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PubMed1998

Suppression of decorin expression and partial induction of anchorage-independent growth by the v-src oncogene in human fibroblasts.

To determine its role in cell transformations, the v-src oncogene was introduced into the human foetal diploid fibroblasts MRC-5 and into MRC-SV1, a simian virus 40 (SV40)-transformed cell line derived from them. Infected cells were found to contain stably integrated intact proviruses, as determined by Southern blot analysis. Although highly expressed, v-src did not change the morphology or growth patterns of MRC-5 cells and failed to induce foci or alter their saturation densities. However, overexpression of v-src reduced the plating efficiencies of MRC-5 and induced anchorage-independent growth in a low but significant number of cells. Northern blot analysis showed that v-src selectively abolished the expression of decorin, a small dermatan/chondroitin sulphate proteoglycan that interacts with extracellular-matrix components and modulates collagen-fibril formation and the activity of transforming growth factor (TGF) beta1. Addition of herbimycin A, a potent pp60src tyrosine-kinase inhibitor, resulted in the reexpression of decorin in MRC-5 carrying v-src. There were no changes in the expressions of fibronectin, procollagen type I, or tissue plasminogen activator, an activator of extracellular-matrix-degrading enzymes. Moreover, v-src did not alter the expressions of the epidermal-growth-factor receptor or TGFbeta1 or reduce the growth-factor requirements of MRC-5 fibroblasts. MRC-5 and MRC-SV1 expressing v-src remained non-tumourigenic when injected into nude mice. Constitutive expression of v-src did not alter the mRNA levels of c-jun and junB, suggesting that the effects of the oncogene are not mediated by AP-1. Decorin gene expression has been shown previously to be maximal in quiescent cells and virtually absent in transformed ones. Our data indicate that the ability to synthesise decorin can be suppressed in human fibroblasts without their becoming transformed, and that the relations between decorin synthesis and growth controls need further clarification.

Animals↗
PubMed1995

Identification and characterization of a novel synthetic peptide substrate specific for Src-family protein tyrosine kinases.

Using a random combinatorial peptide library method [Wu, J., Ma, Q. N. & Lam, K. S. (1994) Biochemistry 33, 14825-14833] a novel peptide, YIYGSFK, was identified as a substrate for p60c-src protein tyrosine kinase. Mass spectrometric analysis showed that tyrosine-3 from the N-terminus was the phosphorylation site. Kinetic studies showed that the Km of YIYGSFK for p60c-src was 55 microM, about 6.4-fold lower than a peptide derived from p34cdc2 [cdc2(6-20), KVEKIGEGTYGVVYK], which had been reported to be a specific and efficient substrate for the Src-family protein tyrosine kinases. Comparison of the specificity of YIYGSFK and cdc2(6-20) as a substrate for various Src-family and non-Src-family protein tyrosine kinases suggests that YIYGSFK is a much more specific and efficient substrate for the Src-family protein tyrosine kinases.

Amino Acid Sequence↗
PubMed1999

Expression of a src-type protein tyrosine kinase gene, AcSrc1, in the sea urchin embryo.

By screening a cDNA library and 3'-rapid amplification of cDNA ends, the cDNA for a non-receptor type protein tyrosine kinase from the sea urchin Anthocidaris crassispina was analyzed. The deduced protein (AcSrc1) with the highest identity of about 60% to mammalian Src family kinases shows the characteristic features of the Src family. AcSrc1 mRNA is maternally expressed in unfertilized eggs, while zygotic expression is first detected in blastulae and continues through the pluteus stage. Zygotic mRNA expression, visualized by in situ hybridization, is detected specifically in archenteron at the gastrula stage, while it is restricted in plutei to the midgut and hindgut, suggesting specific roles for AcSrcl in the formation and/or functions of the digestive tract. Meanwhile, western blot analysis has shown that the AcSrc1 protein is constantly expressed throughout embryogenesis. By immunostaining, it was found that the protein (distributed evenly in the cytoplasm of unfertilized eggs) is translocated to the membrane after fertilization. All through the following development, AcSrcl was localized to the peripheries of different embryonic cells, although at a relatively low level of localization at the boundaries between adjacent cells.

Amino Acid Sequence↗
PubMed1993

Members of the src family of nonreceptor tyrosine kinases share a common mechanism for membrane binding.

The src family of nonreceptor protein tyrosine kinases share extensive sequence homology, except for 80 NH2-terminal amino acids, thought to comprise a "unique" domain. This region is presumed to mediate interactions specific to each kinase. Recently, we identified three NH2-terminal lysine residues, crucial for pp60v-src membrane association. Surprisingly, these lysines are conserved among several src family members. Since their mechanism of membrane association is unknown, it was of interest to determine whether other tyrosine kinases also utilize their NH2-terminal domain. Here, we demonstrate that pp60v-src, p62c-yes, and p59fyn polypeptides compete with each other for membrane binding, whereas p56lck, which lacks the NH2-terminal lysine motif, has no effect. Moreover, myristylated peptides corresponding to the NH2 termini of src, yes, lyn, and fyn inhibit membrane association of pp60c-src, p62c-yes, and p59fyn. Our results suggest that src family members share a common mechanism for membrane binding, and they provide a molecular explanation for the ability of other src family members to complement pp60c-src function.

Alkylation↗
PubMed1987

Resistance to the cytolytic action of lymphotoxin and tumor necrosis factor coincides with the presence of gap junctions uniting target cells.

The resistance of target cells to the cytolytic action of lymphotoxin (LT) and recombinant tumor necrosis factor (rTNF) has been investigated by using clonally derived cell lines with defined gap junction-mediated, intercellular communication properties. Gap junction-competent Chinese hamster ovary cells are normally insensitive to the action of LT/TNF. However, treatment with 12-o-tetradecanoylphorbol-13-acetate, which promotes the loss of gap junctions, or culturing at low cell density to reduce intercellular contacts, significantly increased their sensitivity to LT/TNF. The LT/TNF-sensitive murine CL-1D and L929 cell lines, which in normal culture conditions are unable to form gap junctions, were not changed in their susceptibility to LT/TNF after treatment with phorbol ester or low culture density. However, the formation of gap junctions by CL-1D can be promoted by treatment with 8-bromo-cyclic adenosine monophosphate (1 mM), and this treatment completely suppressed the ability of LT and rTNF to kill CL-1D. Additionally, the LA25-normal rat kidney cell line, which is infected with a temperature-sensitive mutant of Rous sarcoma virus (LA25), is gap junction-competent and resistant to the effects of LT at the restrictive temperature (39 degrees C). However, when shifted to the permissive temperature (33 degrees C), LA25-normal rat kidney cells express the pp60v-src viral gene product, lose their ability to form gap junctions, and become sensitive to the lytic activity of LT. The results demonstrate that the expression of the retroviral pp60v-src, a tyrosine protein kinase, is sufficient to render cells susceptible to the lytic effects of LT and rTNF. Collectively, these experiments demonstrate a strong correlation between the resistance of target cells to the action of LT/TNF and their ability to cooperate metabolically through gap junctions. The results do not completely exclude the possibility that other mechanisms, such as LT receptor modulation, are also occurring under these experimental conditions. These data also suggest that a possible physiologic function of the stable cytotoxic lymphokines is to induce cytolysis/cytostasis of cells that have lost gap junctional contact, such as those in the process of mitosis or metastasis that have separated from the main tissue mass.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2006

The role of Src in solid and hematologic malignancies: development of new-generation Src inhibitors.

c-Src was the first protooncogene described and was among the first molecules in which tyrosine kinase activity was documented. c-Src has been defined as a common modular structure that participates in much of the crosstalk between the cytoplasmic protein tyrosine kinases and tyrosine kinase receptors. Understanding the structure and function of this important class of protein kinases and elucidating the molecular signaling events mediated by c-Src are important not only for identifying the critical pathways but also for designing new strategies to block or inhibit the action of these kinases. Despite the large amount of information available on c-Src, its precise functions in cancer remain to be elucidated. Recently, there has been renewed interest in c-Src as a molecular target for cancer therapy, and multiple c-Src inhibitors are entering clinical trials. In this review, the authors describe the function and expression of c-Src in human malignancies and the novel c-Src inhibitors and their potential applications for cancer treatment.

Antineoplastic Agents↗
PubMed1982

Transit of pp60v-src to the plasma membrane.

The protein kinase (pp60v-src) encoded by the transforming gene (v-src) of Rous sarcoma virus is synthesized on free polyribosomes and then translocated to the plasma membrane of infected cells. Neither the mechanism of the translocation nor the physiological significance of the membrane localization has been elucidated. We have explored these problems by pursuing previous observations of a complex between pp60v-src and two cellular proteins with molecular weights of 50,000 and 89,000. We found the complex located entirely in the cytoplasm, where it appears to form immediately after the synthesis of pp60v-src. While in the complex, pp60v-src has little detectable kinase activity and is phosphorylated predominantly on serine. After transfer from the complex to the plasma membrane, pp60v-src becomes phosphorylated on tyrosine as well as serine and acquires kinase activity. Under restrictive conditions, temperature-sensitive pp60v-src is produced in normal quantities, but translocation to the plasma membrane is diminished. As an apparent consequence, the cytoplasmic complex accumulates to abnormal abundance. Alternatively, temperature-sensitive pp60v-src that has been synthesized and translocated to the plasma membrane under permissive conditions appears to be released from the membrane and returns to the cytoplasmic complex when the infected cells are shifted to the restrictive temperature. We conclude that the cytoplasmic complex may be the vehicle by which pp60v-src reaches the plasma membrane. It is possible that other proteins may follow a similar route to the membrane. Binding to plasma membrane appears to be a discrete step in the biogenesis of pp60v-src and may be essential to the function of the protein.

Animals↗
PubMed1995

p80/85 cortactin associates with the Src SH2 domain and colocalizes with v-Src in transformed cells.

Expression of oncogenic variants of pp60src leads to dramatic changes in cytoskeletal organization characteristic of transformation. Activated Src associates with the cytoskeletal matrix, resulting in tyrosine phosphorylation of specific cytoskeletal substrates. We have previously shown that stable association of Src with the cytoskeletal matrix is mediated by the Src SH2 domain in a phosphotyrosine-dependent interaction. In this report, we demonstrate that one of the cytoskeletal binding partners of Src is p80/85 cortactin. The association was observed in lysates of transformed cells but was not seen in normal fibroblasts. The interaction could be reconstituted in vitro using transformed cell extracts and a glutathione S-transferase (GST) fusion protein containing the Src SH2 domain but not with GST-Src SH3 or with GST-Src SH2 containing a point mutation in the FLVRES sequence. Confocal microscopy revealed that cortactin redistributed and colocalized with v-Src and a Src SH3 deletion mutant in transformed cells. However, in cells expressing a Src SH2 deletion mutant, the redistribution of cortactin and colocalization with Src did not occur. Furthermore, biochemical fractionation of transformed cells indicated that a significant increase in cortactin distribution to the cytoskeletal fraction occurred, which correlated with a shift in the tyrosine-phosphorylated form of the protein. Cortactin fractionated from cells expressing kinase-defective or myristylation-defective Src mutants did not exhibit this shift. These data suggest a molecular mechanism by which tyrosine phosphorylation of cortactin and association with the Src SH2 domain influence the cytoskeletal reorganization induced in Src-transformed cells.

Animals↗
PubMed2001

Activated SRC oncogene phosphorylates R-ras and suppresses integrin activity.

One of the prominent effects of the Src kinase is to reduce cell adhesion. The small GTPase, R-Ras, affects cell adhesion by maintaining integrin activity, and the ability of R-Ras to do so can be regulated by phosphorylation of a tyrosine residue located in its effector domain by an Eph receptor kinase (Zou, J. X., Wang, B., Kalo, M. S., Zisch, A. H., Pasquale, E. B., and Ruoslahti, E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13813-13818). Here we show that Src regulates cell adhesion through R-Ras and integrins. Reduced substrate attachment of 293T cells transfected with the cDNA for an activated form of Src (v-Src) was accompanied by phosphorylation of endogenous R-Ras. v-Src also phosphorylated R-Ras in vitro. An activated form of Src similar to one that has been found in human cancers, Src527, shared with v-Src the ability to phosphorylate R-Ras. Stronger R-Ras phosphorylation was seen in Madin-Darby canine kidney cells cells transformed with temperature-sensitive v-Src at the permissive temperature than at the non-permissive temperature, and R-Ras and Src co-immunoprecipitated at the permissive temperature. Mutation analysis showed that the Src phosphorylation site in R-Ras was tyrosine 66, the position critical to the ability of R-Ras to support integrin activity. Finally, activated R-Ras in which tyrosine 66 is mutated to phenylalanine rendered cells partially resistant to the effects of Src on cell adhesion. Regulation of cell adhesion by Src through R-Ras may be at least partially responsible for the reduced adhesion and the resulting increased invasiveness of Src-transformed cells.

Animals↗
PubMed1995

Direct and specific interaction of c-Src with Neu is involved in signaling by the epidermal growth factor receptor.

Transgenic mice expressing either the activated or wild type neu oncogene heritably develop metastatic mammary tumors. Tumor development in this transgenic mouse model correlates with activation of the Neu tyrosine kinase. Recently, we have shown that these Neu-induced mammary tumors possess elevated c-Src tyrosine kinase activity. Here, we demonstrate that c-Src requires tyrosine phosphorylated Neu for its ability to associate with Neu in vivo and this association is likely the result of a direct physical binding of c-Src SH2 domain to the tyrosine phosphorylated Neu. By contrast, the c-Src SH2 domain did not interact directly with tyrosine phosphorylated EGFR. Moreover, in established cell lines expressing elevated levels of EGFR, EGF stimulation results in transphosphorylation of Neu and formation of complexes between c-Src and tyrosine phosphorylated Neu. Taken together, these observations suggest that activation of c-Src by these two closely related EGFR family members results from a direct and specific interaction of c-Src with tyrosine phosphorylated Neu.

Animals↗
PubMed1993

Morphological transformation, tumorigenicity and src-specific cytotoxic T-lymphocyte-mediated tumor immunity induced by murine 3T3 cells expressing src oncogenes encoding novel non-myristylated N-terminal domains.

We previously reported the development of a src-specific tumor regression system in chickens in which preinfection with rASV1702, a mutant of Rous sarcoma virus (RSV) encoding non-myristylated src product with a novel N-terminal domain, results in the immune suppression of challenge tumors induced by RSV. In order to adapt this system to the mouse, we have developed NIH3T3 and Balb/c3T3 (B3T3) cell lines that express 1702src, v-src, c-src, and other src variants, either by transfection or by infection with packaged recombinant Moloney virus (MLV) vectors. The sequence of the 1702 src cDNA, produced by reverse transcription-polymerase chain reaction (RT-PCR), confirmed the previously suggested 1702src N-terminal domain structure, fusing six amino acids from Pr76gag and 39 amino acids of env signal peptide sequence to Ala-76 of src. These cell lines were characterized for src expression and activity, cell compartmentalization of src product, tyrosine phosphorylation substrate specificity and transforming and tumorigenic potential. Based on these parameters, murine cell lines expressing 1702src were characteristically similar to chicken embryo fibroblasts (CEFs) infected with rASV1702. Finally, B3T3/1702src expressors, or membrane fractions of these cells, induced src-specific tumor protection in syngeneic mice against v-src-transformed tumor challenges. Splenic lymphocytes isolated from Balb/c mice inoculated with B3T3/1702src cells showed in vitro cytotoxicity against B3T3/v-src cells but not against untransformed B3T3 cells. Antibodies specific for Lyt2, mouse CD3 and H-2Dd blocked this cytotoxicity, whereas those specific for L3T4 did not, suggesting MHC class I-restricted, CD8-mediated cell killing. These data indicate that Balb/c3T3-expressed 1702src induces a cellular anti-tumor immunity based on src-specific tumor rejection antigens.

3T3 Cells↗
PubMed2000

Wild-type and mutant forms of v-src differentially alter neuronal migration and differentiation in vivo.

The effects of three different forms of v-src on brain cell development were determined in vivo. Recombinant retroviral vectors encoding the marker lacZ (control) and either wild-type v-src or SH2 or SH3 domain-deleted forms of v-src (deltaSH2 or deltaSH3, respectively) were used to infect neuronal progenitor cells in the embryonic chicken midbrain (optic tectum; OT). Embryos were injected in the OT with retroviral concentrates on embryonic day (E) 3 and sacrificed at E6, E9, and later in development. Patterns of cell proliferation, migration, and differentiation of lacZ-marked clonal cell progeny were then analyzed. Relative to lacZ-only controls, cell clone size at E6 was significantly increased for v-src-, unchanged for deltaSH2-, and smaller for deltaSH3-injected embryos. At E9, deltaSH2 cell clones were significantly larger than controls, suggesting increased survival from normal programmed cell death. Radial neuronal migration was impaired for v-src and deltaSH3 clones, whereas tangential neuronal migration was enhanced along fiber tracts in v-src and deltaSH2 clones. Moreover, radial glial cell development and differentiation was hindered in v-src and deltaSH3 clones. These experiments demonstrate that ectopic v-src signaling alters proliferation, migration, survival, and differentiation of developing brain cells and suggest that src signaling pathways are involved in these developmental processes. Furthermore, certain effects of v-src on brain cells require specific src homology domains.

Animals↗
PubMed1995

Src-transformation of mouse fibroblasts induces a Ca(2+)-activated K+, current without changing the T-type Ca2+ current.

Membrane currents of src-transformed NIH3T3 mouse fibroblasts were analyzed in comparison with their non-transformed counterparts using the patch-clamp technique. Normal NIH3T3 cells exhibit two types of Ca2+ currents and a membrane current of ohmic behaviour (current amplitude 135 pA at +30 mV) that can partially be blocked by Cd2+. Src-transformed NIH3T3 cells show an additional membrane current that becomes activated after the establishment of the whole-cell configuration with a maximum amplitude of 1040 pA at +30 mV within 30-60 s. This current then inactivates irreversibly within 5-10 min. The additional current is highly K(+)-selective and Ca(2+)-dependent but voltage-independent. It can be blocked by charybdotoxin (IC50 = 20 nM) and by internal tetraethylammonium (TEA; IC50 = 2.9 mM), but it is not sensitive to external TEA (up to 30 mM). Single-channel analysis revealed only one K+ channel type with a conductance of 37 pS at negative potentials and 18 pS at positive potentials (in symmetrical 145 mM K+ solutions), a voltage-independent open-state probability of 0.6 and the same pharmacological properties as the macroscopic KCa current. The properties of the KCa current and the underlying channels of src-transformed NIH3T3 cells are identical to those observed in ras-transformed NIH3T3 cells. In contrast, src- or ras-transformation affects differently the voltage-dependent, transient (T-type) Ca2+ current. While ras-transformation of NIH3T3 cells suppresses their T-type Ca2+ current, this current remains unchanged in src-transformed NIH3T3 cells.

3T3 Cells↗
PubMed1996

Endogenous c-src as a determinant of the tumorigenicity of src oncogenes.

We have compared the tumorigenicity of two src oncogenes, v-src and c-src(527), whose respective protein products pp60v-src and pp60c-src(527) show a different spectrum of amino acid substitutions vis-à-vis the c-src protooncogene-encoded product pp60c-src. Whereas the extent of primary tumor growth induced by c-src(527) was quite similar in the two chicken lines tested, the extent of v-src-induced tumor growth showed a marked line dependence. As examined with a line of chickens that shows immune-mediated regression of v-src-induced tumors, a weaker tumor immunity, as correlated with a greater level of primary tumor growth, resulted from inoculation of c-src(527) DNA than of v-src DNA. These observations indicated that the v-src-specific amino acid substitutions define a major tumor antigenicity. That a separate src-associated antigenicity is also targetable by the tumor immune response followed from the finding that the level of protective immunity against the growth of c-src(527) DNA-induced tumors was augmented under conditions of the prior regression of v-src DNA-induced tumors. As this latter antigenicity may include one or more c-src(527)-encoded peptides that are equivalent to c-src-encoded self peptides, these observations suggest that a host tolerance to pp60c-src can be broken so as to permit a tumor immune response based on recognition of self peptides of pp60c-src(527).

Animals↗