Following receptor engagement, the formation of tyrosine phosphorylation-dependent multimeric complexes involving adapter and effector proteins plays an important role in signal transduction. Upon cell activation through different stimuli, the adapter protein Cbl becomes phosphorylated on tyrosine residues, producing its interaction with SH2-containing signaling molecules (2
). In differentiated adipocytes, insulin stimulates Cbl tyrosine phosphorylation and its association with the adapter protein Crk (36
). Moreover, insulin induces translocation of the Cbl/CAP complex to lipid rafts, where CAP directly binds to the hydrophobic protein flotillin. The insulin-dependent localization of phospho-Cbl to subdomains of the plasma membrane results in the generation of signaling pathways involved in GLUT4 translocation (3
). One major pathway involves the activation of TC10 by C3G that is complexed with Cbl/Crk (6
In adipocytes Cbl phosphorylation seems to be catalyzed by the insulin receptor rather than by Src family tyrosine kinases such as Fyn. Previous studies have also shown that Cbl is able to associate with tyrosine-phosphorylated EGF and PDGF receptors (5
). However, there has been no evidence that the insulin receptor binds to Cbl directly upon insulin stimulation, indicating the requirement of an adapter protein to assist in the complex formation. CAP does not appear to target Cbl directly to the insulin receptor for phosphorylation. In fact, coexpression of CAP with Cbl in 3T3-L1 cells had no effect on Cbl phosphorylation in response to insulin. Therefore, we suspected that 3T3-L1 adipocytes might express a separate adapter protein that is directly involved in the tyrosine phosphorylation of Cbl. Experiments described here revealed that tyrosine-phosphorylated APS is the adapter that couples Cbl to the insulin receptor for phosphorylation. Like CAP, APS is expressed primarily in skeletal muscle, heart, and adipose tissue and in differentiated 3T3-L1 adipocytes (28
). We demonstrate that APS facilitates tyrosine phosphorylation of Cbl on tyrosines 371, 700, and 774. This phosphorylation event is required for the recruitment of Crk to the CAP/Cbl complex and for the subsequent activation of GLUT4 translocation.
APS belongs to the Lnk adapter protein family, which includes Lnk and SH2-B. Members of this protein family contain a PH domain, an SH2 domain, a C-terminal tyrosine-containing motif, and several proline-rich motifs. While Lnk functions predominantly in T-cell receptor (TCR) activation (12
), APS and SH2-B are involved in signaling by various receptors for growth factors such as insulin, insulin-like growth factor 1, PDGF, and nerve growth factor (31
). Although both APS and SH2-B are substrates of numerous tyrosine kinases, APS is the preferential target for the insulin receptor kinase in differentiated adipocytes (16
). Expression of APS in CHO cells overexpressing insulin receptor was previously shown to induce a rapid ubiquitination of the insulin receptor upon ligand binding, presumably through coupling Cbl to the receptor (1
). Our results demonstrate that Y371
is one of the major sites for APS-facilitated phosphorylation in response to insulin. Phosphorylation of Y371
was suggested to play an important role in the suppression of EGF receptor signaling by Cbl (18
). However, preliminary data indicate that expression of APS has no effect on insulin receptor stability or MAP kinase signaling in 3T3-L1 adipocytes, suggesting that this may not be a mechanism for the down regulation of the insulin receptor in physiologically relevant insulin-responsive cells. Indeed, earlier studies indicated that the insulin receptor does not undergo degradation via the ubiquitination pathway and, furthermore, that down regulation of the insulin receptor is a slow process (12 to 24 h) compared to that of EGF and PDGF receptors (29
The inhibition of tyrosine phosphorylation of endogenous Cbl and GLUT4 translocation through overexpression of the APS/Y618
F dominant negative mutant is consistent with its role as an upstream signaling intermediate in the CAP/Cbl pathway. However, it was somewhat surprising that low- and high-level expression of the wild-type APS protein conferred opposite biological effects. It is possible that a multiprotein complex is present in 3T3-L1 adipocytes required for the coordinate regulation of Cbl phosphorylation by insulin, in which APS is responsible for the direct targeting of Cbl to the insulin receptor. The expression levels of proteins involved in such complexes are usually delicately regulated in cells. Therefore, excess expression of a single component like APS may simply interfere with the stoichiometry of the complex formation, resulting in the inhibition instead of enhancement of Cbl phosphorylation. Indeed, it was demonstrated in a recent study that APS was capable of forming multimeric structures and may exist in large protein complexes in vivo (31
). Similarly, ectopic overexpression of Lnk in Jurkat T cells caused inhibition of anti-CD3-induced TCR activation of NF-AT transcription activity (19
). On the other hand, earlier studies suggest a positive role of endogenous Lnk in TCR signaling, because upon TCR activation Lnk becomes tyrosine phosphorylated and signals to the PI 3-kinase, phospholipase Cγ1, and Ras pathways through its multifunctional tyrosine phosphorylation site (12
). In addition, overexpression of either MP1 or JIP1, two adapter proteins that promote the activation of different MAP kinases, has been shown to have inhibitory effects on the respective kinase pathways in transfected cells (38
Our study indicates that APS and CAP are localized to the plasma membrane and specifically associate with each other independently of insulin stimulation. The physiological significance of the APS-CAP interaction remains unclear. Deletion of the SoHo domain of CAP increased binding of APS but decreased binding of Cbl. Therefore, the SoHo domain appears to play an important role in controlling the overall conformation of CAP and the binding specificity of its SH3 domains for different proteins. Since the SoHo domain interacts with the lipid raft protein flotillin, it would be interesting to determine whether and how the association with flotillin influences the interaction of CAP with APS and Cbl, respectively.
Since the C-terminal SH3 domain of CAP is used by both APS and Cbl for binding, it seems unlikely that CAP would directly bridge the formation of a ternary complex including APS and Cbl. However, a multimeric complex containing these proteins may still be present, considering the possible involvement of additional molecules. Although APS is sufficient to facilitate Cbl phosphorylation in a coexpression experiment where both APS and Cbl exist in excess amounts, CAP seems to serve an indispensable role in the control of tyrosine phosphorylation of endogenous Cbl in vivo (3
). We have previously shown that expression of a CAP mutant deficient in flotillin/lipid raft association (ΔSoHo) inhibits insulin-stimulated tyrosine phosphorylation of Cbl and the translocation of GLUT4 in 3T3-L1 adipocytes (15
). This apparent dominant negative effect may result from mistargeting of Cbl or APS and is consistent with a model in which Cbl phosphorylation by the insulin receptor is coordinated by a complex of proteins rather than directed by APS alone. In this regard, it is important to note that APS, CAP, and Cbl all are large multifunctional adapters, each of which possesses the ability to bind multiple proteins simultaneously.