Tyrosine phosphorylation of p190 RhoGAP has been reported to play a role in mediating both the disassembly of actin stress fibers induced by EGF (6
) and the interaction of p190 with p120 RasGAP (3
). A first step in understanding the potential relationship between these two events is to determine the complexity and identity of p-Tyr residues on p190 and to characterize the nature of the association between p190 and p120. Given the size of the p190 molecule, the large number of potential p-Tyr sites, and the suggestion from previous mutagenesis studies that at least two sites would be phosphorylated to the same extent (9
), it was surprising to find only one major site of tyrosine phosphorylation (Y1105) on p190 (Fig. and Table ). However, this result was reproduced whether p190 was derived from v-Src or EGF receptor-transformed cells, from wt
c-Src overexpressers, or from normal control fibroblasts, stimulated or not with EGF (Fig. ). Furthermore, both MS analysis (Fig. ) and mutagenesis (Fig. and ) failed to support a role for phosphorylation of Y1087 (the second site proposed by Hu and Settleman [9
]). Together, these results confirm that only one major site of tyrosine phosphorylation (Y1105) exists on p190.
Y1105 was further shown to be a preferred substrate of c-Src both in vitro and in vivo (Fig. and ) and to be the sole mediator of the p-Tyr-dependent association of p190 with p120 (Fig. and ), thus directly linking c-Src to the regulation of p190-p120 complex formation. Indeed, in 10T1/2 fibroblasts that overexpressed K+ or K− c-Src, the level of p190 complexed with p120 generally correlated with the overall level of p190 tyrosine phosphorylation (Fig. ). Further analysis also revealed that the level of p-Y1105 was a close measure of total p190 tyrosine phosphorylation (p-Y1105 represented 95% of the total p-Tyr in p190 from K+ c-Src cells and 80% in p190 from Neo control cells, while the minor site represented 5 and 20%, respectively [Fig. and Table ]). In addition, all of the tyrosine-phosphorylated p190 was found in complex with p120. Together these data are consistent with a model in which formation of the p190-p120 complex is regulated by a single p-Tyr–SH2 interaction.
However, further investigation revealed that such a model may be an oversimplification. Results from three different experiments (Fig. to ) provided evidence for a p-Tyr-independent mechanism of association as well as a p-Tyr-dependent mechanism. While p-Tyr-independent complexes were less abundant than the p-Tyr-dependent complexes, they still represented a significant proportion (10 to 20% of the p-Tyr-dependent complexes). Furthermore, quantitative analysis indicated that under circumstances of relatively high stoichiometry of p190 tyrosine phosphorylation (such as in K+ c-Src overexpressers [Fig. ]), the ratio of tyrosine-phosphorylated p190 to the amount of p190 protein complexed with p120 was greater than 1 (i.e., 3.5), suggesting that some p-Y1105 was not bound by the SH2 domain of p120 and was free to carry out other functions. Since all of the tyrosine-phosphorylated p190 is complexed with p120, we reasoned that under such circumstances, p190 also must be binding to p120 in a p-Tyr-independent manner. Thus, a more complex picture emerges regarding the mechanisms regulating p190-p120 interaction, as well as the possibility that p-Y1105 has more than one role to play in p190 function.
Although the nature of the p-Tyr-independent interaction is unclear, several models could explain our data while not contradicting the published report that both SH2 domains of p120 are required for association (3
). One possibility is that p190 and p120 are bridged by an as yet unidentified tyrosine-phosphorylated protein that binds one of the SH2 domains of p120, while p-Y1105 of p190 binds the other SH2 domain. In support of this model, we have observed several cellular proteins that coprecipitate with p190 and become phosphorylated on tyrosine when the immune complexes are subjected to an in vitro kinase assay. SH3-polyproline interactions could play a role in the association between p190 and its coprecipitating proteins, because close examination of the amino acid sequence of p190 reveals 13 PXXP motifs scattered throughout the molecule but localized predominantly to the middle domain and the extreme carboxyl terminus. Lastly, because p190 is so heavily phosphorylated on serine, it has been suggested that p-Ser residues contribute to the binding of p190 to p120 (10
). Indeed, there are numerous examples of non-p-Tyr (p-Ser) interactions with SH2 domains in signaling proteins (7
We therefore carried out an experiment to test the possibility that p-Ser mediates p190-p120 interactions. In this experiment, transiently expressed, metabolically 32P-labeled wt p190 was immunoprecipitated from COS-7 cell extracts, subjected to treatment with alkaline phosphatase or protein phosphatase 2a, and subsequently examined for levels of coprecipitating p120. Our data indicate that under these conditions, p190 was labeled almost exclusively on p-Ser (<0.5% p-Tyr) and underwent >90% dephosphorylation upon incubation with phosphatase. Compared to p190 that was subjected to mock dephosphorylation, binding of p120 to p190 was completely unaffected by removal of the p-Ser (data not shown). While these experiments are preliminary, they suggest that p-Ser alone or p-Ser–SH2 interactions are not major participants in the p-Tyr-independent mechanism of association between p120 and p190.
What then might the nature of the interaction be, and what purpose might the p-Tyr-independent pool of p190/p120 serve? Does the p-Tyr-independent pool constitute a signaling entity separate from and parallel to the p-Tyr-dependent pool, or does it exist in dynamic equilibrium with the p-Tyr-dependent pool? For example, does the p-Tyr-independent pool represent a low-affinity interaction between p190 and p120 that becomes high affinity upon phosphorylation of Y1105? These questions are subjects for future investigation, but given the current evidence, we favor the hypothesis that the p-Tyr-independent interaction involves a third protein (possibly binding to p190 through an SH3-polyproline interaction and to p120 through a p-Tyr– or p-Ser–SH2 mechanism) and that this interaction is involved in establishing a low-affinity complex between p190 and p120, which acquires high-affinity status upon phosphorylation of Y1105. Indeed, if the p-Tyr-independent complex represents a low-affinity interaction, then the 10 to 20% figure may be an underestimation of its abundance, due to the difficulty in maintaining association during isolation.
The quantitative analysis depicted in Fig. is, however, in part supportive of the dual, direct p-Tyr–SH2 model. It shows that overall, the amount of p120 found in association with p190 correlates with the level of p190 tyrosine phosphorylation. In addition, the phosphopeptide analysis revealed the presence of two p-Tyr-containing peptides, peptides 3 and 8. However, the differences in the phosphotyrosine levels of these two peptides (4:1, respectively, in Neo control cells and 20:1 in K+ c-Src cells) indicate that only a minority of p190 molecules are capable of interacting by this mechanism under steady-state conditions. Under certain transient circumstances (such as growth factor activation or adhesion/motility) when the two sites may be differential targets for phosphatases, with different turnover rates, the amounts of complex formed by this mechanism could vary.
Quantitation of the level of p190 associated with p120 and the level of p-Tyr in p190 in K+
c-Src cells suggested additional roles for Y1105 other than mediating the interaction with p120. Previous experiments (6
) showed that the level of p190 tyrosine phosphorylation correlates with the extent of EGF-induced (i) subcellular redistribution of p190, (ii) actin stress fiber disassembly, and (iii) mitogenesis, suggesting possible roles for tyrosine phosphorylation of p190 in regulating actin cytoskeleton contributions to mitogenesis. These contributions would most likely be mediated through Rho, a small GTPase known to regulate actin stress fiber assembly (25
). In this context, tyrosine phosphorylation of p190 may modulate its own enzymatic (GTP binding/hydrolysis or RhoGAP) activities through phosphorylation-induced conformational changes. Alternatively, p-Y1105 could serve as a docking site for SH2-containing signaling proteins other than p120. This raises the possibility of competition between p120 and other proteins for binding to p-Y1105, which could offer dynamic linkages to other signaling pathways, including those that regulate the actin cytoskeleton. Overall, the identification and characterization of the p-Tyr sites on endogenous p190 provides us the framework to model previous results into a more complete picture of the potential mechanisms that are involved in regulating p190 function.
The increase in p190 tyrosine phosphorylation upon overexpression of wt
c-Src, coupled with the dominant negative effect of kinase deficient c-Src overexpression on p190 tyrosine phosphorylation, strongly suggests that p190 is a direct substrate of c-Src in vivo (4
). It remains a formal possibility, however, that c-Src affects p190 tyrosine phosphorylation indirectly, through a downstream tyrosine kinase. The data presented here, which demonstrate that c-Src selectively phosphorylates Y1105 in vitro (Fig. ), argue for a direct phosphorylation of p190 at Y1105 by c-Src in vivo. Direct phosphorylation is also supported by the work of Songyang et al. (30
), who report that c-Src preferentially phosphorylates synthetic peptides containing the sequence EEIY, with isoleucine in the −1 position being the most critical. Y1105 is located in a peptide with the sequence EENIY1105
, similar to the preferred synthetic sequence and with the isoleucine appropriately positioned.
Several investigators have reported increases in p190 tyrosine phosphorylation in response to polypeptide growth factors, suggesting that p190 may also be a substrate of tyrosine kinase receptors. Ellis et al. (8
) showed that in Rat1 cells overexpressing the human EGF receptor, p190 tyrosine phosphorylation gradually increased over a 60-min time course of EGF stimulation, with only a small increase detected after 2 min. Tyrosine phosphorylation of p190 has also been shown to increase in response to mast cell growth factor and granulocyte-macrophage colony-stimulating factor stimulation of the myeloid cell line M07e (16
) and in v-Fms (oncogenic form of the receptor for colony-stimulating factor 1)-transformed fibroblasts (31
). In contrast to these reports, we have not detected an EGF-induced increase in the tyrosine phosphorylation of p190 in cells expressing endogenous levels of the EGF receptor. However, we have seen a modest increase in the overall tyrosine phosphorylation of p190 and specifically phosphorylation of Tyr 1105 (Fig. ) in cells overexpressing the receptor after 30 min of EGF treatment. Whether this increase in phosphorylation is mediated by the receptor directly or indirectly is not known. Several studies indicate that Src family members play an integral role in EGF and colony-stimulating factor 1 signaling (12
), suggesting that p190 may be phosphorylated by c-Src following EGF stimulation. Indeed, the approximately twofold increase in Y1105 phosphorylation in EGFR and EGFR/K+
c-Src cells following EGF stimulation (Fig. ) is consistent with phosphorylation by endogenous and overexpressed c-Src, respectively.