike that from proteins eluted from gels, was independent of the size of the protein. The detection of 32P-labeled, phosphotyrosine-containing proteins in sodium dodecyl sulfate-polyacrylamide gels has been shown to be substantially improved by incubation of the gel in 1.0 N KOH for 2 h at 55 degrees C. Base hydrolysis of proteins bound to Immobilon proved to be faster and more sensitive than hydrolysis of proteins in gels. Less than 10% of bound protein was lost from Immobilon during the 2-h incubation at 55 degrees C in 1.0 N KOH. The autoradiographic image after alkaline hydrolysis of proteins on Immobilon was sharper than that obtained after hydrolysis of proteins in the gel. In addition, unlike base-treated gels, the dimensions of the Immobilon filter were unaffected by treatment with base.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1989

Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines.

CD45 is a family of high molecular weight leukocyte cell surface glycoproteins. Recently, two related subregions of the cytoplasmic domain of CD45 have been shown to have 30-40% amino acid identity with a human placental protein phosphotyrosine phosphatase, and CD45 isolated from human spleen was found to exhibit intrinsic protein phosphotyrosine phosphatase (EC 3.1.3.48) activity. In the present studies, we demonstrate that each of the known isoforms of murine CD45 has an equivalent basal level of protein phosphotyrosine phosphatase activity and establish that this enzymatic activity is associated with the cytoplasmic domain of the glycoprotein. Studies with three independent sets of well-characterized parental CD45+, mutant CD45-, and revertant CD45+ lymphoma cell lines indicate that loss of CD45 increases the phosphorylation of the src-related leukocyte-specific tyrosine protein kinase p56lck on tyrosine-505, a putative negative regulatory site. This suggests that CD45 may play a role in leukocyte growth regulation by altering the kinase activity of p56lck.

Animals↗
PubMed1989

The sequence of chicken c-yes and p61c-yes.

We have deduced the sequence of the protein encoded by the chicken c-yes gene from overlapping cDNA clones. The predicted protein, p61c-yes, contains 541 amino acids and has a molecular weight of 60,911 with the amino terminal methionine residue. Chicken p61c-yes differs from Y73 virus p90gag/v-yes in three respects. First, the carboxy-terminal eight amino acids of p61c-yes are replaced by three amino acids in p90gag/v-yes, which are encoded by the avian leukemia virus env gene. This alteration changes the position and context of a tyrosine residue in p61c-yes. Second, nucleotides which are present as 5' non-translated sequence in the p61c-yes mRNA, are translated in the p90gag/v-yes mRNA. Third, there are fourteen dispersed nucleotide differences in Y73 v-yes which result in six amino differences between the body of p90gag/v-yes and p61c-yes. Chicken p61c-yes differs from human p61c-yes at 43 residues, and from chicken pp60c-src at 122 residues.

Amino Acid Sequence↗
PubMed1989

Analysis of the activity and phosphorylation of the lck protein in lymphoid cells.

p56lck, the tyrosine protein kinase encoded by the lck gene, is expressed at a 40-fold elevated level in the LSTRA cell line. This is associated with increased tyrosine phosphorylation of cellular proteins. We have asked here whether the increased tyrosine protein phosphorylation is due to an altered activity of the protein or to the unusually high level of p56lck. In vitro protein kinase assays showed that neither the specific activity nor the affinity of p56lck for two different substrates was abnormal in LSTRA cells. Additionally, analysis of the phosphorylation of p56lck in LSTRA and other cell lines showed that the protein was phosphorylated extensively at a negative-regulatory site, Tyr 505, in all of the cells examined. Since the primary structure of the p56lck expressed at a high level in LSTRA cells is the same as that found in normal thymus and we found no evidence of activation of the protein by dephosphorylation, it appears that high levels of p56lck can induce increased tyrosine protein phosphorylation in lymphoid cells. In contrast, high levels of the closely related protein, p60c-src have no significant effect on tyrosine protein phosphorylation in fibroblasts. The regulation of the protein kinase activity of p56lck in lymphoid cells may therefore differ from the regulation of p60c-src in fibroblasts.

Animals↗
PubMed1989

Protein kinases.

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Humans↗
PubMed1988

Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine protein kinase, p56lck, reveals its oncogenic potential in fibroblasts.

p56lck, a cellular tyrosine protein kinase (EC 2.7.1.112) of the src family, is expressed in essentially all T cells and in some B cells. Expression in nonlymphoid cells is observed only rarely. We have found that mutation of a carboxyl-terminal phosphorylation site, tyrosine-505, reveals an oncogenic activity of this protein. Infection of fibroblasts with a retrovirus encoding wild-type p56lck is without consequence. In contrast, infection with a virus encoding the mutant protein leads to greatly increased phosphorylation of cellular proteins on tyrosine, morphological transformation, and anchorage-independent growth. This suggests that the tyrosine protein kinase activity and the oncogenic potential of p56lck are normally suppressed in vivo by phosphorylation of tyrosine-505. Since similar results were obtained previously with an analogous mutant of c-src, our results suggest that the protein kinase activity of all members of the src family of cytoplasmic tyrosine protein kinases will prove to be regulated by tyrosine phosphorylation at a conserved residue near the carboxyl terminus. Because p56lck is normally expressed only in lymphoid cells, it was possible that p56lck would be without effect in other tissues. The transformation of fibroblasts by mutant p56lck shows that this lymphoid protein can interact productively with nonlymphoid polypeptide substrates.

Animals↗
PubMed1988

Transcriptional activation of lck by retrovirus promoter insertion between two lymphoid-specific promoters.

p56lck, a member of the src family of cytoplasmic tyrosine protein kinases, is expressed primarily in lymphoid cells. Previous RNase protection data demonstrated the existence of at least two lck mRNAs (type I and type II) with different 5' untranslated regions in most T cells. These have been found here to arise from two separate promoters. S1 nuclease analysis and primer extension were used to locate the site of initiation of type I lck mRNA. The nucleotide sequence of the region upstream of this start site contains no classical promoter motifs. A cDNA clone of type II lck mRNA was isolated. The promoter of this mRNA must be more than 10 kilobases upstream of the type I promoter region. In two murine thymoma cell lines, LSTRA and Thy19, lck is expressed at elevated levels as a result of Moloney murine leukemia virus retrovirus promoter insertion. p56lck is encoded in these cells by a hybrid virus-lck mRNA containing the 5' untranslated region of Moloney virus mRNA. The structures and the sites of integration of the proviruses upstream of lck in these cells were examined by molecular cloning and Southern analysis. A truncated and rearranged provirus, flanked by 554 nucleotides (nt) of duplicated cellular sequences, was found 962 nt upstream of the start site for type I lck mRNA in LSTRA cells. What appears to be a Moloney mink cytopathic focus-forming provirus was found between 584 to 794 nt upstream of the start site for type I lck mRNA in Thy19 cells. Thus in both tumor cell lines, viral DNA is present between the promoters for type I and type II lck mRNAs. Comparison of the sequences of the 5' ends of the lck and c-src genes suggests that divergence of these two genes involved exon shuffling and that a homolog of the neuronal c-src(+) exon is not present in lck.

Animals↗
PubMed1988

Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine.

Antibodies to phosphotyrosine were used to identify proteins phosphorylated on tyrosine in chicken embryo fibroblasts transformed by the oncogenes, v-src, v-yes, v-fps, v-ros, or v-erb-B. These antibodies allowed detection of a minimum of 50, 44, and 47 bands in immunoblots of cellular lysates from fibroblasts transformed by v-src, v-yes, or v-fps respectively. Eight of these bands were also detected in fibroblasts transformed by v-ros and v-erb-B, suggesting that the cellular transformation induced by v-ros, v-erb-B, v-src, v-yes, and v-fps may be achieved by the phosphorylation of some of the same proteins. The viruses SD10 and SD11 are point mutants of the Prague-C strain of Rous sarcoma virus that encode non-myristylated p60v-src. They do not induce the transformation of chicken fibroblasts. Approximately thirty bands were detected by antibodies to phosphotyrosine in fibroblasts transformed by wild-type Prague-RSV-C, only four of which were not substrates nonmyristylated mutant p60v-src. This suggests that the phosphorylation of a large subset of the substrates of p60v-src is not sufficient for transformation.

Animals↗
PubMed1988

Most of the substrates of oncogenic viral tyrosine protein kinases can be phosphorylated by cellular tyrosine protein kinases in normal cells.

The cellular transformation induced by viral tyrosine protein kinases may result from the excessive phosphorylation of the normal polypeptide substrates of endogenous cellular tyrosine kinases, from the phosphorylation of proteins that are not normal substrates of cellular tyrosine protein kinases in uninfected cells, or from the phosphorylation of proteins of each type. To differentiate between these possibilities, antibodies to phosphotyrosine were used with immunoblotting to compare the substrates of p60v-src, the transforming tyrosine protein kinase of Rous sarcoma virus (RSV), with those of cellular tyrosine protein kinases. Specifically, the substrates of p60v-src were compared with those of (1) p60c-src, (2) the tyrosine protein kinases activated by the binding of platelet-derived growth factor and (3) normal cellular tyrosine protein kinases in fibroblasts treated with sodium orthovanadate, an inhibitor of phosphatases. Comparison of the patterns observed on the immunoblots with the pattern of phosphotyrosine-containing proteins isolated by immunoaffinity chromatography with antiphosphotyrosine antibodies demonstrated that the proteins detected by Western blotting did indeed contain phosphotyrosine. Cells transformed by a variant of c-src activated by a single point mutation had an almost identical pattern of tyrosine protein phosphorylation as cells transformed by v-src. The several mutations and carboxyl-terminal substitution that differentiate p60v-src from p60c-src appear therefore to affect the enzymatic activity, but not the polypeptide substrate specificity, of the viral protein. In cells transformed by v-src, 27 of the 35 phosphotyrosine-containing proteins were also phosphorylated on tyrosine in normal uninfected fibroblasts treated with sodium orthovanadate. The phosphorylation of the large majority of the substrates of p60v-src can therefore occur in uninfected cells. Nine of the substrates of p60v-src were also phosphorylated by the viral tyrosine protein kinases encoded by the oncogenes, v-abl, v-fps, v-fes, and v-fgr. Together these data are consistent with the idea that viral tyrosine protein kinases induce transformation largely by intervening in cellular regulatory pathways that are normally controlled by tyrosine protein phosphorylation.

Animals↗
PubMed1987

Myristoylated alpha subunits of guanine nucleotide-binding regulatory proteins.

Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the alpha subunits of Gs (stimulatory) (alpha 45 and alpha 52), a 41-kDa subunit of Gi (inhibitory) (alpha 41), a 40-kDa protein (alpha 40), and the 36-kDa beta subunit. No protein that comigrated with the alpha subunit of Go (unknown function) (alpha 39) was detected. In cells grown in the presence of [3H]myristic acid, alpha 41 and alpha 40 contained 3H label, while the beta subunit did not. Chemical analysis of lipids attached covalently to purified alpha 41 and alpha 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein beta and gamma subunits and of Gt (transducin) subunits (alpha, beta, and gamma) failed to reveal fatty acids. The fatty acid associated with alpha 41, alpha 40, and alpha 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.

Animals↗
PubMed1987

Two lck transcripts containing different 5' untranslated regions are present in T cells.

p56lck is a new member of the src family of cellular tyrosine protein kinases. It is expressed constitutively at a low level in normal T cells and at an elevated level in the LSTRA and Thy19 Moloney murine leukemia virus-induced thymoma cell lines. It is possible that the expression of p56lck at an elevated level contributes to the transformation of these thymoma cells. The structure of the mRNAs encoding p56lck was examined by using an RNase protection assay. Both a chimeric lck mRNA containing the 5' untranslated region of Moloney virus mRNA and a normal lck mRNA were found in Thy19 and LSTRA cells. The chimeric lck transcript was 4- to 10-fold more abundant than the normal transcript. Transcription arising from a viral promoter is therefore responsible for the elevated levels of lck mRNA in these two cell lines. Surprisingly, uninfected murine T cells were also found to contain lck transcripts with differing 5' untranslated regions. One species of mRNA was colinear with the region of the chromosome just upstream of the initiation codon for p56lck. The other appeared to arise through splicing of an unidentified 5' untranslated exon to a sometimes cryptic splice acceptor just upstream of the region encoding p56lck. These data suggest that lck is expressed through the use of at least two different promoters. The promoters could be subject to different forms of regulation.

Animals↗
PubMed1987

Chicken proto-oncogene c-ros cDNA clones: identification of a c-ros RNA transcript and deduction of the amino acid sequence of the carboxyl terminus of the c-ros product.

Three overlapping cDNA clones that hybridized to a v-ros probe were isolated from a cDNA library constructed from chicken kidney mRNA. Sequence analysis of these clones showed that they were all derived from c-ros mRNA. Using hybridization probes synthesized from the cDNA clones, a c-ros mRNA transcript of approximately 8.3 kb was detected in chicken kidney RNA, but not in chick embryo fibroblast RNA. The amino acid sequence predicted from the cDNA sequence indicates that the carboxyl terminus of the chicken c-ros protein contains 58 amino acids which are not present in v-ros. The predicted amino acid sequence of the chicken c-ros protein differs by 25% from that of its closest known human counterparts within the tyrosine protein kinase catalytic domain, and by 66% downstream of this domain. Despite these differences, both the chicken and human amino acid sequences share a potential site of tyrosine phosphorylation near their carboxyl termini that is absent from v-ros.

Amino Acid Sequence↗
PubMed1986

Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation.

Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV.

Actins↗