Here we demonstrate a novel function of Bcr as a negative regulator of Ras signaling. To achieve this effect, the Bcr kinase phosphorylates AF-6, binds to its PDZ domain, and allows AF-6 to interact with Ras and keep Ras away from downstream signaling (the proposed model is depicted in Fig. ). This mechanism may be responsible for maintaining the cell in a nonproliferative state where the Bcr kinase is constitutively active.
FIG. 7. Proposed model depicting the effect of Bcr on Ras-dependent stimulation of ERK via AF-6. (A) In quiescent cells the constitutively active Bcr phosphorylates AF-6 (step 1), which leads to the interaction of the PDZ domain of AF-6 with the PDZ-binding motif (more ...)
We recognized by sequence analysis that the C terminus of Bcr contains a motif typical for ligands of PDZ domains, S-T-E-V, and indeed, the C terminus of Bcr binds to the PDZ domain of AF-6. Several other ligands are known to bind to the PDZ domain of AF-6, such as cell adhesion molecules and signal transducers. AF-6 colocalizes with these binding partners at sites of cell-cell contact. We show that AF-6 colocalizes with Bcr at the plasma membrane in confluent epithelial cells. Also, in primary tissue, such as brain tissue, Bcr and AF-6 are expressed in similar regions (4
). Moreover, both proteins can be coprecipitated from mouse brain lysate (Fig. ). These data suggest a biological role for the interaction between Bcr and AF-6 at the cellular membrane.
We show that Bcr phosphorylates AF-6. Since no homolog of Bcr exists in invertebrates, Bcr-dependent phosphorylation and regulation of AF-6 must be specific for vertebrates. The importance of phosphorylation for the interaction between Bcr and AF-6 was demonstrated here by comparison of the BcrWT and the kinase-defective mutant BcrΔNT and showed that the PDZ domain of full-length AF-6 is accessible only for efficient binding after phosphorylation. We mapped the major phosphorylation site of Bcr on AF-6 to Thr 893, a site that is conserved in AF-6 of human and mouse but not in AF-6 homologues of fly and worm (Fig. ). This supports the notion that this Bcr-dependent phosphorylation of AF-6 is specific for vertebrates. AF-6 and AF6-T893V did not differ in their ability to bind to BcrWT. This indicates that in addition to phosphorylation of AF-6 at Thr 893 there has to be a further phosphorylation event which regulates phosphorylation-dependent binding of AF-6 to BcrWT. However, the mutant BcrV1271A, which cannot bind to the PDZ domain of AF-6, reveals differences. In the case of AF-6, which can be phosphorylated at Thr 893, the interaction is reduced. In the case of AF6-T893V, which can no longer be phosphorylated at this site, the interaction to BcrV1271A is much stronger. Thus, under certain conditions phosphorylation of AF-6 at Thr 893 may play a regulatory role, e.g., by affecting the specificity of the binding to the PDZ domain or to another binding site.
The phosphorylation site Thr 893 is located in a linker-like central part of AF-6 and is close to the PDZ domain. Phosphorylation in the proximity of a PDZ domain for regulation of ligand binding is a novel phenomenon. In other cases, the binding between ligand and PDZ domain is also regulated by phosphorylation of the ligand. This is shown for the S/T-X-Φ motif interacting with class I PDZ domains. Phosphorylation of S/T in this motif regulates the binding affinity of, e.g., the E6 protein of the human papilloma virus to the PDZ protein hDlg (18
). This demonstrates the relevance of phosphorylation for the interaction of PDZ domains with their ligands.
Bcr is not the only kinase that phosphorylates AF-6, thereby regulating binding of its ligand to the PDZ domain. Receptor tyrosine kinases of the Eph receptor family have been shown to phosphorylate AF-6 (13
). Bcr and Eph receptors have in common that they are both negative regulators of proliferation. The Bcr kinase has a function in quiescent cells where it is constitutively active. Eph receptors, when activated by ephrins, lead to inhibition of proliferation and induction of differentiation (28
). In both cases AF-6 is involved in maintenance of a nonproliferative state. We propose this in a model (Fig. ).
AF-6 has previously been shown to compete with Raf for binding to active Ras in vitro (15
). In addition, overexpression of the Ras-binding domain of AF-6 inhibits Ras/Raf-dependent c-fos promoter stimulation (48
). Here we show that overexpression of the full-length AF-6 protein can interfere with Ras-dependent signaling and inhibits activation of ERK2. This could be the result of a residual low-affinity binding of overexpressed AF-6 independently of its phosphorylation state. We show that BcrWT but not the mutant BcrV1271A, which is defective in binding to AF-6, reduced Ras-dependent activation of ERK2. We attribute this to direct binding of BcrWT to endogenous AF-6 under conditions where AF-6 is phosphorylated and the Ras-binding site is accessible. This requires the kinase function of Bcr and its ability to bind to the PDZ domain of AF-6. The Bcr kinase is active in nonproliferating cells and inhibits low basal signal activity, which we mimic here by low expression of active Ras (Fig. and ). However, when the Bcr kinase activity is downregulated by tyrosine phosphorylation, Bcr is not able to reduce Ras-dependent signaling (20
) (Fig. ). In the developing system of the Drosophila
eye, the AF-6 homolog Canoe also modulates the Ras activity (27
), although it does so by other regulators, since a homolog of human Bcr does not exist in Drosophila
. Additional signaling mechanisms may have evolved to control the more complex organization in vertebrates.
Ras is not the only GTPase that interacts with AF-6. Other GTPases, such as Rap1A, Rit, and Rin, also bind to the Ras-binding domain of AF-6 (1
). Rap1A and AF-6 colocalize at the plasma membrane of epithelial cells, and overexpression of Rap1 maintains their epithelial morphology (1
). In contrast, high expression of active Ras gives rise to disruption of cell-cell contacts. Thus, AF-6 might regulate different cellular responses, such as cell adhesion, differentiation, or morphogenesis, through interaction with Ras or Rap1A (52
). Whether Bcr also modulates a functional interaction between Rap1A and AF-6 is under investigation.
We identified a trimeric complex between Bcr, AF-6, and Ras. Alternatively, Bcr can also be linked to the Ras pathway through the adaptor protein 14-3-3, yet the biological significance has not been clearly shown. Indeed, Bcr phosphorylates 14-3-3 and also interacts via 14-3-3 with the Ras effector Raf (3
). However, this interaction does not depend on a PDZ domain-containing protein. Thus, there are two substrates of Bcr, AF-6 and 14-3-3, both of which might regulate the Ras/Raf/MEK/ERK pathway, one affecting Ras and the other one affecting Raf. Here we demonstrate the function of Bcr as a negative regulator of Ras, which acts via AF-6 in a PDZ domain-dependent manner.
Bcr contains additional functional domains besides its kinase domain, e.g., the GAP domain, which regulates Rho/Rac-dependent signaling. Rho-dependent signaling is involved in organization of the actin cytoskeleton at sites of cell-cell contact (9
). AF-6 also associates with the cytoskeleton through binding to profilin, which is a regulator for actin polymerization and participates in cortical actin assembly (1
). Thus, the Bcr/AF-6 complex could play an important role in regulating signaling pathways at sites of cellular junctions, but it may as well be important for the structural organization of the cell.