Pp60 V-src Research Articles

Pp60v-src reactivation inhibits serum-induced accumulation of inositol phosphates and phosphatidylethanol in tsNRK.

In tsRSV-infected NRK (tsNRK) cells, pp60(v-src) reactivation by temperature-shift from a nonpermissive temperature, 39 C, to a permissive one, 32 degrees C, induced the production of inositol phosphates (IPt) and phosphatidylethanol (PEt). This was accompanied by an increase in membrane-associated protein kinase C (PKC) activity in the absence of exogenous growth factors. However, with serum-stimulation, the amounts of IPt and PEt at 32 degrees C were less than those at 39 degrees C. Pretreatment with PKC inhibitors, Ro-31-8220 and staurosporine, enhanced the accumulation of IPt but not of PEt at 32 degrees C. The tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) was increased either by serum or by pp60(v-src) reactivation. These results suggest that serum transduces its signal through PLCgamma1 mediation, and that pp60(v-src), possibly through the PKC mediation, negatively affects serum-induced PLCgamma1 activation.

Pp60v-src Induction of Cyclin D1 Requires Collaborative Interactions between the Extracellular Signal-regulated Kinase, p38, and Jun Kinase Pathways: A ROLE FOR cAMP RESPONSE ELEMENT-BINDING PROTEIN AND ACTIVATING TRANSCRIPTION FACTOR-2 IN pp60v-src SIGNALING IN BREAST CANCER CELLS

The cyclin D1 gene is overexpressed in breast tumors and encodes a regulatory subunit of cyclin-dependent kinases that phosphorylate the retinoblastoma protein. pp60(c-src) activity is frequently increased in breast tumors; however, the mechanisms governing pp60(c-src) regulation of the cell cycle in breast epithelium are poorly understood. In these studies, pp60(v-src) induced cyclin D1 protein levels and promoter activity (48-fold) in MCF7 cells. Cyclin D1-associated kinase activity and protein levels were increased in mammary tumors from murine mammary tumor virus-pp60(c-src527F) transgenic mice. Optimal induction of cyclin D1 by pp60(v-src) involved the extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase members of the mitogen-activated protein kinase family. Cyclin D1 promoter activation by pp60(v-src) involved a cAMP response element-binding protein (CREB)/activating transcription factor 2 (ATF-2) binding site. Dominant negative mutants of CREB and ATF-2 but not c-Jun inhibited pp60(v-src) induction of cyclin D1. pp60(v-src) induction of CREB was blocked by the p38 inhibitor SB203580 or by mutation of CREB at Ser133. pp60(v-src) induction of ATF-2 was abolished by the c-Jun N-terminal kinase inhibitor JNK-interacting protein-1 or by mutation of ATF-2 at Thr69 and Thr71. CREB and ATF-2, which bind to a common pp60(v-src) response element, are transcriptionally activated by distinct mitogen-activated protein kinases. Induction of cyclin D1 activity by pp60(v-src) may contribute to breast tumorigenesis through phosphorylation and inactivation of the retinoblastoma protein.

Early effects of PP60v-src kinase activation on caveolae

Members of the nonreceptor tyrosine kinase family appear to be targeted to caveolae membrane. We have used a Rat-1 cell expressing a temperature sensitive pp60(v-src) kinase to assess the initial changes that take place in caveolae after kinase activation. Within 24-48 h after cells were shifted to the permissive temperature, a set of caveolae-specific proteins became phosphorylated on tyrosine. During this period there was a decline in the caveolae marker protein, caveolin-1, a loss of invaginated caveolae, and a 70% decline in the sphingomyelin content of the cell. One of the phosphorylated proteins was caveolin-1 but it was associated in coimmunoprecipitation assays with both a 30 kDa and a 27 kDa tyrosine-phosphorylated protein. Finally, the cells changed from having a typical fibroblast morphology to a rounded shape lacking polarity. In light of the recent evidence that diverse signaling events originate from caveolae, pp60(v-src) kinase appears to cause global changes to this membrane domain that might directly contribute to the transformed phenotype.

Cns1 is an essential protein associated with the hsp90 chaperone complex in Saccharomyces cerevisiae that can restore cyclophilin 40-dependent functions in cpr7Delta cells.

Saccharomyces cerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR) and the oncogenic tyrosine kinase pp60(v-src). In addition, it has recently been shown that Cpr7 plays a major role in negative regulation of the S. cerevisiae heat shock transcription factor (HSF). To better understand functions associated with Cpr7, a search was undertaken for multicopy suppressors of the cpr7Delta slow-growth phenotype. The screen identified a single gene, designated CNS1 (for cyclophilin seven suppressor), capable of suppressing the cpr7Delta growth defect. Overexpression of CNS1 in cpr7Delta cells also largely restored GR activity and negative regulation of HSF. In vitro protein retention experiments in which Hsp90 heterocomplexes were precipitated resulted in coprecipitation of Cns1. Interaction between Cns1 and the carboxy terminus of Hsp90 was also shown by two-hybrid analysis. The functional consequences of CNS1 overexpression and its physical association with the Hsp90 machinery indicate that Cns1 is a previously unidentified component of molecular chaperone complexes. Thus far, Cns1 is the only tetratricopeptide repeat-containing component of Hsp90 heterocomplexes found to be essential for cell viability under all conditions tested.

NSAID-Induced Apoptosis in Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts is Dependent on v-src and c-myc and is Inhibited by bcl-2

Mounting epidemiological and experimental evidence implicates nonsteroidal antiinflammatory drugs as anti-tumorigenic agents. Our previous work showed that nonsteroidal antiinflammatory drug treatment of src-transformed chicken embryo fibroblasts caused apoptosis -- a mechanism by which these drugs might exert their anti-tumorigenic effect. The present studies employ a sensitive technique for detecting single- and double-stranded DNA cleavage (the comet assay) to quantitate apoptosis. By this method pp60 v-src, which antagonizes apoptosis in many cell systems, was found to induce apoptosis in 11–23% of serum-starved fibroblasts. However, treatment with diclofenac following pp60 v-src activation produced a much stronger response beginning within 6 hours of treatment that resulted in 100% lethality. During cell death, cyclooxygenase-2 but not cyclooxygenase-1 mRNA was found to be uniformly increased by all apoptotic drugs tested. Examination of the expression of apoptosis-associated genes showed that c-rel and p53 (found in normal or v-src-transformed chicken embryo fibroblasts at moderate levels), and bcl-2 (present at an extremely low level) were largely unchanged by treatment with eight different nonsteroidal antiinflammatory drugs. However, over-expression of human bcl-2 inhibited diclofenac-mediated apoptosis by 90%, demonstrating directly that bcl-2 expression can regulate nonsteroidal antiinflammatory drug induction of cell death. The proto-oncogene c-myc is known to cause apoptosis in chicken embryo fibroblasts when artificially overexpressed in cells deprived of trophic factors. We found that nonsteroidal antiinflammatory drug treatment following pp60 v-src activation persistently induced myc protein and mRNA by more than 20-fold above that evoked by pp60 v-src activation alone. Moreover, transfection of antisense c-myc oligonucleotides reduced drug-induced myc expression by 80% and caused a concomitant 50% reduction in cell death. These findings suggest that nonsteroidal antiinflammatory drug-induced apoptosis proceeds through a src/myc dependent pathway which is negatively regulated by bcl-2.

Focal adhesion kinase

Focal adhesion kinase (FAK) is a member of a growing family of non-receptor protein tyrosine kinases. Though originally identified as a putative substrate for the oncogenic tyrosine kinase pp60 v-src , it is now well-established that FAK tyrosine phosphorylation is induced by adhesion of cell surface integrins to extracellular matrix and by a variety of other extracellular factors including the ligands for receptor tyrosine kinases and for seven transmembrane domain G-protein-coupled receptors. FAK can associate with multiple cellular components including other focal adhesion-associated proteins and signalling molecules. FAK is localized to focal adhesions and is centrally implicated in the regulation of cell motility and adhesion. Knocking out the FAK gene in mice prevents normal embryonic development and is associated with loss of mesenchymal cell motility. Recent findings further suggest novel tissue-specific functions for FAK, particularly in the brain. These findings implicate FAK in the aberrant migration of cells in a variety of diseases and suggest that FAK may be a novel target for therapeutic strategies.

Inhibitions of protein kinase C and proto-oncogene expressions in NIH 3t3 cells by apigenin

Apigenin, a low-toxic and non-mutagenic plant flavonoid, suppresses 12- O-tetradecanoyl-phorbol-13-acetate (TPA)-mediated tumour promotion of mouse skin. TPA has the ability to activate protein kinase C (PKC) and induce proto-oncogene expression. Our study shows that apigenin inhibits PKC by competing with ATP, and exhibits an ic 50 value of 10 ± 0.5 μM. Apigenin also reduces the level of TPA-stimulated phosphorylation of cellular proteins. Of the protein tyrosine kinases tested, the fibroblast growth factor (FGF) receptor was most strongly affected by apigenin (IC 50 20 μM), and pp60 v-src most weakly affected ( ic 50 > 200 μM). Treatment of NIH 3T3 cells with 100 ng/ml TPA and 10, 50 and 100 μM apigenin resulted in 50, 80 and 10096 suppression of TPA-induced C-JUN expression, respectively. Treatment of TPA with 10 μ,M apigenin inhibited TPA-induced C-FOS expression. TPA-stimulated cell growth was suppressed by 25 μM apigenin. Our results provide some evidence for understanding apigenin's inhibitory effects of TPA-mediated tumour promotion.

Fatty acyl transfer by human N-myristyl transferase is dependent upon conserved cysteine and histidine residues.

N-Myristyl transferase (Nmt) catalyzes attachment of myristate onto the N terminus of suitable proteins. In order to identify amino acids important for catalytic functions, human Nmt and mutants representing all six conserved cysteine and histidine residues (Cys-169, Cys-214, His-131, His-171, His-218, and His-293) were expressed in Escherichia coli and analyzed for their ability to bind and transfer myristic acid. N-Terminal histidine-tagged fusion proteins displayed varying abilities to form an association with radiolabeled myristic acid indicative of an acyl-enzyme intermediate. When co-expressed with an acceptor substrate protein, pp60v-src, the mutants showed differential incorporation of radiolabeled myristic acid into v-Src protein. In vitro experiments monitoring transfer of myristyl CoA to a peptide homologous to the N terminus of pp60 v-src gave results similar to those obtained in vivo. Our studies showed that mutation at Cys-169, His-171, and especially His-293 interfered with formation of an acyl-enzyme intermediate, while human Nmts containing mutations at Cys-169, His-218, or His-293 showed greatly attenuated abilities to form acylated product. We propose a model for the Nmt reaction mechanism in which Cys-169 serves as the fatty acid attachment site for a covalent myristyl enzyme intermediate, while His-171 acts as a general acid/base and His-293 as a specific acid/base during acyl-enzyme intermediate formation. His-218 could then act as an acid or base needed to catalyze transfer of the acyl group from the acyl-enzyme intermediate to a polypeptide substrate. This working model will be useful for the design of regulators of Nmt function.

Inhibitors of Protein Tyrosine Kinase pp60v-src: Sterol Sulfates from the Brittle Star Ophiarachna incrassata

Bioactivity-guided fractionation of the extracts of the green brittle star Ophiarachna incrassata using a protein tyrosine kinase pp60v-src inhibition assay led to the isolation of a new sterol sulfate [2] together with four known ones (1,3-5). All five compounds were found to inhibit protein tyrosine kinase pp 60v-src.

Pp60 ν- src kinase overexpression leads to cellular resistance to the antiproliferative effects of tumor necrosis factor

While some tumor cells are sensitive to the antiproliferative effects of tumor necrosis factor (TNF), others are resistant. The molecular basis for cellular resistance to TNF is not completely understood. Previously we have shown that transfection of cells with an oncogene HER2/neu/erb B2, a receptor tyrosine kinase, leads to resistance to the anticellular effects of TNF [(1988) Proc. Natl. Acad. Sci. USA 85, 5102-5106]. In the present study, we demonstrate that the overexpression of another oncogenic tyrosine kinase, pp60 v-src also induces resistance to TNF. In contrast to HER2, however, pp60 v-src transfection of cells did not lead to down-modulation of TNF receptors but rather to decreased intracellular glutathione levels. The pp60 v-src -induced cellular resistance to TNF could be abrogated by interferon-γ. Thus, these results indicate that the resistance of certain tumors to TNF may also be due in part to the overexpression of pp60 v-src oncogene.

Chaperone functions of the heat shock proteins associated with steroid receptors

Mammalian steroid receptors exist in hormone-free cells in a heterocomplex that contains the three heat shock proteins hsp90, hsp70 and hsp56. Some protein kinases, including pp60 v-src and v-Raf, exist in similar cytosolic heterocomplexes containing hsp90 and a 50 kDa protein of unknown function, pp50. The four proteins—hsp90, hsp70, hsp56 and pp50—exist together in a heterocomplex independent of the presence of steroid receptors and protein kinases. Both the receptor and the protein kinase hetorocomplexes can be formed by a protein folding-heterocomplex assembly system in reticulocyte lysate that carries out an hsp70-dependent attachment of the proteins to the preformed heat shock protein complex. Association of receptors with this structure occurs at the termination of receptor translation and is critical for maintenance of the receptors in a transcriptionally inactive state in the absence of hormone. We discuss how this preformed protein folding structure may be involved in the subsequent targeted trafficking of steroid receptors through the cytoplasmic space to the nucleus.

Increased tyrosine kinase activity in pp60 c-src immunoprecipitate from platelet activating factor stimulated human platelets: : In vitro phosphorylation of a synthetic peptide

The involvement of pp60 c-src tyrosine kinase was studied in human platelets stimulated with platelet activating factor (PAF). Immunoprecipitation of pp60 c-src from platelets followed by immunoblot with pp60 v-src monoclonal antibody revealed four protein bands of 60, 50 and 29 kDa as detected by enzymographic web. The phosphorylation of these bands was increased in the pp60 c-src immunoprecipitate from PAF stimulated platelets. To assay the tyrosine kinase activity, we used a 13 amino acid synthetic peptide (ArgArgLeuIleGluAspAlaGluTyrAlaArg;Gly) which contains sequences similar to the phosphorylation site on pp60 c-src. Incubation of the pp60 c-src immunoprecipitate with the peptide and [ 32P]ATP caused phosphorylation of this peptide in vitro. This peptide phosphorylation was not observed when normal mouse IgG-bound protein(s) was used instead of pp60 c-src immunoprecipitate. The peptide phosphorylation was markedly increased by pp60 c-src immunoprecipitate obtained from PAF treated platelets. Lyso-PAF had no effect on the phosphorylation. PAF antagonists CV-6209 and WEB-2086 blocked PAF stimulated phosphorylation. This indicated structurally specific and PAF receptor dependency of this response. These results provide direct evidence that PAF stimulation of human platelets increased tyrosine kinase activity in pp60 c-src immunoprecipitate.

A growth-dependent post-translational modification of annexin VI

Annexin VI (p68, 67-kDa calelectrin) is a member of a family of Ca 2+/phospholipid-binding proteins, that includes p35 (annexin I) and p36 (annexin II), the major cellular substrates for phosphorylation by the epidermal growth factor receptor and pp60 v-src tyrosine kinase activities, respectively. We report here that like annexins I and II, annexin VI is is phosphorylated in vivo, but that in contrast, annexin VI phosphorylation is associated with cell growth. In both Swiss 3T3 fibroblasts and human T-lymphoblasts the pattern of phosphorylation followed an almost identical profile. In particular, annexin VI was not phosphorylated in quiescent cells, but was phosphorylated on serine and to a lesser extent threonine, several hours following cell stimulation. Furthermore, annexin VI also incorporated phosphate in a growth-dependent manner, in a form other than a phosphoamino-acid. The phosphate was visualised following acid hydrolysis of immunoprecipitated annexin VI, as part of a complex having high mobility on 2-D thin-layer electrophoresis. The identity of this complex is not known. The results suggest that a post-translational modification other than direct protein phosphorylation may influence the activity of annexin VI and provide evidence linking cell growth with regulation of annexin VI function.

Evaluation of new sesquiterpene quinones from two Dysidea sponge species as inhibitors of protein tyrosine kinase

Our program to discover inhibitors of enzymes thought to be important in tumorigenesis motivated a study of marine sponge-derived sesquiterpene quinones (hydroquinones) against pp60 v-src protein tyrosine kinase (PTK). Five new metabolites were isolated from the sponge Dysidea avara including melemeleone A (3), melemeleone B (4), 18-methoryavarone (6a), popolohuanone-C (8), and popolohuanone-D (9). These were accompanied by two known compounds, (+)-avarol (2a) and (+)-19-methoryavarone (7)

V-Src Induces elevated levels of diglyceride by stimulation of phosphatidylcholine hydrolysis

When Rat-1 cells bearing the ts LA29 mutant of Rous sarcoma virus (Rat1 LA29) are shifted from restrictive to permissive temperature, the pp60 v-Src tyrosine kinase is activated and there is an increase in the cellular level of sn1,2-diacylglycerol (DRG) within 30 min which is not accompanied by increased inositol phospholipid hydrolysis. Temperature shift also increases the hydrolysis of phosphatidylcholine (PC), as determined by an increase in the generation of water soluble choline metabolites. Transphosphatidylation studies have shown that this occurs at least in part via a phospholipase D (PLD) catalysed pathway.

Marine sponge polyketide inhibitors of protein tyrosine kinase

The marine polyketide natural product, halenaquinone, was shown to be an irreversible inhibitor of pp60 v-src, the oncogenic protein tyrosine kinase encoded by the Rous sarcoma virus. This compound had an IC 50 of approximately 1.5 μM against pp60 v-src and also inhibited the ligand-stimulated kinase activity of the human epidermal growth factor receptor with an IC 50 of approximately 19 μM. Halenaquinone blocked the proliferation of a number of cultured cell lines, including several transformed by oncogenic protein tyrosine kinases. Halenaquinol, xestoquinone, halenaquinol sulfate, and several simple synthetic quinone analogs were also shown to inhibit pp60 v-src.

A novel antibody against a synthetic NH 2-terminal myristoyl glycyl src peptide can detect pp60 v-src, the transforming protein of Rous sarcoma virus

Novel antibodies were raised against a synthetic NH 2-terminal myristoyl(Myr-) tetrapeptide ( N-Myr-Gly-Ser-Ser-Lys) which is characteristic of an NH 2-terminal portion of 60 v-src, the transforming protein of Rous sarcoma virus. Antisera raised against N-Myr-Gly-Ser-Ser-Lys-haemocyanin reacted with 125I-albumin conjugates with N-Myr-Gly-Ser-Ser-Lys. The immunoreaction was competed for by haemocyanin as well as albumin conjugated with this N-Myr-peptide, while underivatized proteins or an NH 2-terminal octapeptide (Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys) had no effect. N-Myr-Gly-Ser-Ser-Lys-( 125I)tyramine was also recognized by the antibody. The reaction was competed for by N-Myr-Gly-Ser-Ser-Lys, but not by Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys. These results suggest a high affinity of the antibody for an N-Myr-peptide moiety. The major ( 3H)myristate-labelled protein of an apparent molecular weight of 60,000 was detected from chick embryo fibroblasts transformed by Rous sarcoma virus (tsNY68). This protein was demonstrated to possess N-Myr-Gly-Ser-Ser-Lys by the immunoprecipitation and HPLC analyses. Furthermore, the entire circumference of the transformed cells was stained by the antibody upon an immunofluorescent microscopic observation. Thus, these results taken together indicate that the haptenic antibody raised against the myristoyl peptide is useful to detect pp60 v-src protein.

Antibodies to an NH 2-terminal myristoyl glycine moiety can detect NH 2-terminal myristoylated proteins in the retrovirus-infected cells

Novel antibodies were raised against a synthetic NH 2-terminal myristoyl glycine moiety which is characteristic of N -myristoyl-proteins. Antisera raised against N -myristoyl-Gly-hemocyanin reacted with N -myristoyl-Gly-[ 125I]albumin. The immunoreaction was competed for by albumin conjugated with N -myristoyl-glycine, while underivatized albumin had no effect. Of the [ 3H]myristate-labeled proteins detected, pp60 v-src , which is a transforming protein of Rous sarcoma virus, and p19 gag and p17 gag , which are core proteins in the human T-cell leukemia virus and the human immunodeficiency virus, were identified as N -myristoylated proteins by the radioimmunoprecipitation analyses with the antibody.

Studies in pig heart tissue on various 60 000 Da phosphoproteins

Pig heart tissue have been shown to contain 3 different 60 000 Da phosphoproteins. Different purification procedures were used in order to separate them, suggesting that the 3 phosphoproteins differ in their environmental parameters. The 2 major ones appear essentially as peripheral phosphoproteins that are associated with cellular membranes through ionic forces, whereas the third minor phosphoprotein behaves as an integral plasma membrane protein. The three phosphoproteins also differ in their relative amount of phosphorylated serine, threonine and tyrosine residues after in vitro protein kinase assay. Evidence that the 3 phosphoproteins are related arises from the similarity between their respective phosphopeptide maps after partial hydrolysis with proteases, an experiment that also points out relatedness in primary structure between them and the transforming protein of Rous sarcoma virus, pp60 v-src. The 3 phosphoproteins, however, do not appear to be immunologically related to pp60 v-src since none of them is immunoprecipitated by sera that precipitate pp60 v-src. The possibility that the three 60 000 Da phosphoproteins under study represent 3 differentially localized and phosphorylated products of c- src and/or c- src related genes is discussed.

A rat mutant cell clone showing temperature-dependent transformed phenotypes with functional expression of the src gene product

The cellular mutant B814 isolated from a Fischer rat cell line shows temperature-sensitivity of focus formation on infection with Moloney murine sarcoma virus (Mo-MSV) and Rous sarcoma virus (RSV). An RSV-transformed clone (S814-2) isolated from B814 cells shows temperature-sensitive transformed phenotypes for morphology, growth in soft agar, and glucose uptake. The expression, phosphorylation, and tyrosine kinase activity of pp60 v-src in S814-2 were not affected at the nonpermissive temperature, and virus rescued from this clone had wild-type transforming ability, suggesting that a cellular factor altered in S814-2 is responsible for the cellular steps of transformation after the function of pp60 v-src In addition, the cellular 36K protein, a possible candidate as a target of pp60 v-src, was phosphorylated at the nonpermissive temperature in S814-2, indicating that phosphorylation of the 36K protein is not correlated with transformed phenotypes.