(Received for publication, November 17, 1994)
From the
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.
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 ()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
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.
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 atmosphere at 37 °C.
Monoclonal antibody (mAb) 327 producing hybridoma cells were kindly
provided by Dr. Joan S. Brugge (University of Pennsylvania).
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.
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
-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.
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.
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.