c-Src Tyrosine Kinase Activity Research Articles

Measurement of the Protein Tyrosine Kinase Activity of c-src Using Time-Resolved Fluorometry of Europium Chelates

A nonradioactive, sensitive assay method to evaluate the activity of protein tyrosine kinases is described. This method utilizes europium chelate-labeled anti-phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, dissociation-enhanced fluorescence. Recombinant c-src was used to demonstrate that substrate phosphorylation was dependent on incubation time, enzyme concentration, and the amount of substrate used to coat the microtiter plate wells. A series of proprietary c-src inhibitors was evaluated in competition assays, and demonstrated a rank order of potency which was identical to that determined by other assay methods. Substrate phosphorylation was also demonstrated to be dependent on the concentration of ATP present during the kinase reaction. Because the kinase assay can be performed with different ATP concentrations (unlike with assays utilizing radioactive ATP analogs), the assay described can be used to distinguish compounds that compete for the ATP or substrate binding sites of the kinase.

Activation and Translocation of c-Src to the Cytoskeleton by Both Platelet-derived Growth Factor and Epidermal Growth Factor

We have examined the subcellular distribution and catalytic activity of c-Src tyrosine kinase after stimulation of A172 glioblastoma cells with peptide growth factors. Treatment of resting cells with platelet-derived growth factor resulted in an increase (3.5-fold) in the amount of c-Src protein associated with the cytoskeleton. In addition, an increase in specific c-Src kinase activity was observed in the cytoskeleton as well as in the cytosol and the membrane fraction. Similar effects on both c-Src redistribution and activity were seen after stimulation with epidermal growth factor. These data show that, like other signal transducing components, c-Src also becomes activated and associated to the cytoskeleton in response to growth factor stimulation.

Identification of a hormonally regulated protein tyrosine phosphatase associated with bone and testicular differentiation.

Absence of the tyrosine kinase activity of c-src and c-fms results in impairment of bone remodeling. Such dysfunction underscores the importance of tyrosine phosphorylation, yet the role of protein tyrosine phosphatases in bone metabolism remains unexamined. We have isolated the cDNA for a novel receptor-like tyrosine phosphatase expressed in bone and testis named osteotesticular protein tyrosine phosphatase (OST-PTP). The deduced 1711-residue protein possesses an extracellular domain with 10 fibronectin type III repeats and a cytoplasmic region with two catalytic domains. In primary rat osteoblasts, the 5.8-kilobase OST-PTP transcript is up-regulated in differentiating cultures and down-regulated in late stage mineralizing cultures. In addition, a presumed alternate transcript of 4.8-5.0 kilobases, which may lack PTP domains, is present in proliferating osteoblasts, but not detectable at other stages. Parathyroid hormone, a modulator of bone function, as well as cyclic AMP analogues, increase OST-PTP mRNA 5-8-fold in UMR 106 cells. In situ hybridization of adult rat testis revealed stage-specific expression of OST-PTP. OST-PTP may function in signaling pathways during bone remodeling, as well as serve a broader role in cell interactions associated with differentiation in bone and testis.

C-src tyrosine kinase activity: A marker of dysplasia in ulcerative colitis

c-src tyrosine kinase activity: A marker of dysplasia in ulcerative colitis

ELEVATED PROTEIN-TYROSINE-PHOSPHATASE ACTIVITY IN HUMAN COLON-CARCINOMA

We have examined the activity of protein tyrosine phosphatase in various tumor cell lines and surgically resected human colon carcinoma specimens. The protein tyrosine phosphatase activity of rat fibroblasts increases with the transformation by v-src gene. Likewise human colon carcinoma and human neuroblastoma cell lines, in which the activation of c-src tyrosine kinase was reported, show elevated protein tyrosine phosphatase activity in most cases. We compared the protein tyrosine phosphatase activity of eight human colon cancer tissues with that of normal mucosa of the same patient. The results show that the protein tyrosine phosphatase activity in tumor tissue was increased, suggesting the biological significance of protein tyrosine phosphatase in colon cancer.

An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis.

The tyrosine kinase activity of c-Src is stimulated during mitosis by dephosphorylation of its regulatory tyrosine residue. This is associated with increased accessibility of its Src homology-2 (SH2) domain for binding a phosphotyrosine-containing peptide. But physiological targets of activated c-Src in mitosis have not yet been identified. Here we report that a 68K protein (p68) becomes tyrosine-phosphorylated and physically associates with Src during mitosis in mouse fibroblasts. p68 independently binds the Src SH2 and SH3 domains in vitro and both domains are required for p68 phosphorylation and binding in vivo. p68 is closely related to the p62 protein that is associated with the Ras GTPase-activating protein (GAP) and selectively binds, directly or indirectly, polyribonucleotides. Because the Src SH3 domain also binds heterogeneous nuclear ribonucleoprotein K, these results raise the intriguing possibility that c-Src may regulate the processing, trafficking or translation of RNA in a cell-cycle-dependent manner.

Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity.

Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20 to 30% of human breast cancers. Although the activation of Neu receptor tyrosine kinase appears to be a pivotal step during mammary tumorigenesis, the mechanism by which Neu signals cell proliferation is unclear. Molecules bearing a domain shared by the c-Src proto-oncogene (Src homology 2) are thought to be involved in signal transduction from activated receptor tyrosine kinases such as Neu. To test whether c-Src was implicated in Neu-mediated signal transduction, we measured the activity of the c-Src tyrosine kinase in tissue extracts from either mammary tumors or adjacent mammary epithelium derived from transgenic mice expressing a mouse mammary tumor virus promoter/enhancer/unactivated neu fusion gene. The Neu-induced mammary tumors possessed six- to eightfold-higher c-Src kinase activity than the adjacent epithelium. The increase in c-Src tyrosine kinase activity was not due to an increase in the levels of c-Src but rather was a result of the elevation of its specific activity. Moreover, activation of c-Src was correlated with its ability to complex tyrosine-phosphorylated Neu both in vitro and in vivo. Together, these observations suggest that activation of the c-Src tyrosine kinase during mammary tumorigenesis may occur through a direct interaction with activated Neu.

Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity.

Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20 to 30% of human breast cancers. Although the activation of Neu receptor tyrosine kinase appears to be a pivotal step during mammary tumorigenesis, the mechanism by which Neu signals cell proliferation is unclear. Molecules bearing a domain shared by the c-Src proto-oncogene (Src homology 2) are thought to be involved in signal transduction from activated receptor tyrosine kinases such as Neu. To test whether c-Src was implicated in Neu-mediated signal transduction, we measured the activity of the c-Src tyrosine kinase in tissue extracts from either mammary tumors or adjacent mammary epithelium derived from transgenic mice expressing a mouse mammary tumor virus promoter/enhancer/unactivated neu fusion gene. The Neu-induced mammary tumors possessed six- to eightfold-higher c-Src kinase activity than the adjacent epithelium. The increase in c-Src tyrosine kinase activity was not due to an increase in the levels of c-Src but rather was a result of the elevation of its specific activity. Moreover, activation of c-Src was correlated with its ability to complex tyrosine-phosphorylated Neu both in vitro and in vivo. Together, these observations suggest that activation of the c-Src tyrosine kinase during mammary tumorigenesis may occur through a direct interaction with activated Neu.

Activation of the c-Src tyrosine kinase is required for the induction of mammary tumors in transgenic mice.

Transgenic mice expressing the polyomavirus (PyV) middle T oncogene in the mammary epithelium develop multifocal mammary tumors that metastasize with high frequency. The potent transforming activity of PyV middle T antigen can, in part, be attributed to its ability to associate with and to activate a number of c-Src family tyrosine kinases (c-Src, c-Yes, and Fyn). As a first step toward assessing the role of individual c-Src family tyrosine kinases in PyV middle T antigen-induced mammary tumorigenesis, we have crossed transgenic mice carrying the mouse mammary tumor virus (MMTV)/PyV middle T antigen fusion gene with mice bearing a disrupted c-src proto-oncogene. In contrast to the rapid tumor progression seen in the original MMTV/PyV middle T antigen strains, mice expressing the transgene in the absence of functional c-Src rarely developed mammary tumors. After long latency, these mice did eventually develop abnormal hyperplastic mammary tissue. This growth disturbance was correlated with elevated expression of the PyV middle T antigen and the activation of the PyV middle T antigen-associated c-Yes tyrosine kinase. However, transgenic mice expressing the PyV middle T antigen in the mammary epithelium of wild-type or Yes-deficient mice developed multifocal mammary tumors with comparable kinetics. Taken together, these findings suggest that c-Src tyrosine kinase activity is required for PyV middle T antigen-induced mammary tumorigenesis and also illustrate an in vivo genetic approach to the dissection of mitogenic signal transduction pathways.

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.

Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis.

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.

Myristylation Is Required for Tyr-527 Dephosphorylation and Activation of pp60c-src in Mitosis

The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.

Increased tyrosine kinase activity of c-Src during calcium-induced keratinocyte differentiation.

In cultured human epidermal keratinocytes, induction of differentiation by Ca2+ and ionophore treatment was found to result in rapid elevation of c-Src tyrosine kinase activity and inactivation of the c-Yes tyrosine kinase. Activation of c-Src kinase was accompanied by tyrosine dephosphorylation, which might be explained by a rapid increase in intracellular protein-tyrosine phosphatase activity. Ca(2+)-induced differentiation was also associated with altered tyrosine phosphorylation of several cellular proteins and correlated with a marked redistribution of intracellular phosphotyrosine from membrane and adhesion sites to the nucleus. Some of the c-Src protein was also found in the nucleus after Ca2+ treatment, and Ca(2+)-activated c-Src bound to three cellular proteins (120 kDa, 65 kDa, and 34 kDa). In agreement with these results, immunohistochemistry on human epidermis revealed an increase in c-Src expression and tyrosine phosphorylation in cells undergoing differentiation, which strongly suggests a possible role of non-receptor tyrosine kinases in epithelial cell maturation.

Relationship between C-SRC Tyrosine Kinase Activity and the Control of Glucose Transporter Gene Expression

It has been shown previously that the rate of glucose transport in fibroblasts is accelerated by oncoproteins such as v-src, ras, and the transforming protein of feline sarcoma virus. This induction of glucose transport is associated with, and presumably caused by, induction of Hep-G2/rat brain glucose transporter gene expression. To determine the mechanism underlying the induction of glucose transporter gene expression by the v-src oncogene we studied cell lines that overexpress the normal counterpart of the v-src protein (c-src), or various mutants of the c-src protein. In these mutants, the tyrosines at positions 416, 527, or 519, or various combinations of these, have been replaced by phenylalanine by site directed mutagenesis, resulting in mutated c-src proteins that possess varying tyrosine kinase activity and transforming potential. Cells that overexpress the c-src protein show no changes in glucose transporter gene expression. However, when Tyr 527 in the COOH terminus of the c-src protein is replaced with Phe, the tyrosine kinase activity and transforming potential of the protein are increased and the protein acquires a potent ability to increase levels of glucose transporter mRNA and protein, as well as the rate of 2-deoxy-D-glucose uptake. This ability is abolished by the double mutation of Tyr to Phe in positions 416 and 527, which reduces the tyrosine kinase activity of the 527 single mutant. Thus, the ability of src proteins to induce expression of the glucose transport system is linked to the tyrosine kinase activity of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of pp60c‐src tyrosine kinase activity during secretion in stimulated bovine adrenal chromaffin cells

High levels of the proto-oncogene product, pp60c-src, have been found in developing and adult neural tissues as well as in certain fully mature cells of the hematopoietic lineage, e.g., platelets and myelomonocytes. Adrenal medullary chromaffin cells exhibit characteristics of both types of cells, i.e., they are derived from the neural crest and carry out exocytosis in response to specific stimuli. Earlier studies have shown that pp60c-src localizes not only to the plasma membrane of chromaffin cells but also to the membranes of chromaffin granules, the secretory vesicles of these cells that store catecholamines and other secretory products. To investigate the possible involvement of pp60c-src in exocytosis, cultured bovine chromaffin cells were analyzed for changes in c-src tyrosine kinase activity in response to stimulation by several secretagogues. Results of in-vitro immune complex kinase assays showed that pp60c-src, derived from cells that had been stimulated for various lengths of time, exhibited decreased auto- and transphosphorylating activities as compared to pp60c-src immunoprecipitated from control cells. The greatest reduction in activity was observed 10 min post-stimulation, while normal levels were regained 2-6 hr after secretagogue treatment. Western immunoblot analysis of the immunoprecipitated pp60c-src revealed that approximately 50% less c-src protein was present in immune complexes prepared 10 min after stimulation as compared to those prepared from mock-stimulated controls, resulting in a specific autophosphorylating activity that was 42-47% of control and little or no reduction in the transphosphorylating specific activity. In experiments in which the rate of secretion of [3H]-norepinephrine from cells preloaded with this compound was compared to the rate of modulation of pp60c-src activity, 50% of the maximal reduction in pp60c-src activity occurred within 2-4 min while 50% maximal release of [3H]-norepinephrine occurred within 1-3 min. Taken together, these results suggest that pp60c-src may play some role (direct or indirect) in the exocytotic process.

Effect of chemically well-defined sphingosine and its N-methyl derivatives on protein kinase C and src kinase activities

In view of the possible effects of the sphingoid base on protein kinases, and the fact that the sphingoid bases used in previous studies were not chemically well-defined, we have studied the effects of chemically well-defined sphingosines and their derivatives on kinase activity. Both (4E)-D- and (4E)-L-erythro-sphingenine showed a weak inhibitory effect, and (4E)-L-threo-sphingenine had a moderate inhibitory effect. In contrast, (4E)-N,N-dimethyl-D-erythro-sphingenine and the sphingosine preparation from a commercial source showed a strong inhibitory effect on PK-C in A431 cells as well as on purified PK-C. Synthetic (4E)-D-erythro-sphingenine and several samples of natural sphingosine inhibited v-src or c-src tyrosine kinase activity measured with polyglutamate-tyrosine (4:1) as substrate. N-Acetylated or N-methylated sphingosines did not inhibit src kinase activity, but rather produced a consistent 1.5-2-fold stimulation of such activity.

Protein kinase C promotes the phosphorylation of immunoprecipitated middle T antigen from polyoma-transformed cells

Stimulation of protein kinase C in polyoma virus-transformed cells increased the phosphorylation of tyrosine residues of the viral middle T (mT) antigen in mT:pp60 c-src complexes precipitated by anti-mT antibodies. This increase might have been due to a stimulation of the complex's pp60 c-src tyrosine kinase activity or to an increased ability of the mT protein to be phosphorylated by pp60 c-src . These observations suggest that cellular protein kinase C might control the ability of polyoma virus to transform its host cell.