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Molecular Cancer Research 1:428-437 (2003)
© 2003 American Association for Cancer Research

Cancer Genes and Genomics

Src-Dependent Association of Cas and p85 Phosphatidylinositol 3'-Kinase in v-crk-Transformed Cells1

Rebecca B. Riggins1, Regina M. DeBerry2, Maziar D. Toosarvandani1 and Amy H. Bouton1

1 Department of Microbiology and Cancer Center, University of Virginia Health System, Charlottesville, VA and
2 United States Patent and Trademark Office, Arlington, VA

Requests for reprints: Amy H. Bouton, Department of Microbiology and Cancer Center, University of Virginia Health System, PO Box 800734, Charlottesville, VA 22908. Phone: (434) 924-2513; Fax: (434) 982-1071. E-mail: ahb8y{at}virginia.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Cellular changes associated with oncogenic transformation are generally caused by deregulation of signal transduction pathways. We show that, in cells transformed by the v-crk oncogene, the adapter protein Cas forms a stable complex with the p85 regulatory subunit of phosphatidylinositol 3'-kinase (PI3K) coincident with the appearance of Cas-associated PI3K activity. The interaction between Cas and p85 PI3K appears to be driven primarily by Src-dependent tyrosine phosphorylation of Cas, and mapping studies indicate that the carboxyl terminus of Cas is necessary and sufficient for binding to p85 PI3K. One of the cellular effects of v-Crk expression is to promote DNA synthesis in the presence of low serum. This effect is potentiated in Cas-null fibroblasts when wild-type Cas is expressed, but not when a Cas variant is expressed that lacks the carboxyl-terminal p85 PI3K binding region. This suggests that the association of Cas with p85 PI3K may play a role in uncoupling growth regulatory pathways through v-Crk.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Communication with the external environment is crucial for the development and survival of multicellular organisms. Many of the signaling cascades that participate in this process involve regulated protein-protein interactions, mediated in part by adapter proteins that serve as molecular scaffolds. p130CAS (Cas) is an adapter protein the function of which is implicated in multiple signal transduction pathways, including those involved in integrin-mediated cell adhesion, migration, cellular transformation, mitogenic signaling through cell surface receptors, and differentiation (for review, see Refs. 1, 2). Cas was originally identified as a tyrosine-phosphorylated protein of Mr 130,000 in cells expressing the oncogenes v-crk or v-src (3–6). It contains several motifs that mediate protein-protein interactions, including a Src-homology 3 (SH3) domain, a substrate-binding domain consisting of multiple putative Src-homology 2 (SH2) binding sites, and a carboxyl terminus that contains a bipartite binding site for the protein tyrosine kinase (PTK) Src (6).

To date, numerous binding partners of Cas have been identified (for a review, see Ref. 1). While the functional significance of many of these associations is unclear, interactions between Src and Cas have been convincingly shown to play a crucial role in v-Src-mediated cellular transformation. Cas is an established substrate and binding partner of v-Src as well as its non-oncogenic counterpart c-Src (7–9). Cas is heavily tyrosine phosphorylated in v-src-transformed cells and fibroblasts isolated from Cas-/- embryos are resistant to transformation by v-Src (10). Moreover, the association of Cas with non-oncogenic c-Src results in an increase in c-Src kinase activity, coincident with the acquisition of growth properties characteristic of transformed cells (11, 12).

A second binding partner of Cas is v-Crk (4, 5). This oncoprotein is a viral fusion protein that is composed of SH2 and SH3 domains derived from cellular c-Crk proteins. Although it does not contain catalytic domains, transformation by v-Crk involves activation of cellular PTKs (4, 5). There is considerable evidence that suggests both Cas and c-Src function in pathways leading to transformation by this oncoprotein. c-Src activity is increased 3- to 4-fold in v-crk-transformed cells, coincident with Cas hyperphosphorylation and redistribution to membrane fractions of the cell (6, 13–15). Recent studies have shown that transformation by v-Crk enhances cell survival through a pathway involving phosphatidylinositol 3'-kinase (PI3K) and AKT (16, 17). The relationship between elevated c-Src activity, increased phosphorylation of Cas, and PI3K-mediated cell survival is currently unknown. However, several lines of evidence suggest that Cas may contribute to cellular events that involve PI3K. For example, the amino-terminal SH2 domain of the p85 regulatory subunit of PI3K can bind to Cas isolated from adherent cells (18, 19). Furthermore, this regulatory subunit has been reported to associate with Cas during the process of adenovirus entry (20).

PI3K can become activated upon recruitment to the plasma membrane via interactions between the SH2 domains contained within the p85 regulatory subunit and phosphotyrosine (pTyr) residues located on membrane-bound receptors (21). We hypothesized that PI3K-dependent survival pathways may be activated in v-crk-transformed cells through an analogous mechanism that involves PI3K recruitment to the membrane by Cas, which is highly phosphorylated and membrane associated in these cells. In this study, we show that Cas and p85 PI3K do in fact form a molecular complex in cells transformed by v-Crk, and that PI3K activity is associated with Cas under these conditions. These interactions are dependent on the kinase activity of Src and the phosphorylation state of Cas. Consistent with this finding, the SH2 domains of p85 PI3K are capable of directly binding to Cas isolated from v-crk-transformed cells. We also provide evidence that Cas-p85 PI3K interactions in v-crk-transformed cells are mediated through the carboxyl terminus of Cas. However, the proline-rich motif located in this region, which was shown to be necessary for Cas-p85 PI3K association during adenovirus entry (20), appears to be dispensable for this interaction in v-crk-transformed cells. Finally, we demonstrate that Cas-p85 PI3K interactions may contribute to a deregulation of proliferative pathways in the presence of v-Crk. Taken together, these data are consistent with a model in which Src family kinases are activated in the presence of v-Crk, inducing phosphorylation and relocalization of Cas to the plasma membrane. Subsequent interactions between p85 PI3K and Cas could then effectively recruit PI3K activity to the membrane, resulting in activation of cell growth and survival pathways. In this way, Cas-p85 PI3K interactions may play a critical role in v-Crk-induced transformation and cell survival.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Cas-p85 PI3K Complex Formation in v-crk-Transformed Cells
Transformation by v-Crk is coincident with activation of cellular PTKs and phosphorylation of several proteins including Cas (Fig. 1A, lanes 2 and 4) (6, 14). Phosphorylation of Cas leads to the generation of multiple docking sites for SH2-containing proteins and a redistribution of Cas from soluble to membrane fractions (6). We hypothesized that PI3K may be recruited to the membrane in cells expressing v-Crk through an interaction between the SH2 domains present in the p85 regulatory subunit and pTyr residues located on Cas. To determine whether these two molecules do in fact associate, Cas and p85 PI3K immune complexes were isolated from parental and v-crk-transformed 3Y1 cell extracts and examined for the presence of the reciprocal proteins (Fig. 1B). p85 PI3K was not detected in Cas immune complexes generated from parental 3Y1 cell extracts (lane 2), nor was Cas detected in p85 immune complexes (lane 4). In contrast, both proteins were present in reciprocal immune complexes generated from 3Y1-Crk cell extracts (lanes 6 and 8). Densitometric analysis indicated that approximately 1.5% of total cellular Cas was present in p85 PI3K immune complexes isolated from 3Y1-Crk cell extracts, and about 2–3% of total cellular p85 PI3K was detected in Cas immune complexes. The species of Cas that was found to be associated with p85 PI3K exhibited reduced electrophoretic mobility (upper panel, lane 8), characteristic of hyperphosphorylated Cas (6), suggesting that the pool of Cas associated with p85 PI3K in cells expressing v-Crk was heavily tyrosine phosphorylated.



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  		FIGURE 1. Cas-p85 PI3K binding correlates with Cas-associated PI3K activity in 3Y1-Crk cells. (A) Transformation by v-Crk is coincident with tyrosine phosphorylation of Cas. Twenty-five micrograms of 3Y1 and 3Y1-Crk cell extract were separated by SDS-PAGE (lanes 3 and 4) and immunoblotted for pTyr (top panel) and Cas (bottom panel). Three hundred micrograms of the same cell extracts were immunoprecipitated for Cas (lanes 1 and 2), divided in half, separated by SDS-PAGE, and immunoblotted for pTyr (top panel) and Cas (bottom panel) to confirm that the tyrosine-phosphorylated bands present at Mr 130,000 were in fact Cas. (B) Transformation by v-Crk is coincident with an association between Cas and p85 PI3K. One milligram of 3Y1 and 3Y1-Crk cell extract was incubated with the designated antibodies or normal rabbit serum (Ig), and immune complexes were recovered by protein A-Sepharose beads. One half of each immunoprecipitation was separated by 8% SDS-PAGE and immunoblotted with either Cas (top panel) or p85 PI3K antibodies (bottom panel). Total cell lysate (50 µg) was also analyzed (lanes 1 and 5). (C) PI3K activity is associated with Cas in 3Y1-Crk cells. The second half of each immunoprecipitation was analyzed for the presence of PI3K activity by TLC. The positions representing the origin and phosphatidylinositol 3-phosphate (PIP) product are indicated.

 
To determine whether PI3K activity was also associated with Cas in 3Y1-Crk cells, a portion of the immune complexes analyzed by immunoblot in Fig. 1B was subjected to an in vitro lipid kinase phosphorylation assay (Fig. 1C). While only negligible amounts of total PI3K activity were found to be associated with Cas in 3Y1 cells (lane 1), approximately 3% of total PI3K activity was present in Cas immune complexes isolated from 3Y1-Crk cells (lane 4). These data indicate that the Cas-p85 PI3K interactions detected in 3Y1-Crk cell extracts correlated with Cas-associated PI3K activity in these cells.

Cas Association With p85 PI3K Requires Src Activity
It has recently been reported that c-Src is one of the major PTKs responsible for Cas phosphorylation (8). Considering that hyperphosphorylated forms of Cas were found to associate with p85 PI3K in 3Y1-Crk cells (Fig. 1), we hypothesized that Cas-p85 PI3K interactions may be dependent on c-Src activity. To determine whether this was the case, 3Y1-Crk cells were treated with the pharmacological Src family kinase inhibitor PP2. p85 PI3K was then immunoprecipitated from these cell extracts and immunoblotted for Cas (Fig. 2A). The amount of Cas present in p85 PI3K immune complexes was significantly reduced following PP2 treatment (top panel, lanes 3–5), whereas the level of total Cas and p85 PI3K remained essentially unchanged (bottom panels). This decrease in Cas association was coincident with an inhibition of Cas phosphorylation in PP2-treated cells (Fig. 2B, top panel, lanes 3–5). However, the reduction in Cas phosphorylation was more modest than the dramatic decrease in Cas-p85 PI3K association that was observed following PP2 treatment. This is likely due to the fact that additional PTKs, particularly c-Abl, are also activated by v-Crk (21). Interestingly, phosphorylation by Src occurs initially at sites within the Cas carboxyl terminus, whereas c-Abl may be more likely to phosphorylate tyrosine residues located in the substrate-binding YXXP domain (Refs. 8, 22, and data not shown). On the basis of these data, it appears that Cas-p85 PI3K interactions can be largely inhibited in v-crk-transformed cells under conditions in which Src kinase activity is blocked, irrespective of the fact that phosphorylation of tyrosine residues by PP2-insensitive kinases such as c-Abl is maintained. This finding is supported by the fact that Cas mutants lacking the carboxyl-terminal Src phosphorylation sites but retaining the tyrosine residues in the substrate-binding YXXP domain are still heavily phosphorylated in cells expressing v-Crk (data not shown).



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  		FIGURE 2. Cas-p85 PI3K association is dependent on a PP2-sensitive kinase. (A) PP2 treatment results in decreased Cas-p85 PI3K association. 3Y1-Crk cells were treated with 10 µM PP2 for 0.5, 2, or 4 h. Five hundred micrograms of cell lysate were then immunoprecipitated with p85 PI3K antibodies, and complexes were immunoblotted for Cas (top panel) or p85 PI3K (middle panel). Fifty micrograms of cell lysate were also analyzed to confirm that PP2 treatment did not alter the expression level of Cas (bottom panel). (B) Inhibition of Src by PP2 results in Cas dephosphorylation. In an analogous experiment, 3Y1-Crk cells were treated with PP2 as described above. One milligram of cell lysate was immunoprecipitated for Cas, divided in half, and complexes were immunoblotted for pTyr (top panel) or Cas (bottom panel).

 
To directly test whether Src kinase activity was required for Cas-p85 PI3K association, we reconstituted this interaction in COS-1 cells by co-expressing plasmids encoding myc-tagged Cas together with plasmids encoding either wild-type or kinase-dead c-Src (Fig. 3). p85 PI3K was detected in myc immune complexes in the presence of overexpressed wild-type c-Src, but not when vector or kinase-dead c-Src was expressed (bottom panel, compare lane 5 with lanes 2 and 8). Similarly, Cas was only detected in p85 PI3K immune complexes under conditions in which wild-type c-Src was also present (top panel, compare lane 6 with lanes 3 and 9). The species of Cas that was observed to interact with p85 PI3K under these conditions was again the slower migrating form, consistent with hyperphosphorylation (lane 6). Taken together, these data indicate that Src kinase activity plays an important role in the association of Cas and p85 PI3K.



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  		FIGURE 3. Cas-p85 PI3K association requires Src kinase activity. Plasmids encoding wild-type myc-Cas were transfected into COS-1 cells together with vector (lanes 1–3), wild-type c-Src (lanes 4–6), or kinase-dead c-Src (Src KD) (lanes 7–9). Five hundred micrograms of cell lysate were incubated with either myc mAb 9E10 or polyclonal p85 PI3K antibodies. Immune complexes and 50 µg of total cell lysate were separated by 8% SDS-PAGE and immunoblotted with either Cas (top panel) or p85 PI3K (bottom panel) antibodies.

 
The finding that Cas-p85 PI3K association required Src kinase activity suggests that this interaction may be dependent on tyrosine phosphorylation of Cas. This is supported by the observation that the species of Cas found in association with p85 PI3K in v-crk-transformed cells exhibited a reduced electrophoretic mobility. The SH2 domains present in p85 PI3K have been shown to bind to pTyr residues located on receptor PTKs and other phosphoproteins (23). To determine whether Cas-p85 PI3K interactions could be mediated through one of these two SH2 domains, a glutathione S-transferase (GST) fusion protein containing both SH2 domains of p85 PI3K was incubated with extracts derived from 3Y1 or 3Y1-Crk cells (Fig. 4). Cas isolated from 3Y1-Crk-transformed cells and, to a lesser extent, parental 3Y1 cells, was found to associate with the GST-p85 SH2 fusion protein (lanes 3 and 7). Moreover, the Cas isolated from 3Y1-Crk cells that bound to the GST-p85 fusion protein exhibited a reduced electrophoretic mobility, consistent with a high pTyr content. In contrast, Cas did not show any interaction with either GST alone (lanes 2 and 6) or the SH2 domain of tensin, a non-Cas interacting protein (lanes 4 and 8). Collectively, these data support the idea that Cas and p85 PI3K associate under conditions in which Cas is heavily tyrosine phosphorylated, and that this interaction may be mediated through one of the two SH2 domains of p85 PI3K.



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  		FIGURE 4. Cas can associate with the SH2 domains of p85 PI3K. One microgram of the indicated GST-SH2 fusion proteins was immobilized on glutathione-Sepharose beads and incubated with 500 µg of 3Y1 or 3Y1-Crk cell extract. Bound proteins were separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with Cas antibodies. For comparison, 50 µg of total cell protein were also analyzed (lanes 1 and 5).

 
Cas Associates With p85 PI3K Through Its Carboxyl Terminus
Having shown that Cas-p85 PI3K association is dependent on Src activity and can occur through one of the SH2 domains of p85 PI3K, studies were initiated to identify the region of Cas responsible for binding to p85 PI3K. Tyrosine-containing motifs are present in two regions of Cas, the substrate-binding YXXP domain and the carboxyl terminus. To determine whether either of these regions of Cas is required for p85 PI3K association, 3Y1-Crk cells were transfected with plasmids encoding myc-tagged wild-type Cas, a Cas variant lacking the carboxyl terminus (Cas-{Delta}CT), or a Cas variant lacking the substrate-binding domain (Cas-{Delta}YXXP) (Fig. 5). In each case, c-Src was also overexpressed to enhance phosphorylation of the ectopic Cas variants. Under these conditions, p85 PI3K was found to associate with wild-type Cas and Cas-{Delta}YXXP (top panel, lanes 2 and 4), but not with Cas-{Delta}CT (lane 3), even though both Cas-{Delta}YXXP and Cas-{Delta}CT were expressed at similar levels (lower panel). This suggests that sequences found in the Cas carboxyl terminus may be important for binding to p85 PI3K.



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  		FIGURE 5. The Cas carboxyl terminus is required for association with p85 PI3K. 3Y1-Crk cells were cotransfected with plasmids encoding c-Src and myc-tagged wild-type Cas, Cas-{Delta}CT, or Cas-{Delta}YXXP. Five hundred micrograms of cell lysate were immunoprecipitated with p85 PI3K antibodies, and immune complexes were separated by 8% SDS-PAGE and immunoblotted with either myc (top panel) or p85 PI3K (middle panel) antibodies. Fifty micrograms of cell lysate were also analyzed for expression of the Cas constructs (bottom panel).

 
The carboxyl terminus of Cas contains a bipartite binding site for Src (24). The initial interaction between these molecules is thought to occur via the proline-rich motif RPLP642SPP of Cas and the Src SH3 domain. Subsequent phosphorylation of tyrosine residues within an adjacent YDYV motif of Cas then generates a second site of interaction for the Src SH2 domain (24). Because c-Src has also been reported to bind to p85 PI3K (25, 26), p85 PI3K association with Cas could either occur through a direct interaction with tyrosine-phosphorylated residue(s) in the carboxyl terminus of Cas, or via a tripartite complex utilizing c-Src as a molecular bridge. In support of the latter possibility, the proline-rich sequence of Cas to which the Src SH3 domain binds has been implicated in Cas-PI3K interactions that occur during the process of adenovirus entry (20).

To explore the role of c-Src in promoting Cas-p85 PI3K interactions, we sought to determine whether p85 PI3K associated with the carboxyl terminus of Cas under conditions in which c-Src could not bind to Cas. COS-1 cells were co-transfected with plasmids encoding wild-type or kinase-dead c-Src and either the independently expressed myc-tagged Cas carboxyl terminus (Cas-CT), or a variant of Cas-CT that contains an amino acid substitution in the Src SH3-binding site that blocks stable Cas-Src interactions (Cas-CTP642A) (11). In the presence of wild-type c-Src, myc immune complexes derived from Cas-CT-expressing cells contained both p85 PI3K (Fig. 6A, lane 2, top panel) and c-Src (bottom panel). Thus, the carboxyl terminus of Cas is sufficient for binding to p85 PI3K. The association between Cas and p85 PI3K was dependent on c-Src activity, as expression of kinase-dead c-Src did not promote Cas-CT-p85 PI3K interactions (lane 6). c-Src immune complexes isolated from cells expressing wild-type c-Src also contained detectable levels of p85 PI3K (top panel, lane 4), but this was not the case in the presence of kinase-dead c-Src (lane 8). The P642A mutation effectively blocked Src-Cas interactions, as shown by the absence of Src in myc immune complexes generated from Cas-CTP642A-expressing cells (Fig. 6B, bottom panel, lane 2). However, these complexes still contained p85 PI3K (lane 2, top panel). Thus, whereas the P642A substitution prevented the association between Cas and c-Src, it had no such inhibitory effect on Cas-p85 PI3K interactions. Moreover, there was a reduced amount of p85 PI3K in the c-Src immune complex under these conditions (lane 4), suggesting that the inability of c-Src to bind to Cas may affect c-Src-p85 PI3K interactions. Overall, these data indicate that interactions between Cas-CT and p85 PI3K do not appear to require a physical interaction between Cas and c-Src. In fact, the converse may be true, in that c-Src-p85 PI3K interactions may be dependent on the establishment of a stable Cas-Src complex.



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  		FIGURE 6. The proline-rich carboxyl-terminal Src binding site on Cas is not required for p85 PI3K association. Plasmids encoding the myc-tagged carboxyl terminus of Cas (Cas-CT) (A), or a variant containing a mutation in the proline-rich Src binding motif of Cas (Cas-CTP642A) (B), were cotransfected with plasmids encoding wild-type or kinase-dead c-Src into COS-1 cells. Five hundred micrograms of cell lysates were immunoprecipitated with either myc, p85 PI3K, or Src antibodies as indicated, and immune complexes along with 50 µg of whole cell lysate were separated by 8% SDS-PAGE and immunoblotted for p85 PI3K (top panels) or c-Src (bottom panels).

 
Direct Binding Between the SH2 Domains of p85 and Cas
To verify that p85 PI3K and Cas could directly interact without the need for c-Src as a molecular bridge, a Far Western filter binding assay was performed. Proteins isolated from either 3Y1 cells, 3Y1-Crk cells, or COS-1 cells transfected with plasmids encoding independently expressed Cas-CT in the presence or absence of overexpressed wild-type c-Src were separated by gel electrophoresis, transferred to nitrocellulose, and incubated with either GST, GST-p85-SH2, or GST-Crk-SH2 fusion proteins. The GST-p85-SH2 fusion protein was observed to bind to endogenous Cas isolated from v-crk-transformed cells, but not parental 3Y1 cells (Fig. 7, compare lanes 5 and 6). This fusion protein also bound to Cas-CT expressed in COS-1 cells, but only in the presence of overexpressed, wild-type c-Src (compare lanes 7 and 8). The specificity of the p85 PI3K-Cas-CT interaction was confirmed by probing these same lysates with the Crk-SH2 domain, which associates with Cas via the YXXP substrate-binding domain, and not the carboxyl terminus (6). The GST-Crk-SH2 fusion protein bound to endogenous Cas from 3Y1-Crk cells (lane 10), but showed no significant interaction with Cas isolated from 3Y1 cells (lane 9) or Cas-CT expressed in COS-1 cells (lane 12). GST control molecules were not found to bind to either endogenous Cas or Cas-CT (lanes 1–4). These data suggest that the interaction between p85 PI3K and the Cas carboxyl terminus is direct, and while c-Src may not be functioning as a molecular bridge, it appears to be required for Cas-p85 PI3K association.



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  		FIGURE 7. Direct binding of the SH2 domains of p85 PI3K with Cas. Fifty micrograms of lysates derived from 3Y1, 3Y1-Crk, or COS-1 cells transfected with plasmids encoding Cas-CT in the absence or presence of wild-type c-Src were separated by 8% SDS-PAGE and transferred to nitrocellulose. A Far Western filter binding assay was then performed to assess binding to GST (lanes 1–4), GST-p85-SH2 (lanes 5–8), or GST-Crk-SH2 (lanes 9–12). Interactions were detected by immunoblotting with GST antibodies.

 
p85 PI3K Association With Cas Contributes to v-Crk-Dependent BrdUrd Incorporation in Low Serum
Thus far, it has been demonstrated that Cas and p85 PI3K associate in v-crk-transformed cells through an interaction that requires Src activity and the Cas carboxyl terminus. Cas, Src, and p85 PI3K have all been shown to participate in pathways leading to transformation by v-Crk (6, 13–17), but the potential significance of Cas-p85 PI3K interactions in this process has not been investigated. One of the hallmarks of cellular transformation is proliferation in the presence of low serum. To determine if Cas-p85 PI3K interactions were important for this aspect of v-Crk transformation, Cas-null fibroblasts were plated on coverslips and transiently transfected with plasmids encoding v-Crk, or v-Crk in combination with either wild-type Cas that has been shown to associate with p85 PI3K or Cas-{Delta}CT that does not interact with p85 PI3K (see Fig. 5). The cells were then incubated in low-serum media [0.05% fetal bovine serum (FBS)] for 45 h, after which 5'-bromodeoxyuridine (BrdUrd) was added to the media for 3 h. The percentage of transfected cells that entered into S phase during this 3-h window was then determined by immunostaining for both v-Crk and BrdUrd (Fig. 8). In low serum, only 3% of vector-transfected cells were observed to incorporate BrdUrd. v-Crk expression dramatically increased this level to approximately 27%. When wild-type Cas was co-expressed with v-Crk, the number of BrdUrd-positive cells rose to an average of 48%, a statistically significant increase over cells expressing v-Crk alone. Interestingly, co-expression of v-Crk and Cas-{Delta}CT, which could not bind to p85 PI3K, was not able to enhance BrdUrd incorporation under these conditions.



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  		FIGURE 8. v-Crk-mediated BrdUrd incorporation in 0.05% serum is enhanced by Cas, but not Cas-{Delta}CT. Cas-null fibroblasts plated on FN-coated coverslips were transfected with plasmids encoding v-Crk alone or in combination with Cas or Cas-{Delta}CT, incubated in low-serum media for 45 h, then allowed to incorporate BrdUrd for 3 h. The cells were fixed, immunostained for v-Crk and BrdUrd, and visualized by fluorescence microscopy to determine the percentage of transfected cells that had entered S phase (incorporated BrdUrd). Data are presented as the mean ± SD for four independent experiments. See "Materials and Methods" for a discussion of statistical analyses. * denotes a statistically significant difference from v-Crk-expressing cells, and # indicates a statistically significant difference from v-Crk plus Cas co-expressing cells. {square}, vector; {blacksquare}, v-Crk; {blacksquare}, v-Crk + Cas; {blacksquare}, v-Crk + dlCT.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Tyrosine phosphorylation of Cas creates multiple SH2 domain docking sites that are involved in the recruitment of signaling proteins. In this report, we show that one molecule that associates with Cas in this manner is the p85 regulatory subunit of PI3K. Approximately 2–3% of cellular p85 PI3K was found in complex with the hyperphosphorylated form of Cas in v-crk-transformed cells, and this directly correlated with Cas-associated PI3K activity. The interaction between Cas and p85 PI3K was shown to be driven primarily by c-Src-dependent tyrosine phosphorylation of Cas. Because c-Src and p85 PI3K both interact with Cas through its carboxyl terminus, we explored the possibility that Cas and p85 PI3K interactions were indirect and mediated through co-association with c-Src. This does not appear to be the case, however, because Cas-p85 PI3K complexes were detected in the absence of a physical interaction between c-Src and Cas. In addition, filter binding assays indicated that a fusion protein containing the two SH2 domains of p85 PI3K could directly bind to Cas. We propose that the association of Cas with p85 PI3K may play a key role in transformation by v-Crk. This is supported by the finding that expression of wild-type Cas was found to enhance v-Crk-dependent BrdUrd incorporation into Cas-null cells, whereas a Cas molecule deficient for p85 PI3K binding (Cas-{Delta}CT) was incapable of doing so.

Transformation by v-Crk proceeds, at least in part, through activation of Src family kinases, leading to the tyrosine phosphorylation of several cellular proteins including Cas (5, 15, 27). Our data indicate that c-Src may play a catalytic role in the assembly of Cas-p85 PI3K complexes and that this interaction is dependent on phosphorylation of Cas. First, treatment of v-crk-transformed cells with the Src family kinase inhibitor PP2 resulted in an inhibition of Cas phosphorylation coincident with decreased association of Cas and p85 PI3K. Second, the species of Cas found in complex with p85 PI3K in 3Y1-Crk cell extracts exhibited a reduced electrophoretic mobility, characteristic of a hyperphosphorylated form of Cas (6, 14). Third, the interaction between endogenous p85 PI3K and ectopic Cas in COS-1 cells was absolutely dependent on co-expression of catalytically active c-Src. We have previously shown that, when Cas and c-Src are overexpressed in this way, c-Src is activated and ectopic Cas becomes heavily phosphorylated on tyrosine (8, 11, 12). Finally, a fusion protein containing the two SH2 domains of p85 PI3K was shown to interact with Cas in both a GST pulldown assay and a filter binding assay. Taken together, these data suggest that Src kinase activity may target Cas in the context of v-crk-transformation, resulting in phosphorylation of potential binding sites for p85 PI3K. It is interesting to note that, while the Src-specific inhibitor PP2 dramatically decreased Cas-p85 PI3K association, Cas phosphorylation was decreased to a much lesser degree (Fig. 2). This may be explained by the fact that v-Crk transformation also results in the activation of c-Abl, which is capable of phosphorylating Cas (21, 22). This raises the possibility that c-Src and c-Abl are responsible for the phosphorylation of different tyrosine residues within Cas, and that it is Src that specifically targets the p85 PI3K binding sites.

One of the hallmarks of cellular transformation is the ability to grow in low serum. Assays performed in Cas-null cells under these conditions revealed that wild-type Cas, but not Cas lacking the carboxyl terminus, enhanced the incorporation of BrdUrd induced by v-Crk. In contrast to v-Src, which is completely unable to transform cells in the absence of Cas (10), v-Crk was able to promote BrdUrd incorporation in low serum in Cas-null cells. This suggests that v-Crk can promote aspects of cellular transformation independently of Cas. However, when these cells were reconstituted with wild-type Cas, v-Crk-induced BrdUrd incorporation was significantly enhanced. This was not the case with Cas-{Delta}CT, which is deficient in p85 PI3K binding, suggesting that the Cas-p85 PI3K interaction may be important for this aspect of v-Crk transformation. Collectively, these data support a model in which the activation of Src leads to tyrosine phosphorylation of Cas, recruitment of p85 PI3K to the carboxyl terminus of Cas, and an enhancement of serum-independent growth in response to v-Crk expression.

Mapping studies in 3Y1-Crk and COS-1 cells revealed that the carboxyl terminus of Cas was both necessary and sufficient for binding to p85 PI3K. It is well established that the Cas carboxyl terminus contains binding sites for the SH3 and SH2 domains of Src (24, 28). Because c-Src is also reported to interact with p85 PI3K (25, 26), the possibility existed that c-Src could function as a molecular bridge that effectively recruits Cas and p85 PI3K to the same molecular complex. However, this is unlikely because mutation of the proline-rich Src SH3 domain binding site in the carboxyl terminus of Cas (Cas-CTP642A) did not disrupt p85 PI3K binding, while Cas-Src association was completely ablated. In fact, our data suggest that c-Src-p85 PI3K interactions may be dependent on the presence of stable Src-Cas complexes.

Src kinase activity may also play a role in promoting interactions between the p85 subunit of PI3K and the focal adhesion kinase, Fak, in chicken embryo fibroblasts (CEFs) (29). Fak appears to participate in PI3K signaling pathways induced by v-Crk, because activation of the PI3K effector AKT did not occur in Fak-/- mouse embryo fibroblasts expressing v-Crk. In vitro, the NH2-terminal SH2 domain of p85 PI3K was found to associate with Fak. Interestingly, Akagi et al. (29) failed to demonstrate an interaction between Cas and the NH2-terminal SH2 domain of p85 PI3K. This could be due to the absence of the COOH-terminal SH2 domain from the GST fusion protein used in their studies. Alternatively, v-Crk-transformed chicken embryo fibroblasts may exhibit lower levels of tyrosine-phosphorylated Cas than 3Y1 rat fibroblasts. Nonetheless, Cas may still function in Fak-dependent pathways of PI3K/AKT activation in v-crk-transformed cells through its ability to activate Src and thus promote Fak-p85 PI3K interactions.

The finding that Cas and p85 PI3K can interact in the absence of a functional Src SH3 domain binding motif (P642A) is particularly interesting in light of a recent report that adenovirus binding and entry into host cells induced an association between Cas and p85 PI3K that required the same proline-rich motif used by Src (PLP642SPP) (20). In that report, mutations in this region were shown to inhibit interactions with the SH3 domain of p85 PI3K, leading to a concomitant decrease in adenovirus entry. However, this motif was not sufficient for p85 PI3K binding, because adenovirus-induced interactions between p85 PI3K and wild-type Cas were inhibited in the presence of inhibitors of Src family tyrosine kinases. Our data suggest that, at least under conditions in which c-Src is activated, the PLPSPP motif in Cas does not appear to play a significant role in Cas-p85 PI3K binding. This emphasizes the fact that the mechanism of interaction between Cas and p85 PI3K may depend on the nature of the physiological conditions that induce association.

Several in vitro studies have suggested that, in addition to adenovirus entry, Cas-p85 PI3K association may occur in response to signals initiating from the extracellular matrix (ECM) (18, 19). Our work now provides evidence for a third physiological condition in which this complex forms. A recent study found that PI3K was constitutively activated in v-crk-transformed cells (16). The authors suggested that a component of this activation may be mediated by the interaction of one of the p85 PI3K SH2 domains with a protein that becomes tyrosine phosphorylated as a consequence of v-Crk expression. Our data suggest that one of these SH2 ligands may be Cas. A potential consequence of Cas/p85-PI3K association may be the direct activation of PI3K, because disinhibition of PI3K has been reported to occur through the engagement of p85 PI3K SH2 domains (30). A second potential outcome of PI3K association with Cas may be translocation of PI3K to the membrane, because the most highly phosphorylated forms of Cas are present in the membrane fraction of v-crk-transformed cell extracts (6, 14). Membrane targeting of PI3K has been shown to correlate with activation of downstream effectors such as AKT, and as discussed above, AKT has been reported to be constitutively activated in v-crk-transformed cells (17, 31). Finally, Cas may also function to activate Src in v-crk-transformed cells, which could then lead to AKT activation through Fak-p85 PI3K interactions. Collectively, Cas may serve to increase localized PI3K activity in the cell and either directly or indirectly activate downstream effectors such as AKT. Further studies are needed to investigate the link between Cas-p85 PI3K association and AKT-mediated cell survival pathways that function in v-crk-transformed cells.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Antibodies and Reagents
Polyclonal and monoclonal (mAb) Cas antibodies have been described previously (32). p85 PI3K polyclonal antibodies and pTyr mAb 4G10 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Myc mAb 9E10 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-gag mAb and polyclonal antisera recognizing p47gag-Crk (v-Crk), and Src mAbs EC10 and 2–17, were generously provided by S.J. Parsons (University of Virginia, Charlottesville, VA). GST mAb 9D9 was a gift from J.T. Parsons (University of Virginia). Protein A-Sepharose CL-4B, horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse immunoglobulin, and 125I-protein A were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). 125I-goat anti-mouse immunoglobulin was purchased from NEN Life Science Products, Inc. (Boston, MA). Texas Red-conjugated anti-mouse immunoglobulin was purchased from Molecular Probes (Eugene, OR). PP2 was purchased from Calbiochem (San Diego, CA). BrdUrd and fibronectin were purchased from Sigma Chemical Co. (St. Louis, MO). FITC-conjugated anti-BrdUrd was purchased from Chemicon International, Inc. (Temecula, CA).

Cells and Lysis Conditions
3Y1 and 3Y1-Crk cells (4) were a generous gift of Dr. H. Hanafusa (Osaka Bioscience Institute, Osaka, Japan), and the Cas-null fibroblasts (10) were kindly provided by Dr. H. Hirai (University of Tokyo, Tokyo, Japan). Cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were lysed in modified RIPA [150 mM NaCl, 50 mM Tris (pH 7.5), 1% Igepal CA-630, 0.5% deoxycholate] containing protease and phosphatase inhibitors (100 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0.15 unit/ml aprotinin, 1 mM sodium vanadate) (7).

Plasmids
pRK5 constructs containing myc-tagged full-length rat Cas (amino acids 1–874), the carboxyl terminus (Cas-CT; amino acids 544–874), the carboxyl terminus with a proline-to-alanine substitution in the Src SH3-binding site (Cas-CTP642A), a deletion of the substrate-binding YXXP domain (Cas-{Delta}YXXP; deletion of amino acids 119–421), and a deletion of the carboxyl terminus (Cas-{Delta}CT; deletion of amino acids 546–874) have been described previously (11, 33). pcDNA encoding wild-type chicken c-Src and kinase-dead c-Src containing an alanine-to-valine substitution at residue 430 (Src KD) (34) were the kind gifts of S.J. Parsons (University of Virginia). pMEX encoding v-Crk (35) was generously provided by Dr. M. Matsuda (International Medical Center of Japan, Tokyo, Japan).

Protein Expression
Transient transfections were performed as specified by the manufacturer using the following reagents: COS-1 cells were transfected using Superfect (Qiagen, Valencia, CA), LipofectAMINE PLUS (Invitrogen, Carlsbad, CA) was used for 3Y1 and 3Y1-Crk cells, and Fugene 6 was used to transfect Cas-null cells (Roche, Indianapolis, IN). Cells were lysed 24 h (COS-1 cells) or 48 h (3Y1 cells) post-transfection in modified RIPA buffer (see above). Protein concentrations were determined using the BCA assay (Pierce, Rockford, IL).

Immunoprecipitation/Immunoblotting
Immunoprecipitations were performed by incubating the antibodies described above with cell extract for 1–2 h on ice, or overnight at 4°C. Immune complexes were recovered by incubation with protein A-Sepharose beads for 1 h at 4°C, then washed twice in modified RIPA buffer and twice in cold Tris-saline [TN; 150 mM NaCl, 50 mM Tris (pH 7.5)]. The immune complexes were resuspended in Laemmli sample buffer, boiled for 5 min, and separated by 8% SDS-PAGE. Following transfer to nitrocellulose (Schleicher & Schuell, Keene, NH), the membranes were blocked at room temperature in 5% milk diluted in rinse buffer [10 mM Tris, 150 mM NaCl (pH 7.2)] containing 0.05% Tween-20 (RBT) at room temperature, and incubated with antibodies directed against Src, Myc, Cas, p85 PI3K, gag, or GST overnight at 4°C. To detect pTyr-containing proteins, membranes were blocked in 5% BSA in RBT for 2 h at 37–42°C, then incubated with pTyr mAb 4G10 for 1 h at room temperature. Proteins were detected with 125I-anti-mouse immunoglobulin (1 µCi/ml), 125I-protein A (1 µCi/ml), or HRP-conjugated anti-rabbit or anti-mouse immunoglobulin followed by autoradiography or enhanced chemiluminescence (NEN Life Science Products), respectively.

In Vitro GST Pulldown Assay
GST fusion proteins were expressed in Escherichia coli and isolated as described previously (36). One microgram immobilized GST fusion protein was incubated with 500 µg cell extract for 2 h at 4°C and washed three times in modified RIPA buffer and twice in TN. Bound proteins were eluted by boiling in Laemmli sample buffer, separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted as described above.

Far Western Filter Binding Assay
GST fusion proteins were expressed in E. coli as described above, and the assay was performed essentially as described previously (37). Briefly, 50 µg of cell extract were separated by SDS-PAGE and transferred to nitrocellulose. The membranes were denatured and renatured using Guanidine-HCl, probed with 50 µg of fusion protein overnight at 4°C, and immunoblotted with GST mAb 9D9 and HRP-conjugated immunoglobulin as described above.

PI3K Activity
Cells were lysed in modified RIPA buffer and immunoprecipitated with Cas or p85 PI3K antibodies for 2 h at 4°C. Immune complexes were washed three times in modified RIPA buffer, once in TN, and once in 10 mM Tris-HCl (pH 7.4). Half of each immune complex was used for immunoblot and half for the PI3K assay. PI3K assays were performed essentially as described (38). The protein A beads were suspended in 45 µl kinase buffer [10 mM MgCl2, 50 mM Tris-HCl (pH 7.4)], 1 mg/ml sonicated phosphatidylinositol and phosphatidylserine (1:1) dissolved in HEPES buffer [25 mM HEPES (pH 7.4), 1 mM EDTA], and 10–20 µCi [32P]{gamma}-ATP (7000 Ci/mmol). The reaction mixture was incubated at room temperature for 20 min, and stopped by addition of 100 µl 1 N HCl. Phospholipids were extracted with CHCl3/methanol (1:1) and the organic phase was washed with methanol/100 mM HCl, 2 mM EDTA (1:1). Reaction products were spotted on silica Gel-60 F254 plates (Mallinckrodt Baker, Inc. Phillipsburg, NJ) coated with 1% potassium oxalate and 2 mM EDTA in H2O/methanol (3:2), and resolved by chromatography in CHCl3/methanol/H2O/NH4OH (45:35:8.5:1.5) for 30 min. Phosphorylated products were detected by autoradiography.

BrdUrd Incorporation
Cas-null fibroblasts were plated on fibronectin-coated coverslips (20 µg/ml in PBS) overnight before transfection, at which time the coverslips were cultured in DMEM containing 0.05% FBS. Cells were grown under these low-serum conditions for 45 h, at which time BrdUrd (final concentration, 100 mM) was added directly to the media for 3 h. The cells were then methanol-fixed and dried before immunostaining with Gag mAb (1:200) and Texas Red-anti-mouse immunoglobulin (1:500). The coverslips were incubated in 2 N HCl at 37°C for 1 h, followed by neutralization in 2 M borate buffer (pH 8.5) and immunostaining with FITC-conjugated anti-BrdUrd (1:20). Coverslips were mounted on glass slides using VectaShield mounting medium (Vector Laboratories, Burlingame, CA) and visualized using a Nikon fluorescence microscope. Cells expressing v-Crk (red) were scored for BrdUrd-positive nuclei (green). For vector-transfected cells, four to five random fields were counted by phase and fluorescence microscopy to determine the percentage of BrdUrd incorporation. The data are presented as the mean ± SD for four independent experiments. Single-factor ANOVA was performed on the log10 values of percentage incorporation to determine whether statistically significant differences existed between experimental groups. Where differences existed, the Student t test was used to generate P values. Statistical significance was defined as P < 0.05 at a 95% confidence interval.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank past and present members of the Bouton lab as well as Drs. J.T. Parsons, K. Ravichandran, T. Bender, D. Castle, and U. Lorenz for intellectual and technical input.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust (J-556) and the National Science Foundation (MCB-9723820 and MCB-0078022) to A.H.B. Note: R.B.R. and R.M.D. contributed equally to this work. Back

Received October 9, 2002; revised February 27, 2003; accepted March 4, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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