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Molecular Endocrinology 13 (12): 1988-2001
Copyright © 1999 by The Endocrine Society

Pertussis Toxin-Sensitive and -Insensitive Thrombin Stimulation of Shc Phosphorylation and Mitogenesis Are Mediated through Distinct Pathways

William A. Ricketts, Joan Heller Brown and Jerrold M. Olefsky

Program in Biomedical Sciences (W.A.R., J.H.B., J.M.O.) University of California San Diego, La Jolla, California 92037-0673
Department of Pharmacology (J.H.B.) University of California San Diego La Jolla, California 92037-0645
Veterans’ Administration Research Service (J.M.O.), San Diego, California 92161
Whittier Diabetes Program (J.M.O.) La Jolla, California 92093


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of both receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) result in phosphorylation of the adaptor protein Shc, providing sites of interaction for proteins in downstream signal transduction cascades. The mechanism of Shc phosphorylation and its function in G protein signaling pathways is still unclear. By examining Shc phosphorylation in response to thrombin in two cell lines, we have defined distinct pertussis toxin (PTX)-sensitive and -insensitive mechanisms by which GPCRs can stimulate tyrosine phosphorylation of Shc. By mutating the tyrosines in Shc, we show that the three sites of tyrosine phosphorylation, Y239, Y240, and Y317, are necessary for thrombin signaling in both systems. The SH2 (src homology 2) domain of Shc is also critical for signaling, but not required for phosphorylation of Shc. In both cell types, inhibition of src family member kinases by chemical inhibitors or microinjection block Shc phosphorylation and bromodeoxyuridine (BrdU) incorporation in response to thrombin. However, in the PTX-sensitive thrombin pathway, both ß{gamma} function and the epidermal growth factor receptor (EGFR) are necessary for Shc phosphorylation and BrdU incorporation. In contrast, signaling in the PTX-insensitive pathway is not mediated through ß{gamma} or the EGFR. Thus, while phosphorylation and function of Shc appear to be the same in both thrombin pathways, the mechanism of tyrosine kinase activation proximal to Shc is different. The differences in signaling between the two thrombin pathways may be representative of mechanisms used by other PTX-sensitive and -insensitive GPCRs to mediate specific responses. In addition, transactivation of RTKs may be a manner by which GPCRs can amplify their signal.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Signaling by receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) has been viewed as occurring through distinct mechanisms. RTKs possess an intrinsic kinase activity that allows them to activate intracellular signaling components by direct phosphorylation (1). Phosphorylation of the other partner in the receptor dimer can also activate cytoplasmic proteins by providing sites of interaction that localize the proteins near to their activators or effectors (2, 3). GPCR signaling is initially mediated by the heterotrimeric G proteins (4). GPCRs do not have tyrosine kinase activity but, instead, increase the exchange of GDP for GTP on the heterotrimeric G proteins, resulting in the separation of the {alpha}- and ß{gamma}-subunits (5). However, the finding that both pathways can activate a similar set of signal transducers, including src, Ras, and mitogen-activated protein kinase (MAPK), indicated more parallels than originally thought (6, 7, 8, 9, 10, 11).

One of the proteins recently established as being required for signaling by both RTKs and GPCRs is the adaptor protein Shc (12, 13, 14, 15, 16, 17, 18). Shc contains no catalytic domain but does encode an amino-terminal phosphotyrosine binding (PTB) domain and a carboxy-terminal src homology 2 (SH2) domain (19, 20). The PTB and SH2 both interact with phosphotyrosine-containing sequences, but these regions are used differentially to mediate signals in response to specific growth factors (21). Between these two domains is a region termed the collagen homology (CH) domain, which contains three tyrosines, Y239, Y240, and Y317 (20, 22). The best studied of the three tyrosines, Y317, is involved in Ras activation by targeting the Grb2-SOS complex to the membrane (23, 24). Less understood, Y239 and Y240 appear necessary for the induction of c-myc expression (22, 25).

Shc phosphorylation has been well documented as an early signaling event leading to MAPK activation (11, 14). There appear to be multiple pathways to achieve Shc phosphorylation in response to stimuli (16). The classical pathway for Shc activation by a RTK was via interaction of one of the phosphotyrosine interaction domains with the activated RTK and its subsequent phosphorylation on tyrosine 317 (18, 23). It is now clear that Shc is phosphorylated and necessary in GPCR pathways, but the precise mechanism by which GPCR stimulation induces Shc phosphorylation remains unclear (12, 13, 15, 26). Expression of ß{gamma}-subunits of the heterotrimeric G proteins can lead to Shc phosphorylation by members of the src family of kinases (SFKs), but expression of a wild-type or a constitutively activated form of the {alpha}-subunit of G12 can also induce Shc phosphorylation (12, 15). In addition, several lines of evidence support cross-talk and transactivation between RTKs and GPCRs (5, 27). In particular, several groups have implicated the epidermal growth factor (EGF) receptor (EGFR) as a necessary signaling component in response to GPCR activation (28, 29, 30, 31, 32, 33, 34).

We examined Shc phosphorylation by thrombin as a means to further elucidate the mechanisms of tyrosine kinase activation by GPCRs. The experiments were conducted in cell lines that are distinguished by whether thrombin induces Shc phosphorylation and DNA synthesis in a pertussis toxin (PTX)-sensitive or -insensitive manner. Our results demonstrate that there are at least two distinct mechanisms of Shc phosphorylation by G proteins: ligand activation of PTX-insensitive G proteins induces Shc phosphorylation through the {alpha}-subunit and SFK activation, but PTX-sensitive G proteins function through ß{gamma}-subunits and transactivation of the EGFR. Therefore, we conclude that G protein activation of tyrosine kinases occurs differently, depending on the role of {alpha}- and ß{gamma}-subunits in the pathway.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Mitogenic Response to Thrombin Can Be Mediated through a PTX-Sensitive or Insensitive Pathway
Thrombin stimulates DNA synthesis and Shc phosphorylation in both HIRcB and 1321N1. To assess the PTX sensitivity of these responses, we treated cells with 100 ng/ml PTX for 6 h before assaying the effects on subsequent thrombin signaling (Fig. 1Go). Thrombin-stimulated bromodeoxyuridine (BrdU) incorporation in HIRcB cells was inhibited by 84% (P < 0.01), whereas PTX treatment had no effect in 1321N1 cells (P > 0.05) (Fig. 1AGo). Longer treatments with PTX produced similar results (35). PTX treatment was also an effective inhibitor of thrombin-stimulated Shc phosphorylation in HIRcB cells but not in 1321N1 cells. EGF-stimulated Shc phosphorylation was unaffected by PTX treatment in either cell type (Fig. 1BGo). These data demonstrate that thrombin can stimulate Shc phosphorylation and DNA synthesis through both PTX-sensitive or -insensitive pathways.



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  		Figure 1. Differential PTX Sensitivity of the Thrombin Pathways

A, HIRcB and 1321N1 cells were treated with 100 ng/ml PTX for 6 h before stimulation with 0.5 U/ml thrombin. Cells were assayed for BrdU incorporation by immunofluorescence. B, HIRcB and 1321N1 cells were either treated as above except cells were stimulated for 5 min with 0.5 U/ml thrombin and lysed, and Shc proteins were immunoprecipitated. Phosphorylation of Shc was analyzed by Western blotting with a monoclonal antiphosphotyrosine antibody.

 
The Function of Shc Is Similar in Both Pathways
We previously reported that Shc was necessary for thrombin-induced DNA synthesis in 1321N1 cells (12). To determine whether Shc functioned differently in PTX-sensitive and -insensitive thrombin signaling, the different structural elements of Shc were mutated. We constructed mammalian expression vectors encoding full-length FLAG-tagged Shc with point mutations that abolished PTB function (S154P), SH2 function (R401L), or tyrosine phosphorylation at tyrosines 239 and 240 (Y239/240F), tyrosine 317 (Y317F), or all three tyrosines (3YF). These constructs were transfected into either HIRcB cells or 1321N1 cells. After transfection and serum deprivation, cells were treated with 0.5 U/ml thrombin or vehicle. Incorporation of BrdU in cells expressing the FLAG-tagged Shc proteins were then assayed by immunofluorescence 18 h later (Fig. 2Go).



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  		Figure 2. The Shc SH2, Y239/240, and Y317 Are Necessary for Thrombin Signaling

A, HIRcB cells were transfected with Shc proteins containing the indicated point mutations. Cells were stimulated with 0.5 U/ml thrombin, and cells that were expressing the FLAG Shc proteins were assayed by immunofluorescence for BrdU incorporation. B, 1321N1 cells were transfected with the same constructs and assayed for BrdU incorporation. Levels of incorporation are expressed as percent of mock transfected cells stimulated with thrombin. Each bar represents three to five experiments and error bars represent SD.

 
Expression of wild-type p52 Shc did not significantly alter thrombin- stimulated BrdU incorporation (Fig. 2Go, A and B). In cells expressing Shc lacking a functional PTB domain (S154P), thrombin-stimulated BrdU incorporation remained at control (mock transfected) levels. In contrast, expression of Shc with a nonfunctional SH2 domain (R401L) completely blocked thrombin-stimulated BrdU incorporation in HIRcB cells and inhibited BrdU incorporation by 71% in 1321N1 cells. Results from these experiments suggest that the Shc SH2 mutant is unable to signal but interferes with the function of endogenous Shc. Thus, the Shc SH2 and not the Shc PTB participates in mediating both PTX-sensitive and -insensitive thrombin signaling.

Consistent with previous work, mutation at Y317 of Shc, the site implicated in Grb2-SOS association and activation of Ras, blocked mitogenesis in HIRcB and 1321N1 cells by 98% and 64%, respectively. Y239/240 were critical for thrombin signaling since expression of Y239/240F also blocked the mitogenic response to thrombin by 92% in HIRcB cells and 58% in 1321N1 cells. Finally, mutation of all three tyrosines (3YF) markedly attenuated thrombin-induced DNA synthesis in both cell types. The novel finding that Y239/240 is essential for cell cycle events mediated by thrombin in conjunction with Y317 and 3YF data suggests all three tyrosines are necessary for mitogenic signaling by thrombin. In conclusion, all of these elements, the SH2, Y239, Y240, and Y317, are important for both PTX-sensitive and -insensitive thrombin signaling pathways.

The Shc SH2 Does Not Mediate Tyrosine Phosphorylation of Shc
To determine whether the SH2 of Shc is required for its phosphorylation in response to thrombin, we assayed phosphorylation of Shc constructs both in vivo (Fig. 3Go) and in vitro (data not shown). Wild-type or R401L FLAG-tagged Shc was expressed in HIRcB cells and stimulated with thrombin for 5 min, and the amount of tyrosine phosphorylation was determined by Western blotting. There was no detectable difference in thrombin-induced phosphorylation of R401L compared with wild-type Shc. In vitro phosphorylation data of mutant Shc proteins resembled the in vivo situation with phosphorylation of wild-type Shc and R401L Shc being similar (data not shown). Since mutation of the SH2 does not affect phosphorylation, we conclude that the Shc SH2 is not used in the process by which activation of the thrombin receptor leads to Shc phosphorylation. The Shc SH2 does appear to participate in thrombin siganling by mediating complex formation with other phosphotyrosine-containing signaling proteins. In both HIRcB cells and in 1321N1 cells, the GST-SH2 interacted with a number of phosphorylated proteins, several of whose phosphorylation state was altered by stimulating the cells with thrombin (data not shown).



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  		Figure 3. Phosphorylation of Shc Mutants

FLAG-tagged wild-type and R401L Shc were immunoprecipitated with anti-FLAG polyclonal antibodies from transfected HIRcB cells either unstimulated or stimulated with 0.5 U/ml thrombin. Western blotting was done as above.

 
Functions of the EGFR and SFKs Differ between PTX-Sensitive and -Insensitive Thrombin Pathways
Earlier studies have implicated the activation of SFKs and/or the EGFR in GPCR signaling (6, 7, 8, 36, 37). To determine whether SFKs or the EGFR were involved in the thrombin signaling in HIRcB or 1321N1 cells, we assessed the effects of pharmacological inhibitors of these kinases on BrdU incorporation (Fig. 4Go). Pretreatment of HIRcB cells with the EGFR inhibitor AG1478 or the src inhibitor PP1 blocked thrombin-induced BrdU incorporation to basal levels (Fig. 4AGo). In contrast, thrombin-stimulated BrdU incorporation in 1321N1 was inhibited by PP1 (P < 0.01) but was unaffected by AG1478 (95% of stimulated, P > 0.05) (Fig. 4BGo). The EGFR inhibitors A48 and Compound 56 and src inhibitor PP2 gave similar results, but inhibitors of the HER/neu, platelet-derived growth factor, fibroblast growth factor, or insulin receptors had no effect in either cell type (data not shown).



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  		Figure 4. Inhibition of the EGFR and SFKs Have Different Effects on Thrombin- Stimulated BrdU Incorporation in the Two Cell Lines

A, HIRcB cells were treated with DMSO, the EGFR inhibitor AG1478, or the src inhibitor PP1 for 30 min before stimulation. Cells were stimulated with 0.5 U/ml thrombin and DNA synthesis was measured by BrdU incorporation. BrdU was detected by immunofluorescence with an anti-BrdU antibody and FRITC-conjugated antirat antibody. B, 1321N1 cells were treated and assayed in the same manner as the HIRcB cells. Bars represent the average of three experiments with SD.

 
To determine whether similar differences were exhibited with respect to Shc phosphorylation, cells were incubated with dimethylsulfoxide (DMSO; vehicle), A63 (inactive tyrphostin control), AG1478, or PP1 for 30 min before stimulation with thrombin. After stimulation for 5 min with thrombin, the cells were lysed, Shc proteins were immunoprecipitated and Western blotted with a monoclonal antiphosphotyrosine antibody (4G10). As observed for BrdU inhibition, both the EGFR inhibitor and SFK inhibitor blocked thrombin-stimulated Shc phosphorylation in HIRcB cells (Fig. 5AGo). In contrast, Shc phosphorylation in 1321N1 cells was unaffected by the presence of AG1478 but sensitive to PP1 (Fig. 5BGo). Neither inhibitor had an effect on insulin-stimulated Shc phosphorylation in HIRcB cells, but the EGFR inhibitor blocked EGF-stimulated Shc phosphorylation as expected (data not shown), demonstrating the specificity of these compounds. These results indicate that PTX-sensitive and -insensitive thrombin-signaling pathways may differentially utilize the EGFR to mediate signaling, and the role of the EGFR in the two pathways may represent a major mechanistic difference in the signaling properties of PTX-sensitive and -insensitive pathways.



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  		Figure 5. Shc Phosphorylation in HIRcB and 1321N1 Cells Is Differentially Affected by the EGFR and src Inhibitors

A, HIRcB cells were treated with DMSO (vehicle), A63 (inactive tyrphostin), AG1478 (EGFR inhibitor), or PP1 (src inhibitor) for 30 min before stimulation. Cells were stimulated with 0.5 U/ml thrombin for 5 min and lysed, and Shc was immunoprecipitated with polyclonal anti-Shc antibodies. Proteins were separated on a 10% gel by SDS-PAGE and transferred to Immobilon, and phosphorylation of Shc was determined by Western blotting with a monoclonal antiphosphotyrosine antibody (4G10). Membranes were stripped and reprobed with a monoclonal anti-Shc antibody to verify equal loading (data not shown). B, 1321N1 cells were treated and analyzed as above. Bar graphs represent densitometry analysis of three to six experiments. Bars are expressed as percent of stimulated control in the presence of DMSO and include SE.

 
The Kinase Activity of the EGFR Is Necessary for Mediating PTX-Sensitive Thrombin Signaling
To further evaluate the role of the EGFR in response to thrombin, we transfected cells with wild-type EGFR, kinase-inactive EGFR ({Delta}kinase), or HIS-tagged LacZ. Transfected cells were stimulated with EGF or thrombin, fixed after 18 h, and stained to detect BrdU incorporation. In LacZ-expressing HIRcB cells, BrdU increased from 16% to 44% after stimulation with EGF. Overexpression of wild-type EGFR had no effect on basal levels of BrdU incorporation (16% in the basal cells) but produced a slight augmentation in BrdU incorporation when stimulated with EGF (52% compared with 44% in stimulated cells). Overexpression of the {Delta}kinase EGFR inhibited EGF-stimulated BrdU incorporation by 75%.

The effect of the EGFR constructs on thrombin signaling in the HIRcB cells strongly resembled that observed in cells stimulated with EGF (Fig. 6BGo). An augmentation of thrombin signaling was seen in cells overexpressing the wild-type EGFR (an increase of 12%), and expression of the {Delta}kinase EGFR inhibited thrombin-stimulated BrdU incorporation almost to basal levels (24% compared with basal levels of 16%). In the 1321N1 cells (Fig. 6CGo), expression of wild type EGFR did not enhance BrdU incorporation (94% of control stimulation), and expression of the {Delta}kinase EGFR had no effect (93% of control stimulated). These results support the aforementioned hypothesis, based on inhibitor data, that the EGFR is necessary for PTX-sensitive thrombin signaling but does not function in PTX-insensitive thrombin signaling.



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  		Figure 6. PTX-Sensitive Thrombin Signaling Depends upon the EGFR Kinase Activity

A, HIRcB cells were transfected with LacZ (expression marker), wild-type EGFR, and kinase-inactive EGFR ({Delta}kinase). Cells were stimulated with 100 ng/ml EGF and assayed for BrdU incorporation by immunofluorescence. B, HIRcB cells were transfected and assayed as above but were stimulated with 0.5 U/ml thrombin. C, 1321N1 cells were transfected and assayed as above but were stimulated with 0.5 U/ml thrombin. Control cells were stimulated with 1 µg/ml EGF. Bars are the average of three experiments with SD.

 
The SH3 Domain of SFKs Is Necessary for Thrombin Signaling
To further define the functional role of SFKs in thrombin signaling, we microinjected fusion proteins of the src SH3, fyn SH3, a fyn SH3 mutant (W119K, labeled SH3*), which cannot bind target sequences, or the fyn SH2 and measured the effects on BrdU incorporation (Fig. 7Go). The src SH2 inhibited thrombin-stimulated BrdU incorporation in HIRcB cells (32%, P < 0.05) but had no significant effect in 1321N1 cells. In HIRcB cells, the src SH3 and fyn SH3 domains inhibited thrombin-stimulated BrdU incorporation by 54% and 60%, respectively (Fig. 7AGo). Comparable results were observed in the 1321N1 cells (Fig. 7BGo). As controls, we demonstrated that the src SH3 had no significant effect on insulin-stimulated BrdU incorporation, and the fyn SH3* had no effect on thrombin signaling. Although the SH2 domain may be of varied importance between pathways, the involvement of SFK SH3 domains in thrombin signaling is crucial in both PTX-sensitive and -insensitive thrombin signaling.



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  		Figure 7. The SH3 Domain of src or fyn Is Necessary for Both Thrombin-Signaling Pathways

A, HIRcB cells were microinjected with the fyn SH2, fyn SH3, fyn SH3 mutant (W119K), and src SH3 fusion proteins at concentrations of 10 mg/ml. After recovering from injection, cells were stimulated with 0.5 U/ml thrombin or 100 ng/ml insulin (control injection). The effect of injection was measured by an immunofluorescence assay for BrdU incorporation. B, Microinjections were performed in 1321N1 cells, and the effects of injections on BrdU incorporation were determined. Bars represent three to five experiments expressed as percent of control stimulated BrdU incorporation with SD.

 
Thrombin-Activated SFKs Phosphorylate Shc in An SH3-Dependent Manner
SFK activation is a necessary event for thrombin-induced BrdU incorporation and Shc phosphorylation, suggesting that an SFK is the Shc kinase. Since the fyn SH3 has been reported to interact with Shc and recombinant src or fyn were able to directly phosphorylate Shc (Fig. 8AGo), a possible mechanism for the inhibition of BrdU incorporation by the SH3 could be the disruption of Shc phosphorylation (6, 36, 38). Using 1321N1 cell lysates, we addressed this hypothesis by performing kinase assays on FLAG-tagged Shc preincubated with varying amounts of fyn GST-SH3 (Fig. 8BGo). The W119K mutant GST-SH3* was used as a control for nonspecific inhibition. The fyn GST-SH3 inhibited the ability of lysates from thrombin-stimulated cells to phosphorylate Shc, while the W119K mutant had no effect.



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  		Figure 8. Fyn Phosphorylates Shc through an SH3-Dependent Mechanism

A, Wild-type FLAG tagged Shc proteins were expressed in COS7 cells and immunoprecipitated with polyclonal anti-FLAG. This pellet was washed in kinase buffer before addition of 5 µg recombinant src or fyn. Kinase reactions were allowed to proceed for 1 h at 4 C. Proteins were separated on a 10% gel by SDS-PAGE, transferred to Immobilon, and blotted with a monoclonal antiphosphotyrosine antibody (4G10). Controls were 1321N1 lysates from cells stimulated with 0.5 U/ml thrombin for 2 min. B, After immunoprecipitation, FLAG-tagged Shc was incubated with varying concentrations of either GST-SH3 of fyn or GST-W119K SH3 of fyn. Lysates from 1321N1 cells that had been stimulated for 2 min with 0.5 U/ml thrombin were added to the FLAG-tagged Shc. The kinase reaction was incubated for 1 h at 4 C. Samples were run on a 10% gel with SDS-PAGE, transferred to Immobilon, and blotted for phosphotyrosine with a monoclonal antiphosphotyrosine antibody (4G10).

 
PTX-Sensitive Thrombin Signaling Is Dependent on ß{gamma}-Subunits but PTX-Insensitive Thrombin Signaling Is Not
An early event in G protein signaling is the dissociation of {alpha}- and ß{gamma}-subunits after GTP binding (4). Either {alpha} or ß{gamma} can propagate specific signaling events (5, 39). To determine whether PTX-sensitive and -insensitive pathways differed in their dependence on ß{gamma}-subunits, we microinjected a fusion protein of the ß-adrenergic receptor kinase carboxy terminus (ßARK CT), which binds to ß{gamma}-subunits (40). Injection of the ßARK CT into HIRcB cells had no effect on EGF-stimulated BrdU incorporation but blocked PTX-sensitive L-{alpha}-lysophosphatidic acid (LPA) and thrombin signaling by 75% and 76% (Fig. 9AGo). In contrast, injection of the ßARK CT into 1321N1 cells had no significant effect on thrombin signaling (Fig. 9BGo). This shows that a proximal difference in signaling by PTX-sensitive and -insensitive pathways involves the function of ß{gamma}-subunits.



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  		Figure 9. Inhibition of ß{gamma}-Subunits Suppresses PTX-Sensitive Thrombin Signaling but Not the PTX-Insensitive Pathway

A, HIRcB cells were microinjected with 10 mg/ml of a GST fusion with the ßARK CT. After microinjection, cells were stimulated with 100 ng/ml EGF, 1 mM LPA, or 0.5 U/ml thrombin and the effects of injection the GST- ßARK CT on BrdU incorporation were determined. B, 1321N1 cells were treated as above. Bars represent the average of three experiments with the SDs.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The mechanism by which tyrosine kinases are activated by GPCRs is an area of extreme interest. Although lacking the intrinsic kinase activity and sites for protein interaction that the RTKs possess, GPCRs are still able to induce tyrosine phosphorylation of intracellular proteins upon stimulation. PTX-sensitive and -insensitive thrombin signaling pathways both lead to Shc phosphorylation and DNA synthesis. Our results indicate that the function of Shc within these two pathways is similar, but variations in signaling proximal to Shc phosphorylation exist between the two G protein-signaling pathways. Both pathways require a src family member, but PTX-sensitive thrombin signaling proceeds through ß{gamma}-subunits and the transactivation of the EGFR while PTX-insensitive thrombin signaling occurs independently of ß{gamma} and the EGFR. The differential role of ß{gamma}-subunits and the EGFR may represent a fundamental difference between signaling by PTX-sensitive and -insensitive GPCRs.

Shc Performs Multiple Functions in Thrombin Signaling
From the results obtained with the mutant Shc proteins, we conclude that Shc participates in at least three separate facets of thrombin mitogenic signaling through its tyrosine phosphorylation sites and through its SH2 domain.

Mutations at either Y239/240, Y317, or all three tyrosine residues in Shc were able to dramatically reduce thrombin-stimulated BrdU incorporation. Since Y239/240F and Y317F inhibited equally well, we reason that two parallel pathways originate from Shc, and both are necessary for later signaling events, such as DNA synthesis. Y317 has been shown to be necessary for MAPK activation in fibroblasts challenged with thrombin (13). Studies of Y239/240 function implicate this tyrosine phosphorylation site in growth factor-induced expression of c-myc (22, 25). However, the importance of Y239/240 in GPCR signaling has not been previously established. Based on the requirement for both sites of tyrosine phosphorylation, it seems likely that thrombin acts through Shc not only to regulate Ras but also via another pathway such as myc expression, which ultimately work in a concerted manner to promote cell cycle progression.

The Shc SH2 is also necessary for thrombin signaling, but it does not function to mediate phosphorylation of Shc since Shc proteins with a nonfunctional SH2 domain were phosphorylated to the same levels as wild-type Shc in vivo and in vitro. The role of the Shc SH2 in thrombin signaling therefore appears to be in the formation of signaling complexes containing other phosphoproteins. Interactions with the Shc SH2 may be necessary to target proteins to subcellular compartments or to an activating enzyme or substrate (21, 41, 42). We observed similar results with the Shc SH2 in EGF signaling, and others have proposed similar mechanisms for Shc SH2 function (21, 43). Complex formation mediated by the Shc SH2, along with Shc phosphorylation, are required for transducing the thrombin mitogenic signaling.

Activation of Tyrosine Kinases by Thrombin
Tyrosine phosphorylation of cytoplasmic proteins is a necessary signaling event for thrombin-stimulated mitogenesis, but the mechanism(s) by which this occurs remain unclear. Using Shc phosphorylation as a marker for tyrosine kinase activation by thrombin, we found that Shc phosphorylation occurs through both PTX-sensitive and -insensitive pathways, but each pathway employed a different mechanism to obtain this goal. PTX-sensitive thrombin-stimulated Shc phosphorylation and BrdU incorporation were dependent on both the EGFR and an SFK, while PTX-insensitive Shc phosphorylation and BrdU incorporation were only dependent upon a src family member. These results suggest that the transactivation of the EGFR may be a more important component of PTX-sensitive G protein signaling.

Interestingly, when wild-type EGFR was overexpressed in HIRcB cells, a slight increase in both EGF- and thrombin-induced BrdU incorporation was detected as compared with mock transfected cells. This increase did not occur in thrombin-stimulated 1321N1 cells, suggesting that these cells do not transactivate the EGFR even at high EGFR expression levels. This may reflect the different mechanism used for PTX-insensitive thrombin signaling, which is incompatible with PTX-sensitive thrombin signaling. Although transactivation of the platelet-derived growth factor receptor has also been reported as a pathway used by GPCRs (44), inhibitors of the HER/neu, platelet-derived growth factor, fibroblast growth factor, or insulin receptors had no effect on BrdU incorporation in either cell type (data not shown). Thus, transactivation by thrombin occurs either through an EGFR-specific mechanism or independently of the EGFR and other known growth factors.

Several reports of EGFR involvement in GPCR signaling have recently been published, but the mechanism of this transactivation is still incompletely understood (29, 30, 31, 32, 33, 34, 45). Tice et al. (46) have reported that G protein receptor agonists can stimulate src to phosphorylate the EGFR on a novel phosphorylation site, Y845 (46). This site was necessary for LPA-induced BrdU incorporation, and phosphorylation at Y845 can be induced by thrombin (Ref. 47 and S. Parsons, personal communication). Since the EGFR can function as a tyrosine kinase and/or a scaffold protein, transactivation by a GPCR would provide two functions the GPCR cannot perform on its own. In the PTX-sensitive thrombin pathway, the kinase activity of the EGFR was necessary for Shc phosphorylation and DNA synthesis, as determined by both chemical inhibitors and the expression of a kinase-inactive EGFR. Therefore, the kinase activity of the EGFR is necessary for PTX-sensitive thrombin signaling, but an additional role as a scaffold protein cannot be ruled out.

SFK activation and function are necessary for Shc phosphorylation and DNA synthesis in both thrombin-signaling pathways. SFK activation in response to thrombin differs between the PTX-sensitive and -insensitive pathways may differ since microinjection of the src SH2 inhibited BrdU incorporation in HIRcB cells but not in 1321N1 cells. It is possible that microinjecting the src SH2 blocks association with and activation of the EGFR by a src family member in HIRcB cells and, since 1321N1 cells do not transactivate the EGFR, signaling would be unaffected in 1321N1 cells (47). Once activated, the SFKs perform similar functions within the two thrombin mitogenic pathways. The fact that Shc phosphorylation by SFKs has been documented in several cell types, and that the SH3 domain of fyn can disrupt Shc phosphorylation, leads us to conclude that src or fyn is the Shc kinase activated by thrombin in both cell types, and the interaction is mediated by SH3 domain interacting within the proline-rich CH domain of Shc (36, 48, 49, 50).

The Role of {alpha}- and ß{gamma}-Subunits in Thrombin Signaling
To determine whether differences in {alpha}- and ß{gamma}-subunit function play a role in the variations we observed between PTX-sensitive and -insensitive thrombin signaling, we microinjected a fusion protein of the ßARK carboxy terminus (CT). Microinjection of the ßARK CT inhibited thrombin-stimulated BrdU incorporation in HIRcB cells through the PTX-sensitive pathway, presumably by sequestering ß{gamma}-subunits away from their normal effectors (40). In contrast, the PTX-insensitive cascade in 1321N1 cells was unaffected, indicating that the ß{gamma}-subunits do not function within this pathway. ß{gamma}-Subunits may thus be involved in the transactivation of the EGFR, giving rise to the differential tyrosine kinase activation in response to thrombin in the cell types studied.

Activation of tyrosine kinases by ß{gamma}-subunits in the PTX-sensitive thrombin pathway has been recently suggested to occur as a multiple step process. ß{gamma}-Subunits function in localizing the G protein receptor kinases (GRKs) to the activated G protein receptor, the GRKs phosphorylate the GPCR and create sites of interaction for a family of proteins known as the arrestins (51). The arrestins function to down-regulate the GPCRs and also appear to function in forming a signaling scaffold, activating src kinases and leading to the phosphorylation of the EGFR (52). Hence, the role of the ß{gamma}-subunits in PTX-sensitive thrombin signaling may be to recruit the foundation for the signaling scaffold to the receptor.

Since the ß{gamma}-subunits are not necessary for PTX-insensitive thrombin signaling, this pathway must activate tyrosine kinases by a different mechanism. We have previously shown that expression of a constitutively activated G{alpha}12 in 1321N1 cells can mimic several thrombin-stimulated responses, including Shc phosphorylation and AP-1 reporter gene activation (12). Our hypothesis is that only the G{alpha}q or G{alpha}12 subunits function to transmit PTX-insensitive thrombin signals. Activation of tyrosine kinases could be mediated by direct interactions of thrombin-activated G{alpha} subunits in a manner similar to stimulation of phospholipase C (PLC) and Bruton’s tyrosine kinase (53, 54, 55, 56). In addition, second messengers produced by the activation of such enzymes could increase tyrosine kinase activity. For example, Src activation by lipid second messengers has been reported (50).

In conclusion, we have found that Shc tyrosine phosphorylation and its SH2 work in conjunction to mediate both PTX-sensitive and -insensitive thrombin mitogenic signaling. Shc phosphorylation is accomplished by two different mechanisms, depending on the PTX sensitivity of the cell type studied. The difference in PTX-sensitive and -insensitive phosphorylation of Shc was characterized by differential EGFR involvement. We also found that ß{gamma}- subunits were necessary for PTX-sensitive thrombin signaling in the EGFR- dependent HIRcB pathway but did not participate in PTX-insensitive thrombin signaling in 1321N1 cells. An interesting question posed by our data is whether or not the differences we observed are representative of differences between PTX-sensitive and -insensitive GPCRs and signaling across a broad range of cell types and ligands. Our model suggests PTX-sensitive G protein signaling is more dependent on ß{gamma}-subunits than {alpha}-subunits. The role of the G{alpha}-subunits in PTX-sensitive G protein signaling requires additional investigation. Recently, however, Neptune et al. (57) reported that G{alpha}i is not required for chemotaxis in response to stimulation of receptors coupled to G{alpha}i, suggesting that ß{gamma} function is more important than G{alpha} function in their system. These results are consistent with a broad and prominent role for ß{gamma}-subunits in PTX-sensitive pathways.

In PTX-insensitive G protein signaling, our model assumes that ß{gamma}- subunits perform little or no function, while the G{alpha}-subunits are responsible for signal transduction. We hypothesize that ß{gamma} function is minimal, and that EGFRs are not recruited and not involved in downstream signaling. In PTX-insensitive pathways, G{alpha} subunits, such as G{alpha}q, can directly activate enzymes, such as PLC, and the production of second messengers can initiate subsequent downstream events. As mentioned earlier, expression of G{alpha}q or G{alpha}12 subunits can mimic responses of PTX-insensitive pathways, supporting a dominant role for {alpha}-subunits in these pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Lines and Culture Conditions
Rat1 cells overexpressing the human insulin receptor, HIRcBs, were established in our laboratory (58). HIRcB cells were grown in DME/F12 supplemented with 10% FBS, 100 µM methotrexate, 1 mM glutamine, 100 U penicillin, and 100 µg/ml streptomycin. To increase expression of transfected constructs (see below), transfected HIRcB cells were grown in the same media as above but lacking methotrexate. 1321N1 cells were grown in DMEM regular glucose supplemented with 5% FBS, 1 mM glutamine, 100 U penicillin, and 100 µg/ml streptomycin. COS7 cells were grown in DMEM regular glucose supplemented with 10% FBS, 1 mM glutamine, 100 U penicillin, and 100 µg/ml streptomycin. All cell lines were grown at 37 C in a humidified chamber with 5% CO2.

Expression Vectors
The FLAG-tagged Shc expression vector, pRK5 Shc, was a generous gift from Dr. Edward Y. Skolnik (Skirball Institute, New York, NY). Point mutations were introduced into Shc by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutation of serine 154 to proline was introduced with the oligonucleotides 5'-ATC TCT TTC GCG CCC GGT GGG GAT CCG GAC-3' and 5'-GTC CGG ATC CCC ACC GGG CGC GAA AGA GAT-3'. Arginine 401 was mutated to leucine with the oligonucleotides 5'-GAC TTC TTG GTG CTA GAG AGC ACG ACC ACG-3' and 5'-CGT GGT CGT GCT CTC TAG CAC CAA GAC GTC-3'. Mutations of tyrosines 239 and 240 to phenylalanines were accomplished through using oligonucleotides 5'- CCT GAC CAT CAG TTC TTT AAT GAC TTC CCG-3' and 5'-CGG GAA GTC ATT AAA GAA CTG ATG GTC AGG-3'. Finally, the change of tyrosine 317 to phenylalanine was created with the oligonucleotides 5'-GAT GAC CCC TCC TTT GTC AAC ATC CAG AAT-3' and 5'-ATT CTG GAT GTT GAC AAA GGA GGG GTC ATC-3'. The presence of mutations was determined by sequencing (CFAR Sequencing Facility, La Jolla, CA).

All EGFR constructs were obtained from Dr. Gordon N. Gill (University of CA, San Diego). His tagged LacZ was purchased from Invitrogen (La Jolla, CA). Vectors encoding fyn fusion proteins were obtained from Dr. Hamid Band (Harvard University, Boston, MA). The src SH3 fusion protein was obtained from Dr. David D. Schlaepfer (Scripps Research Institute, La Jolla, CA). The ßARK CT fusion protein construct was obtained from Dr. Robert J. Lefkowitz (Duke University, Durham, NC).

Kinase Inhibitors
The inhibitors A63, AG1478, and PP1 were all purchased from Calbiochem (La Jolla, CA). Compounds were resuspended in DMSO and used at concentrations as follows: A63 was used at 2 mM, AG1478 was used at 50 nM, and PP1 was used at 100 nM. Control cells were treated with DMSO and DMSO concentrations never exceeded 0.2% of media volume. Cells were treated for 30 min before stimulation and lysis as described below. PTX was from Sigma (St. Louis, MO). Cells were treated with 100 ng/ml for 6 h before stimulation.

Transfection of Cell Lines
Transfections of all three cell lines were performed with SuperFECT (Qiagen, Valencia, CA) in accordance with manufacturer’s instructions. In brief, cells were plated 1 day before transfection. DNA purified over a CsCl gradient was mixed with the SuperFECT reagent, incubated at room temperature to allow complex formation, and added to cells. Three hours later, the transfection mix was removed by aspiration, cells were washed once with warm PBS Life Technologies, Inc., Gaithersburg, MD), and fresh growth media were added to the cells. Cells were allowed to grow in this media for at least 20 h before being serum deprived for 24 h before experiments.

Cell Stimulation and Cell Lysates
HIRcB cells and 1321N1 cells were stimulated with 0.5 U/ml thrombin (Calbiochem) in the presence of 0.1% fatty acid-free BSA (Sigma) at 37 C. Times of stimulation are indicated in the figure legends. Control cells were exposed to only 0.1% fatty acid-free BSA. In experiments in which other growth factors were used, cells were stimulated with 1 mM LPA (Sigma), 100 ng/ml insulin, or 1 µg/ml EGF (Life Technologies, Inc.). Recombinant human insulin used was a generous gift from Dr. Bruce Frank (Eli Lilly & Co., Indianapolis, IN). After stimulation, cells were washed once in 4 C PBS and lysed in fibroblast solubilization buffer [(FSB) 25 mM HEPES, 8 mM EDTA, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 150 mM NaF, 10 mM Na pyrophosphate, 2 mM Na2VO4, 1 mM phenylmethyl sulfonylfluoride (PMSF), 10% glycerol, and 1% Triton X-100 (pH 7.5)]. Insoluble material was removed by centrifugation and the supernatant used for subsequent experiments.

Immunoprecipitation and Western Blotting
Endogenous Shc proteins were immunoprecipitated to determine the effect of kinase inhibitors on Shc tyrosine phosphorylation. Five micrograms of anti-Shc polyclonal antibodies (Transduction Laboratories, Inc., Lexington, KY) and 50 µl Protein A agarose (Upstate Biotechnology, Inc., Lake Placid, NY) were added to lysates to immunoprecipitate Shc. The pellets were washed twice in FSB and solubilized in sample buffer containing 10% ß-mercaptoethanol (BME).

Samples were separated by SDS-PAGE on a 7.5% acrylamide gel and transferred to Immobilon (Millipore Corp., Bedford, MA). Membranes were blocked in either 5% BSA in TBST [0.5 M Tris, 1.5 M NaCl, 0.1% Tween 20 (pH 7.5)] or 5% nonfat dried milk in TBST. Phosphorylation on tyrosines was detected by Western blotting with a monoclonal antibody to phosphotyrosine, 4G10 (Upstate Biotechnology, Inc.) at 0.5 µg/ml. In the GST-SH2 pull-down experiments (see below), polyclonal antiphosphotyrosine antibodies at 2 µg/ml were used instead. In data not shown, an anti-Shc monoclonal antibody (Transduction Laboratories, Inc.) was used to determine equal protein loading. Detection of FLAG-tagged Shc proteins was done by Western blotting with the monoclonal antibody M2 (Sigma) at 10 µg/ml. Goat polyclonal antimouse and antirabbit IgG antibodies conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, Arlington Heights, IL) at a dilution of 1:1000 were incubated with the membrane and visualized with the SuperSignal chemiluminescence detection reagents (Pierce Chemical Co., Rockford, IL) upon exposure to X-omat AR scientific imaging film (Kodak, Rochester, NY).

Membranes that needed to be probed again to determine levels of a specific protein were treated as follows. Membranes were washed in Tris-buffered saline (TBS) after the initial exposure to enhanced chemiluminescence reagents. Two 10-min washes with stripping buffer (0.5 M acetic acid and 0.5 M NaCl in water) were used to remove antibodies bound to the membrane. After stripping, the membrane was washed three times in TBS to remove excess NaCl and neutralize the acetic acid. Western blotting was then performed as above.

Glutatione-S-transferase (GST) Fusion Proteins
GST fusion proteins were expressed in and recovered from DH5{alpha} or BL21 (GST-src SH3) strains under conditions suggested by the manufacturer of the pGEX vectors (Pharmacia Biotech, Alameda, CA). In brief, bacteria containing the fusion protein plasmids were grown in 1-liter cultures and induced with 0.1 mM IPTG for 4 h. Fusion proteins were purified from bacterial lysates by affinity precipitation with glutathione (GSH)-conjugated agarose beads (Pharmacia Biotech), washed in PBS containing 1 mM PMSF, and eluted from the beads with 10 mM GSH in 100 mM Tris (pH 8.0).

Fusion proteins for microinjection were concentrated in microinjection buffer [5 mM Na phosphate (pH 7.2), 100 mM KCl] by using a Ultrafree-15 centrifugal filter device (Millipore Corp.). Protein concentration was determined by spectrophotometric analysis at OD280 and confirmed by Coomassie blue staining on a 10% acrylamide gel. Fusion proteins were injected at 10 mg/ml into HIRcB or 1321N1 cells as previously described (1, 45). Affinity precipitation experiments were performed using the GST-SH2 of Shc prepared in this manner. For the precipitations, 10 µg of fusion protein and 50 µl GSH-conjugated agarose beads were added to HIRcB and 1321N1 lysates and incubated while rotating for 1 h at 4 C. Beads and associated proteins were collected by centrifugation, washed once in FSB, solubilized in sample buffer containing 10% BME, and analyzed by Western blotting as stated above.

In Vitro Kinase Assays
FLAG-tagged wild-type or mutant Shc proteins were produced for use as a substrate in kinase assays by transfection of COS7 cells with the appropriate expression vector. Cells were serum deprived for 24 h and lysed in KB (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 2 mM Na3VO4 and 1 mM PMSF, 10% glycerol, and 0.1% Triton X-100). FLAG-tagged Shc proteins were immunoprecipitating from COS7 cell lysates with 5 µg of polyclonal antibodies against the FLAG epitope (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 50 µl Protein A-conjugated agarose (Upstate Biotechnology, Inc.) per reaction. Pellets were washed in EBG (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, and 0.01% Triton X-100) containing 2 mM Na3VO4 and 1 mM PMSF. To these pellets, we added 300 µl KB, 200 µl HIRcB or 1321N1 cell lysate, 2 mM MgCl2, 2 mM CaCl2, 2 mM MnCl2, 2 mM ATP, and 2 mM Na3VO4. Cell lysates used in the kinase assay were from cells that were serum deprived, stimulated for 2 min, and lysed in KB, and the detergent-insoluble fraction was removed by centrifugation.

In samples in which purified src or fyn was used in Shc kinase assays, the src or fyn was recombinantly produced (both from Upstate Biotechnology, Inc.). The Shc used as substrate was produced in COS7 cells and isolated as before, but the pellet was resuspended in KB including 2 mM MgCl2, 2 mM CaCl2, 2 mM MnCl2, and 2 mM ATP. Five micrograms of recombinant kinase were added, and both the kinase assay and analysis were done as above. Controls for Shc phosphorylation in this experiment were kinase assays performed in either unstimulated or thrombin-stimulated 1321N1 cell lysates. To assay the effect of the fyn SH3 on Shc phosphorylation, immunoprecipitated FLAG-tagged Shc was incubated in 300 µl KB with the GST-SH3 (fyn), at 5, 10, or 25 µg per reaction, for 30 min before the kinase assay was performed. Two hundred microliters of cell lysate were added, and the kinase assays were done as above. Kinase reactions were allowed to proceed for 1 h at 4 C. The antibody-agarose-FLAG-tagged Shc complex was spun down in a microcentrifuge at 4 C. This pellet was washed once in FSB, solubilized in sample buffer containing 10% BME, and boiled for 2 min. Samples were analyzed as above by Western blotting.

Immunofluorescence
Incorporation of BrdU, a thymidine analog, was used as a marker for cell cycle progression as previously described (1, 45). BrdU (Amersham Pharmacia Biotech) was added to cells 12 h after being stimulated after microinjection, transfection, or treatment with kinase inhibitors. Incorporation of BrdU was allowed to occur for 6 h, whereupon cells were fixed in 3.7% formaldehyde in PBS for 20 min. Coverslips were washed three times in PBS and labeled with rat anti-BrdU (Amersham Pharmacia Biotech) and either a mouse monoclonal antibody to the anti-Xpress epitope (Invitrogen) or the polyclonal anti-FLAG antibody (Santa Cruz Biotechnology, Inc.). Coverslips were washed again in PBS and labeled with donkey antirat antibodies conjugated to rhodamine (tetramethyl rhodamine isothiocyanate) and donkey antimouse or antirabbit antibodies conjugated to fluorescein (fluorescein isothiocyanate) (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA). Analysis was performed on an Axiophot fluorescence microscope (Carl Zeiss, Thornwood, NY).


   ACKNOWLEDGMENTS
 
The authors of this paper would like to thank the investigators mentioned in Materials and Methods for their generous gifts or reagents. We also thank Dr. Salme Taagepera and Dr. Lila Collins for insightful discussions.


   FOOTNOTES
 
Address requests for reprints to: Dr. Jerrold Olefsky, Department of Medicine, Division of Endocrinology and Metabolism, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673.

This work was supported by NIDDK, NIH Grant DK-33651 and GM-36927 (J.H.B.).

Received for publication June 4, 1999. Revision received August 16, 1999. Accepted for publication August 18, 1999.


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 ABSTRACT
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 DISCUSSION
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P. M. Sharma, H.-S. Son, S. Ugi, W. Ricketts, and J. M. Olefsky
Mechanism of SHIP-Mediated Inhibition of Insulin- and Platelet-Derived Growth Factor-Stimulated Mitogen-Activated Protein Kinase Activity in 3T3-L1 Adipocytes
Mol. Endocrinol., February 1, 2005; 19(2): 421 - 430.
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Y. Daaka
G Proteins in Cancer: The Prostate Cancer Paradigm
Sci. Signal., January 20, 2004; 2004(216): re2 - re2.
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 Mol. Cell. Biol.