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PubMed1992

Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.

The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.

Actins↗
PubMed2004

[Expression of the p56lck by colon tumors: a marker of their invasive capacity?].

Since its discovery in murine thymoma in 1982, the p56lck (lymphocyte cellular kinase) has been shown to be a pivotal enzyme to both maturation of thymocytes and activation and proliferation of peripheral lymphocytes. The p56lck sequence appeared highly homologous to that of the oncogene p60c-src as did its exon-intron organisation. These data have suggested the lck gene originates from the ancestral src gene by the exon-shuffling mechanism. However, and in spite of this relationship with the p60src oncogene which is often implicated in human cancers, p56lck does not appear involved in lymphoproliferative diseases, either by overexpression or activating mutations. Nevertheless, its aberrant expression has been reported in some carcinomas (colon, lung and mammary). This unexpected expression of a lymphoid-specific protein in solid tumors remained enigmatic until recent studies. In this review, we report these data and explain the possible mechanisms which could lead to the p56lck ectopic expression. We also discuss of signalling pathways which could be affected by the abnormal presence of the p56lck in these tumoral epithelial cells. In particular, we indicate that p56lck could favor metastases by facilitating loss of cell adhesion.

Colonic Neoplasms↗
PubMed1994

pp60 c-src expression and activity in MG-63 osteoblastic cells modulated by PTH but not required for PTH-mediated adenylate cyclase response.

A role for pp60c-src) tyrosine kinase in bone resorption was recently demonstrated in vivo. However, it is not known whether expression of this ubiquitous tyrosine kinase is essential in osteoblasts or osteoclasts. In this work, we have examined the expression of c-src in osteoblast-like cells. We found that c-src was expressed in the MG-63 osteoblastic osteosarcoma cell line as assessed by immunocytochemistry. When MG-63 cells were treated for 30 minutes with parathyroid hormone (PTH), the levels of tyrosine phosphorylation of c-src were increased in comparison with the untreated control. On the other hand, no changes in total c-src protein were observed after PTH treatment. The increase in c-src tyrosine phosphorylation due to PTH treatment was paralleled by an increase in c-src tyrosine kinase activity. Calcitonin had no effect on c-src phosphorylation, c-src protein level, or tyrosine kinase activity. To determine if c-src tyrosine kinase was required for normal bone cell function and PTH responsiveness, osteoblastic cells were isolated from the calvaria of a src-deficient mouse. The cyclic AMP response to PTH in this cell was identical to that seen in freshly isolated calvarial cells from normal mice. These results suggest that the activity of c-src in MG-63 cells is regulated by PTH as a result of tyrosine phosphorylation. Expression of src is not required for PTH responsiveness in osteoblastic cells as assessed by adenylate cyclase activation. The mode of signal transduction from the PTH receptor to nonreceptor c-src tyrosine kinase is not known at present.

Adenylyl Cyclases↗
PubMed1993

v-Src-induced transformation is inhibited by okadaic acid.

The tumor promoter okadaic acid is a potent inhibitor of the serine/threonine protein phosphatases 1 and 2A. Addition of okadaic acid to v-Src-transformed BALB/c 3T3 cells reverted them to a flat morphology, increased fibronectin levels in the extracellular matrix, reduced saturation density, and inhibited the formation of colonies in soft agar. The ability of v-Src-transformed cells to proliferate in low serum was also inhibited by okadaic acid. These data implicate serine/threonine phosphatases in v-Src-induced transformation.

3T3 Cells↗
PubMed1993

Immune-based resistance to the formation of v-src-induced distal tumors.

Although v-src, the oncogene of Rous sarcoma virus, has been shown to be capable of inducing lethal tumors at visceral sites distal to the primary tumor mass, the mechanisms opposing visceral tumor formation remain to be elucidated. In the present study, we show that visceral tumors, many of which represent a metastasis spawned by the primary mass, are found only in hosts exhibiting reduced levels of tumor immunity. We conclude that it is the weakness of the tumor immune response, rather than a lack of expression of tumor antigen on visceral tumor cells, that is a major underpinning of the formation of v-src-induced visceral tumors.

Animals↗
PubMed1991

c-src structure in human cancers with elevated pp60c-src activity.

We used RNAase protection and restriction fragment length polymorphism assays to detect activating mutations of c-src in a spectrum of human tumours. No mutations were detected at codons 98, 381, 444, and 530. We conclude that mutational activation is not the mechanism of enhancement of pp60c-src-specific kinase activity found in a number of human cancer types.

Base Sequence↗
PubMed1992

Role of p34cdc2-mediated phosphorylations in two-step activation of pp60c-src during mitosis.

Phosphorylation of pp60c-src by p34cdc2 at three amino-proximal serine/threonine residues is temporally correlated with, but insufficient for, mitotic activation of c-Src kinase. The direct cause of activation during mitosis appears to be temporally correlated partial dephosphorylation of Tyr-527, a residue whose phosphorylation strongly suppresses pp60c-src activity. Site-directed mutagenesis of the serine/threonine phosphorylation sites blocks half the mitosis-specific decrease in Tyr-527 phosphorylation and half the increase in pp60c-src kinase activity. We conclude that p34cdc2 partially activates pp60c-src by a two-step process in which its serine/threonine phosphorylations either sensitize pp60c-src to a Tyr-527 phosphatase or desensitize it to a Tyr-527 kinase. Furthermore, additional events, independent of these p34cdc2-mediated phosphorylations, participate in mitotic activation of pp60c-src.

3T3 Cells↗
PubMed1992

MARCKS protein is transcriptionally down-regulated in v-Src-transformed BALB/c 3T3 cells.

Activation of protein kinase C (PKC) by tumor-promoting phorbol esters leads to the phosphorylation of an 80-kilodalton PKC substrate (known as MARCKS) in murine fibroblasts. In BALB/c 3T3 cells stably transformed by v-Src, phorbol esters were unable to induce phosphorylation of MARCKS. Western blot analysis and in vitro kinase assays showed that both PKC protein levels and kinase activity were unchanged in v-Src-transformed relative to the parental nontransformed BALB/c 3T3 cells. However, MARCKS protein levels were reduced in v-Src-transformed cells relative to nontransformed cells. MARCKS RNA levels were also correspondingly reduced in v-Src-transformed cells. Nuclear "run-on" assays showed decreased transcription of MARCKS in v-Src-transformed cells. Thus, the absence of MARCKS in v-Src-transformed cells could be explained by a down-regulation of MARCKS transcription. Inhibiting the protein tyrosine kinase activity of v-Src with herbimycin A restored MARCKS RNA levels, MARCKS transcription, and MARCKS protein, suggesting that down-regulation of MARCKS in v-Src-transformed BALB/c 3T3 cells is a direct effect of v-Src.

3T3 Cells↗
PubMed1996

Increased immunoreactivity and protein tyrosine kinase activity of the protooncogene pp60c-src in preneoplastic lesions in rat pancreas.

This study investigates the possibility that the c-Src protein tyrosine kinase is involved in experimental exocrine pancreatic carcinogenesis. Expression and activity of the protooncogene pp60c-src (c-Src) are investigated in acinar pancreatic (pre-) neoplastic lesions induced in rats by azaserine and compared with normal rat pancreas. Low or absent c-Src immunoreactivity and c-Src tyrosine kinase activity were found in the pancreas of untreated control rats. Compared with these controls, c-Src protein immunoreactivity was increased in "normal" acinar cells and even more in putative preneoplastic atypical acinar cell nodules (AACN) after azaserine treatment. In contrast, more advanced (secondary transformed) acinar cell lesions demonstrated no c-Src immunoreactivity. Rats treated with azaserine showed a 7-fold-higher c-Src tyrosine kinase activity in their pancreas. The level of c-Src tyrosine kinase activity correlated positively with the number of lesions in the pancreas, inasmuch as promotion of azaserine-initiated carcinogenesis by cerulein resulted in a more than 10-fold increase in the number of AACN, which was accompanied by a 6-fold increase in c-Src activity when compared with azaserine treatment alone. c-Src tyrosine kinase activity was responsible, on average, for 40% of the total tyrosine kinase activity in the pancreatic homogenates and was predominantly found in the cytoskeletal subcellular fraction. Furthermore, the transformation from normal to preneoplastic pancreatic tissue in azaserine-treated rats was accompanied by a change in the localization of the c-Src protein. With the use of immunohistochemistry and confocal laser scanning microscopy, the protein was detected in the cytoplasm in morphologically normal pancreatic acini, whereas in AACN it was detected both in the cytoplasm and in the nuclei. It is concluded that the protooncogene c-Src might be involved early in experimental pancreatic carcinogenesis: c-Src probably plays a minor role in pancreatic acinar cells after transformation to malignancy.

Animals↗
PubMed2005

Absence of Fyn and Src causes a reeler-like phenotype.

Nonreceptor protein tyrosine kinases of the Src family regulate the survival, proliferation, differentiation, and motility of many cell types, but their roles in brain development are unclear. Biochemical and in vitro experiments implicate Src and Fyn in the Reelin-dependent tyrosine phosphorylation of Dab1, which controls the positioning of radially migrating neurons in many brain regions. However, genetic evidence that either Src or Fyn mediates Reelin-dependent migrations in vivo has been lacking. Here, we report that, although Src is dispensable and although the absence of Fyn causes an intermediate phenotype, the combined absence of Src and Fyn almost abolishes tyrosine phosphorylation of Dab1 and causes defects in the fetal cortex and cerebellum very similar to those of dab1 mutants of the same age. Neurogenesis is not detectably affected, but the layering of neurons in the cortex is inverted, and the formation of the Purkinje plate is impaired. This implies that Src and Fyn are needed for Reelin-dependent events during brain development.

Animals↗
PubMed1992

Intracellular targeting of pp60src expression: localization of v-src to adhesion plaques is sufficient to transform chicken embryo fibroblasts.

To define the effects of pp60v-src activity at different intracellular sites, we have constructed chimeric molecules which target the pp60v-src kinase to specific intracellular locations. pp60v-src was targeted to the nucleus by insertion of the SV40 large T antigen nuclear localization signal. Nuclear pp60v-src was active as a tyrosine kinase and phosphorylated nuclear proteins at tyrosine. However, cells expressing the nuclear pp60v-src were phenotypically normal by a number of criteria, and nuclear src kinase did not induce the expression of an mRNA (CEF-4) whose induction is characteristic of transformation by wild-type v-src. pp60v-src was targeted to perinuclear membranes by fusion to rat growth hormone and vesicular stomatitis G protein sequences. Cells expressing this chimeric molecule were phenotypically normal by most criteria. However the perinuclear src protein did induce elevated levels of CEF-4 mRNA, indicating that the v-src kinase expressed at this site induces partial transformation. The v-src and activated c-src kinases were targeted to adhesion plaques by fusion to the talin-binding sequence of vinculin. Cells expressing these fusion proteins were transformed by morphological, physiological and biochemical criteria, although the foci induced by these viruses were distinct from those induced by wild-type v-src. A chimeric protein which targeted c-src to adhesion plaques was not transforming. Thus targeting pp60src to adhesion plaques, although not sufficient to activate the transforming capacity of c-src, is sufficient to allow transformation by v-src.

Amino Acid Sequence↗
PubMed1991

Effects of transformation with the v-src oncogene on inositol phosphate metabolism in rat-1 fibroblasts. D-myo-inositol 1,4,5,6-tetrakisphosphate is increased in v-src-transformed rat-1 fibroblasts and can be synthesized from D-myo-inositol 1,3,4-trisphosphate in cytosolic extracts.

Rat-1 fibroblasts transformed with the v-src oncogene show a 6-fold increase in the apparent amount of an inositol polyphosphate which has a high performance liquid chromatography (HPLC) elution characteristic of the D/L-myo-inositol 1,4,5,6-tetrakisphosphate enantiomeric pair (Johnson, R.M., Wasilenko, W.J., Mattingly, R.R., Weber, M.J., and Garrison, J.C. (1989) Science 246, 121-124). By chemical and enzymatic analysis, the structure of this compound produced in both normal and v-src-transformed rat-1 fibroblasts has been determined to be principally D-myoinositol 1,4,5,6-tetrakisphosphate (D-Ins(1,4,5,6)P4). Chronic stimulation with endothelin-1 in the presence of Li+ significantly increased the amount of D/L-Ins(1,4,5,6)P4 only in the v-src-transformed rat-1 cells, suggesting that production of this compound may be remotely coupled to long term agonist-induced phosphatidylinositol turnover. Further evidence for such a link is provided by the progressive loss of D-Ins(1,4,5,6)P4 from the normal cells deprived of serum stimulation. To define a possible synthetic pathway for D-Ins(1,4,5,6)P4, cytosolic extracts of normal and v-src-transformed cells were incubated with [3H]inositol polyphosphates, and the reaction products were identified by HPLC elution and chemical analysis. Although inositol 1,3,4-trisphosphate 6-kinase activity was prominent in extracts of both normal and transformed cells, only the cytosol from v-src-transformed cells ultimately formed measurable amounts of D-Ins(1,4,5,6)P4 from [3H]inositol 1,3,4-trisphosphate. Approximately 6% of 0.1 microM inositol 1,3,4-trisphosphate was converted to D-Ins(1,4,5,6)P4 during a 2-h incubation at 37 degrees C. Inositol pentakisphosphate was identified as a likely intermediate in this conversion, and extracts of both normal and transformed cells converted [3H]inositol 1,3,4,5,6-pentakisphosphate to D-Ins(1,4,5,6)P4. The synthetic pathway described is consistent with the long term regulation of D/L-Ins(1,4,5,6)P4 levels in rat-1 fibroblasts seen in response to src transformation, serum withdrawal, and chronic endothelin treatment, and identifies several new potential interactions between the pathways of inositol polyphosphate metabolism and those of src transformation.

Animals↗
PubMed1993

src-specific immune regression of Rous sarcoma virus-induced tumors.

We have developed an oncogene-specific tumor regression system in chickens. Injection s.c. of chicken wing webs with either rASV1702 or rASV157, mutants of Rous sarcoma virus (RSV) that express nonmyristolyated src product containing novel N-terminal domains, results in noninvasive fibrosarcomas that regress fully. The ability of challenge infections with wild-type RSV to form tumors is suppressed. This protective effect was shown to be specific for determinants encoded or induced by v-src (I. Gelman and H. Hanafusa, J. Virol., 61: 2461-2468, 1989). In the current study, we used SC chickens, inbred for major histocompatibility complex Class I haplotype B2/B2, to investigate whether this protection results from active immunity. Preinoculation of chickens with either replication-defective rASV1702 virus or non-virus-producing syngeneic chicken embryo fibroblasts expressing 1702src conferred protection against challenge infections with RSV. Thus, viremia was not required for this protection. Splenic lymphocytes from rASV1702-infected donors could transfer protective immunity against RSV tumor challenge to naive chickens. These lymphocytes were cytotoxic in vitro against RSV- or rASV1702-infected SC-chicken embryo fibroblasts, but not against SC-chicken embryo fibroblasts infected with helper virus, suggesting a specificity for src-encoded or -induced determinants. In contrast, splenic lymphocytes from RSV-infected chickens transferred protective immunity poorly and exhibited low in vitro cytotoxic potential for src determinants, suggesting possible suppression mechanisms. Finally, murine cell lines expressing 157src or 1702src produced tumors in nude mice that failed to regress. Thus, although cells expressing 157src or 1702src are inherently tumorigenic, the tumors they induce most likely regress due to immune mechanisms. These results suggest that 1702src and 157src induce src-specific tumor Ag that potently prime an oncogene-specific protective cellular immunity.

Animals↗
PubMed1998

Augmentation in the expression of a ribosomal protein associated with transformation.

Using differential hybridization technique, cDNA that was a partial sequence of rat ribosomal protein L41 was isolated from fos- and src-transfected rat fibroblast 3Y1. The cDNA was named #31. Subsequently, the human counterpart(named hm3168) was cloned from a human melanoma cell line(M14) and the amino acid sequence deduced from the cDNA proved to be identical to that of HG-12. The gene expression of hm3168 was inducible by serum-stimulation. It reveals the possible up-regulation of the transcription via fos-mediated signal transduction. Northern-blot and Western-blot analyses showed a large expression of the #31 gene in the rat transformed cell lines.

Animals↗
PubMed1993

Expression of Lyn protein on human malignant lymphomas.

BACKGROUND: lyn is one of the src family genes encoding protein-tyrosine kinases, expressed preferentially in B lymphocytes and monocytes/macrophages. Its gene product, Lyn protein, is thought to participate in cell membrane-associated signal transduction on B lymphocytes by associating physically and functionally with membrane-bound IgM. EXPERIMENTAL DESIGN: To investigate the expression of Lyn on human malignant lymphomas (MLs), 50 ML biopsies, 12 ML samples maintained in mice with severe combined immunodeficiency, and 4 African Burkitt type cell lines were studied with the use of immunohistology, immunochemistry, and Southern blot analysis. RESULTS: Among biopsy specimens, 27 of 27 B-MLs, 5 of 21 T-MLs, and 2 of 2 null-MLs were stained. In severe combined immunodeficiency mouse-maintained B-MLs, unlike biopsied B-MLs, 4 of 11 were found to be unstained. Further analysis disclosed that all 4 of these unstained B-MLs were the Epstein Barr virus transformed B cells proliferating in severe combined immunodeficiency mice and not the original ML cells, suggesting the presence of a specific mechanism down-regulating the Lyn protein in this group. One IgA+ IgM- B-ML and one IgG+ IgM- B-ML were stained by the antibody, indicating the possible existence of molecular mechanisms other than membrane-bound IgM that facilitate Lyn protein expression. Decrease of Lyn expression was also noted in 3 of 4 Epstein-Barr virus-positive African Burkitt's ML lines. Complementary Western blot analysis of 8 immunostained and 4 unstained MLs confirmed the immunohistologic findings. However Southern blot analysis showed that the lyn gene in Lyn-positive and -negative cases were apparently unchanged. CONCLUSIONS: The level of Lyn expression in MLs reflects mainly their normal counterpart, whereas it can be expressed somewhat differently in some cases, especially in Epstein Barr virus-transformed MLs that occur in immunocompromised hosts, on which it is often down-regulated. This is the first report of Lyn expression on human MLs.

Animals↗
PubMed1996

Regulation, substrates and functions of src.

Src is the best understood member of a family of 9 tyrosine kinases that regulates cellular responses to extracellular stimuli. Activated mutants of Src are oncogenic. Using Src as an example, and referring to other Src family members where appropriate, this review describes the structure of Src, the functions of the individual domains, the regulation of Src kinase activity in the cell, the selection of substrates, and the biological functions of Src. The review concentrates on developments in the last 6-7 years, and cites data resulting from the isolation and characterization of Src mutants, crystallographic studies of the structures of SH2, SH3 and tyrosine kinase domains, biochemical studies of Src kinase activity and binding properties, and the biology of transgenic and knockout mouse strains.

Amino Acid Sequence↗
PubMed1993

Correlation between Src family member regulation by the protein-tyrosine-phosphatase CD45 and transmembrane signaling through the T-cell receptor.

Stimulation of tyrosine phosphorylation is an early and important event in antigen-induced T-cell activation. T-cell clones deficient in expression of CD45, a transmembrane protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), are impaired in their ability to respond to either antigen or T-cell receptor cross-linking. Analysis of the CD45-deficient CD8+ T-cell clone L3M-93 demonstrates that the Src family members p56lck and p59fyn show increased immunoreactivity with anti-phosphotyrosine antibody and exhibit decreased kinase activity. The site of increased tyrosine phosphorylation in Src family members was identified by comparison of cyanogen bromide peptide maps. Phosphorylation of the C-terminal phosphopeptide, containing the negative regulatory site of tyrosine phosphorylation, from the CD45-deficient cells was increased 8-fold for p56lck and 2-fold for p59fyn. These data suggest that CD45 dephosphorylates the negative regulatory site of multiple Src family members in the cytotoxic T-lymphocyte clone L3 and show a correlation between the ability to respond efficiently to antigen and the dephosphorylation of Src family members by CD45.

Animals↗