©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation and Translocation of c-Src to the Cytoskeleton by Both Platelet-derived Growth Factor and Epidermal Growth Factor (*)

(Received for publication, November 17, 1994)

Paschal A. Oude Weernink (§) Gert Rijksen

From the Laboratory of Medical Enzymology, Department of Hematology, University Hospital Utrecht, NL-3508 GA Utrecht, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Src is the prototype and the most widely distributed member of the Src family of nonreceptor protein tyrosine kinases(1) . C-Src is the cellular homologue of the transforming protein of Rous sarcoma virus, v-Src. The contribution of c-Src to receptor tyrosine kinase signaling is still unclear. Best documented are the effects of PDGF (^1)receptor activation on c-Src. Treatment of fibroblasts with PDGF results in a small stimulation of c-Src kinase activity(2) . In addition, some 5-10% of the c-Src molecules becomes transiently associated with the PDGF receptor(3) . Two recently identified autophosphorylation sites in the juxtamembrane segment of the PDGF beta receptor (Tyr and Tyr) have been shown to mediate the binding to the SH2 domain of c-Src(4) . The inhibition of PDGF-stimulated entry of cells into S phase by microinjection of catalytically inactive forms of c-Src indicates the requirement of c-Src for PDGF-induced signal transduction (5) . Participation of c-Src in EGF receptor signaling was first indicated by the hyperresponsiveness of cells that overexpress c-Src to EGF as a mitogen(6) . Demonstration that recombinant proteins containing the Src SH2 domain bind to the activated EGF receptor and that endogenous c-Src co-precipitates with tyrosine-phosphorylated EGF receptor suggests that c-Src may also become directly associated to the receptor after EGF stimulation(7) . Recently, we could demonstrate that EGF treatment of EGF receptor overexpressing breast cancer cells resulted in a 2-fold increase of membrane-bound c-Src kinase activity (8) .

Src is attached to the inner face of the plasma membrane by means of an N-terminal myristoyl group. Association with the plasma membrane was shown to be essential for the transforming capacity of v-Src, the src gene product of Rous sarcoma virus(9, 10) . C-Src appears to be bound primarily to the phospholipids in the membrane and can be extracted with mild nonionic detergents. It was recently shown in fibroblasts that c-Src is mainly associated with endosomal membranes (11) . In contrast, the majority of the transforming v-Src has been found to be associated with cytoskeletal proteins and is resistant to detergent extraction(12) . The degree of this cytoskeletal localization correlates with the extent of cell transformation, suggesting that association of Src to the cytoskeleton is indispensable for morphological transformation(12, 13) . In platelets, thrombin-induced aggregation results in cytoskeletal reorganization and association of c-Src with the cytoskeleton(14, 15) . These observations indicate that cytoskeletal association of Src might be an essential part in Src signaling. Indeed, several cytoskeletal proteins have been identified as substrates of Src, including vinculin(16) , ezrin(17) , talin(18) , and p75(19) . We examined therefore whether c-Src also becomes associated to the cytoskeleton in growth factor receptor signaling. We now demonstrate that stimulation of glioblastoma cells with PDGF results in association of c-Src with the cytoskeleton and enhancement of c-Src kinase activity in the cytoskeleton fraction as well as in the cytosol and membrane fraction. Similar effects on c-Src activation were induced by EGF.


EXPERIMENTAL PROCEDURES

The human glioblastoma cell line A172 was obtained from the American Type Culture Collection (Rockville, MD) and was routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mML-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO(2) atmosphere at 37 °C. Monoclonal antibody (mAb) 327 producing hybridoma cells were kindly provided by Dr. Joan S. Brugge (University of Pennsylvania).

Growth Factor Stimulation and Cytoskeletal Isolation

Confluent cultures of A172 cells (5.10^6 cells in two 100-mm dishes per assay) were incubated for 48 h in Dulbecco's modified Eagle's medium supplemented with 0.5% fetal calf serum. The cells were stimulated for different time intervals with EGF (100 ng/ml; Collaborative Research, Bedford, MA) or PDGF-BB (25 ng/ml; Saxon Biochemicals GMBH, Hannover, Federal Republic of Germany), after which the dishes were placed on ice and rapidly washed with ice-cold PBS, including 100 µM sodium orthovanadate. Preparation of cytoskeleton fractions was essentially according to Hamaguchi and Hanafusa(12) . The cells were extracted on ice in 0.5 ml of CSK buffer for 3 min with gentle rocking, and a second time with 0.5 ml of fresh buffer for 1 min. CSK buffer contained 10 mM PIPES, pH 6.8, 250 mM sucrose, 150 mM KCl, 3 mM MgCl(2), 1 mM EGTA, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 50 units/ml aprotinin. The insoluble material remaining on the dish was scraped and solubilized in 1 ml of RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM NaH(2)PO(4), 5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 1 mM sodium orthovanadate, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 50 units/ml aprotinin). These fractions were centrifuged at 50,000 times g for 30 min and the supernatants used for immunoprecipitation.

Subcellular Fractionation by Differential Centrifugation

Alternatively, cells were separated in a cytosol, a membrane, and a cytoskeleton fraction. Cells were washed with ice-cold PBS, including 5 mM EDTA and 100 µM sodium orthovanadate. The cells were harvested by scraping in 800 µl of hypotonic lysis buffer (containing 10 mM HEPES, pH 7.5, 10 mM NaCl, 1 mM KH(2)PO(4), 5 mM NaHCO(3), 1 mM CaCl(2), 0.5 mM MgCl(2), 5 mM EDTA, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 50 units/ml aprotinin), transferred to precooled tubes, and lysed by ultrasonication (two bursts of 5 s on ice). Ultrasonication was proven not to alter the subcellular distribution of c-Src as compared with alternative lysis methods (freeze-thawing cycles or mechanical shear). The lysates were centrifuged at 50,000 times g for 60 min to collect cytosolic fractions. The pellets were extracted in 400 µl of lysis buffer supplemented with 1% Triton X-100 for 30 min. The solubilized membrane fractions were collected by centrifugation at 50,000 times g for 60 min. The remaining Triton-resistant material is referred to as the cytoskeleton fraction. These pellets were solubilized in 400 µl of RIPA buffer for 30 min, and any remaining insoluble material was removed by centrifugation. Prior to immunoprecipitation the detergent concentrations of the cytosol and membrane fractions were adjusted with concentrated RIPA buffer.

Immune Complex Kinase Assay and Immunoblotting

Src was immunoprecipitated from the isolated fractions with mAb 327 (5 µg/ml). Immune complexes were collected after overnight incubation with protein A-Sepharose beads by centrifugation at 12,000 times g for 5 min. The beads were rapidly washed three times with HNTG buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol), supplemented with 1 mM sodium orthovanadate and once with kinase buffer containing 20 mM HEPES, pH 7.5, 5 mM MgCl(2), 3 mM MnCl(2), and 1 mM sodium orthovanadate. For autokinase reactions the final pellets were resuspended in 40 µl of the kinase buffer, warmed to 20 °C, and incubated with 250 kBq of [-P]ATP (DuPont NEN) for 5 min. Kinase reactions toward enolase were carried out in the presence of acid-denatured enolase (3 µg) and additional 3 µM unlabeled ATP. Phosphorylation reactions were terminated by the addition of 70 µl of preheated 2 times concentrated electrophoresis sample buffer. The samples were heated at 95 °C for 5 min and the proteins separated by SDS-PAGE on 8% gels. Transfer of proteins to a polyvinylidene difluoride membrane was performed overnight with a Bio-Rad Trans-Blot apparatus. Following two rinses in PBS for 5 min, the filters were blocked in PBS containing 5% BSA (fraction V, essentially fatty acid-free, Sigma) for 1 h at room temperature. After washing with PBS, 0.1% BSA, the wet filters were covered with food wrap and incorporation of P was analyzed in a PhosphorImager (Molecular Dynamics) using the ImageQuant software. The same filters were successively probed with mAb 327 (1 µg/ml, 16 h), with rabbit anti-mouse Ig (Nordic Immunology, 1:1000, 1 h) and with I-labeled protein A (Amersham, 30 kBq/ml, 1 h), all diluted in PBS, 0.1% BSA. Finally, the filters were thoroughly washed with PBS, 0.1% BSA containing 0.3% Tween 20 (eight times for 10 min), PBS (twice for 5 min) and dried. Quantification of the I signal was performed in the PhosphorImager while shielding the P signal with a plastic covering.


RESULTS

PDGF Increases c-Src Activity at the Cytoskeleton

In this study we used A172 glioblastoma cells which were shown to possess both the PDGF beta-receptor (20) and the EGF receptor. (^2)Cells were serum-deprived for 48 h and treated with PDGF (25 ng/ml) for 10 min. Isolation of detergent-resistant fractions was performed as described under ``Experimental Procedures.'' Src was immunoprecipitated using mAb 327 and assayed for autokinase activity. Proteins were separated by SDS-PAGE, transblotted to PVDF filters, and the level of autophosphorylation was visualized and quantitated in a PhosphorImager (Fig. 1B). After immunoblotting with mAb 327 and I-labeled protein A, the amount of Src protein was analyzed (Fig. 1A). In our phosphorylation experiments we make use of the recently introduced isotope P. Since the maximum emission energy of P is significantly lower than that of P, simultaneous detection of I-labeled antibodies is now possible while shielding the beta-irradiation of the phosphorylated proteins. This enables us to analyze the amount of Src protein as well as the Src tyrosine kinase activity on one and the same PVDF filter. Fig. 1B shows that treatment of the cells with PDGF results in an increase of c-Src activity recovered in the cytoskeleton. Quantification of the P signal in the Src band reveals a 3.1-fold increase in the autophosphorylation rate. This increase in Src activity can partly be explained by a concomitant increase (1.4-fold) in the amount of c-Src protein present in the cytoskeleton (Fig. 1A).


Figure 1: PDGF-induced increase of c-Src activity at the cytoskeleton. Serum-deprived glioblastoma cells were treated with PDGF (25 ng/ml) for 10 min. The cells were extracted with 1% Triton X-100, and the remaining cytoskeleton fraction was scraped in RIPA buffer and immunoprecipitated with anti-Src mAb 327. The precipitates were assayed for autokinase assay, separated by SDS-PAGE, and transferred to PVDF filters. The filters were probed with mAb 327 and I-labeled protein A (A) or imaged directly for phosphate-labeled bands in a PhosphorImager (B). The position of c-Src is indicated by the arrow.



PDGF Induces Association of c-Src with the Cytoskeleton

Recently, PDGF was reported to induce c-Src translocation from the plasma membrane to the cytosol in an isolated plasma membrane system(21) . The translocation was accompanied by an exclusive activation of the soluble c-Src population. This observation prompted us to additionally investigate the c-Src activity and distribution in the cytosol and membrane fractions. Therefore we further fractionated the Triton-soluble fraction in a cytosol and a membrane fraction as described under ``Experimental Procedures.'' Serum-deprived cells were treated with PDGF for several time intervals up to 3 h. At each time point, the cells were lysed, fractionated in a cytosol, a membrane, and a cytoskeleton fraction, and assayed for the presence and tyrosine kinase activity of c-Src. Fig. 2shows a typical result of these experiments. Most of the c-Src protein is recovered in the membrane fraction. Only trace amounts of c-Src are present in the cytoskeleton fraction. Upon stimulation with PDGF, there is a marked increase in the amount of c-Src attached to the cytoskeleton (Fig. 2, left panel). The relocalization to the cytoskeleton does not appear to be a very early response but is observed 20 min following PDGF treatment. Analysis of the corresponding immune complex kinase assays shows a concomitant increase of the c-Src tyrosine kinase activity in the cytoskeleton fraction (Fig. 2, right panel).


Figure 2: Subcellular distribution and autokinase activity of c-Src following PDGF stimulation of glioblastoma cells. Cells were stimulated with PDGF for the times indicated and subsequently lysed and fractionated in a cytosol (A), a membrane (B), and a cytoskeleton fraction (C) as described under ``Experimental Procedures.'' C-Src was immunoprecipitated from each fraction with mAb 327 and assayed for autophosphorylation using P-labeled ATP as a phosphate donor. After SDS-PAGE and transfer of the proteins to PVDF filters, phosphate incorporation was analyzed in a PhosphorImager (right panel). The distribution of c-Src protein was imaged after immunoblotting with I-labeled mAb 327 while shielding the P beta-irradiation (left panel). The position of c-Src is indicated by the arrow.



We quantified the effects of PDGF on both the subcellular distribution as well as the tyrosine kinase activity of c-Src by scanning a series of blots on the PhosphorImager and calculated specific enzyme activities. Fig. 3shows the distribution of c-Src over cytosol, membrane, and cytoskeleton after PDGF treatment. Increased binding of c-Src to the cytoskeleton is observed as early as 2 min following growth factor treatment; it reaches a maximum at 20 min and only very slowly declines up to the last time point (3 h). At 20 min the amount of c-Src associated to the cytoskeleton is increased 3.5-fold as compared with nonstimulated cells. The increase in the recovery of c-Src in the cytoskeleton fraction (Triton X-100-insoluble) runs parallel to a decrease of c-Src in the membrane fraction (Triton X-100-soluble).


Figure 3: PDGF induces association of c-Src with the cytoskeleton. Cells were stimulated with PDGF for several time intervals, and the subcellular distribution of c-Src protein was determined by immunoblotting with I-labeled mAb 327.



PDGF Increases c-Src Specific Activity in All Three Subcellular Fractions

Besides a redistribution of c-Src, PDGF induces an increase in the specific c-Src kinase activity in all three subcellular fractions (Fig. 4). The specific c-Src tyrosine kinase activity was calculated by standardizing the P incorporation into c-Src itself or, alternatively, into enolase for the amount of c-Src protein as assessed by immunoblotting with I-labeled mAb 327. In all experiments the autokinase reactions and enolase assays gave comparable kinase activation factors. Membrane-bound Src is activated about 4-fold; its activation reaches a maximum 60 min after PDGF treatment and continues for at least 3 h (Fig. 4). Both cytosolic and cytoskeleton associated c-Src are activated about 2-fold; the activation of these two c-Src pools appear to be more transient as compared with c-Src from the membrane fraction. Table 1summarizes the effects of PDGF treatment (20 min) on both c-Src protein distribution and c-Src-specific enzyme activity and shows the total c-Src activity, i.e. the resultant of c-Src protein redistribution and effect on c-Src specific activity. Most striking is the increase in cytoskeletal associated c-Src kinase activity by PDGF (6.3-fold).


Figure 4: PDGF increases the c-Src specific kinase activity in all three subcellular fractions. Cells were stimulated with PDGF for several time intervals and the subcellular distribution of c-Src protein as well as c-Src autokinase activities were determined. The specific c-Src tyrosine kinase activity was calculated by standardizing the phosphate incorporation into c-Src for the amount of recovered c-Src protein.





Also EGF Induces Activation and Redistribution of c-Src

Interestingly, similar effects on c-Src activation are observed after stimulation of A172 glioblastoma cells with EGF. Like PDGF, EGF (20 min; 100 ng/ml) also induces an increase in c-Src tyrosine kinase activity in all three subcellular fractions, albeit with lower orders of magnitude. The increase in total c-Src activity is 1.9-fold (cytosol), 1.5-fold (membrane), and 2.4-fold (cytoskeleton). In addition, also an increase in the amount of c-Src protein recovered in the cytoskeleton fraction is observed (data not shown).


DISCUSSION

Thrombin-induced platelet activation has been reported to result in an increase in c-Src kinase activity (22, 23) and association of c-Src with the cytoskeleton(14, 22) . Our results indicate that similar events occur in receptor tyrosine kinase signaling in nucleated cells. The purpose of the localization of activated c-Src at the cytoskeleton in response to growth factor stimulation is not clear. Several structural and particularly cytoskeletal proteins have been identified as substrates for Src family kinases, suggesting that Src kinases are required for changes in the cytoskeletal network(1) .

Recently, PDGF-induced translocation of c-Src from the plasma membrane to the cytosol has been described(21) . However, in that study most experiments were performed in a cell-free plasma membrane system and the presence of c-Src in the cytoskeleton fraction was not investigated. Our experiments do not confirm a release of c-Src to the cytosol upon growth factor stimulation.

The intriguing question is now by what mechanism PDGF and EGF induce activation and translocation of c-Src to the cytoskeleton. It has been suggested that amino-terminal serine phosphorylation is necessary but not sufficient for dissociation of c-Src from the plasma membrane(21, 24) . Recently it was reported that the SH2 domain, but not the SH3 domain, mediates cytoskeletal association of v-Src(25) . Interestingly, in the same study c-Src was shown to gain the ability to associate with the cytoskeleton upon removal of the entire C terminus, including the catalytic domain. The acquisition of cytoskeletal binding could be contributed to the loss of Tyr, the negative regulatory phosphorylation site of c-Src(25) . In vivo, c-Src is highly phosphorylated at Tyr, and folding of this phosphotyrosine residue into the SH2 domain of the same molecule is believed to down-regulate the tyrosine kinase activity of c-Src(26) . It is attractive to hypothesize that PDGF and also EGF induce unfolding of the c-Src molecules, resulting in an increase in the catalytic activity and, in addition, in the exposure of the cytoskeletal binding site.

There is now compelling evidence that part of the EGF receptor population itself is directly associated to the cytoskeleton(27, 28) . Also the activities of various other components involved in signal transduction, including phosphoinositide kinases, diacylglycerol kinase, and phospholipase C-1, are increasingly associated to the cytoskeleton upon EGF stimulation(29) . In hepatocytes, EGF-induced translocation of phospholipase C-1 to the cytoskeleton is highly correlated with its tyrosine phosphorylation and increased catalytic activity(30) . It has been hypothesized that the cytoskeleton provides a matrix where several signaling events are orchestrated. Our results now show that also c-Src is one of the components to become activated and associated to the cytoskeleton in response to both PDGF and EGF stimulation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Hematology, Laboratory of Medical Enzymology, University Hospital Utrecht, P. O. Box 85500, NL-3508 GA Utrecht, The Netherlands. Tel.: 31-30-507602; Fax: 31-30-511893.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; EGF, epidermal growth factor; SH, Src homology; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PVDF, polyvinylidene difluoride; RIPA, radioimmune precipitation buffer; PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
P. A. Oude Weernink and G. Rijksen, unpublished observation.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.