We previously identified SH2-B and APS as substrates of Trk kinases. Both proteins associate with tyrosine-phosphorylated Trk receptors and mediate neurotrophin signaling (15
). In the present study, we provide evidence that both SH2-B and APS exist in cells as homomultimers and/or heteromultimers. SH2-B and APS likely exist as homo- and/or heteropentamers, as determined by size exclusion chromatography experiments. The SH2-B multimerization domain is located in its amino terminus; the amino-terminal 243 amino acids are both necessary and sufficient for multimerization of SH2-B. In support of the idea that the multimerization of SH2-B contributes to TrkA signaling, the amino-terminal multimerization domain of SH2-B is necessary for SH2-B-mediated augmentation of NGF-dependent TrkA autophosphorylation as well as morphological differentiation of PC12 cells.
It has recently been shown that SH2-Bβ, a splice variant of SH2-B, interacts with JAK2 and is a potent activator of JAK2 kinase (18
). We found that overexpression of SH2-B does not activate TrkA kinase in the absence of its ligand, NGF. Rather, SH2-B dramatically enhances NGF activation of autophosphorylation of TrkA. Furthermore, the duration of TrkA phosphorylation is prolonged in cells stably expressing SH2-B compared to parental PC12 cells and cells expressing M8, which cannot multimerize. In accordance with these observations, SH2-B, when coexpressed with a TrkA mutant, F8, supports NGF induction of morphological differentiation of PC12nnr5 cells. F8 itself cannot support morphological differentiation of the cells (15
Our study provides evidence that the amino-terminal multimerization domain of SH2-B is critical for NGF signaling. Although overexpression of full-length SH2-B enhances both the magnitude and duration of NGF activation of phosphorylation of TrkA, we observed no enhancement of NGF-induced TrkA kinase activity in PC12 cells expressing a truncated SH2-B mutant, M8, which lacks the amino-terminal multimerization domain. Moreover, only SH2-B mutants that can form multimers confer NGF-induced morphological differentiation of PC12nnr5 cells when coexpressed with F8. The simplest interpretation of these data is that SH2-B multimerization is required for NGF signaling. An alternative possibility is that some function of the amino-terminal motif other than multimerization contributes to SH2-B-mediated signaling. For example, both a proline-rich motif and a tyrosine residue (Y55) lie within the context of a putative phosphotyrosine binding site, an N/HPXY motif, in the amino-terminal region of SH2-B. Deletion of the SH2-B amino terminus, such as in M8, may disrupt potential interactions between SH2-B and putative signaling molecules that associate with these motifs. However, SH2-B mutants carrying either a deletion of the amino-terminal proline-rich motif (M9) or Y55 mutated to phenylalanine (M3) still mediate NGF-induced neurite outgrowth of PC12nnr5 cells when coexpressed with F8 (Fig. A). No other obvious protein-binding motifs are readily identified in the amino-terminal multimerization domain of SH2-B. Therefore, the multimeric nature of SH2-B may be critical for SH2-B-mediated augmentation of TrkA autophosphorylation and signaling.
The mechanism by which SH2-B and APS homo- or heteromultimers potentiate TrkA kinase activation remains unclear. At least three possibilities exist. One possibility is that by interacting with TrkA via the tyrosine residues within the kinase core domain, which are critical for activation and maintenance of TrkA kinase activity (4
), SH2-B and APS can protect these critical tyrosine residues from being dephosphorylated by tyrosine phosphatase(s) and thus enhance and prolong TrkA kinase activity. We have previously shown that SH2-B and APS interact with a TrkA variant lacking all the conserved tyrosines except the three phosphotyrosine residues within the catalytic loop of the TrkA kinase domain (15
). SH2-B was also found to be a substrate of the insulin receptor (IR), and the interaction between SH2-B and IR occurs between the SH2 domain of SH2-B and phosphotyrosines within the catalytic loop of the IR (12
). The amino acids surrounding the catalytic loop tyrosines in Trk and IR kinases are very similar. Interestingly, the JAK-binding protein JAB, a protein that binds via its SH2 domain to a phosphotyrosine within the catalytic loop of the kinase domain of JAK2, has been shown to regulate JAK2 kinase activity (23
). However, we do not favor the idea that SH2-B functions by preventing receptor dephosphorylation because our data indicate that M8, which effectively interacts with phospho-TrkA (Fig. A and data not shown), fails to potentiate NGF-dependent TrkA kinase activity and fails to promote morphological differentiation of PC12 cells. A second possibility is that SH2-B and APS multimers, when associated with activated TrkA receptors, can stabilize TrkA receptor dimers and prevent them from dissociating, thus prolonging TrkA autophosphorylation. A third possibility is that SH2-B and APS pentamers, by interacting with multiple phosphorylated TrkA receptor dimers, induce clustering of receptor dimers. Either of the latter two models is consistent with the observation that mutant M8, which cannot multimerize, fails to support enhancement of NGF-induced TrkA autophosphorylation and signaling. Moreover, these latter two possibilities are not mutually exclusive.
In summary, we have demonstrated that SH2-B and APS exist in cells as homomultimer and/or heteromultimer complexes and that an amino-terminal SH2-B domain that is necessary and sufficient for multimerization is critical for SH2-B function. Determination of the precise mechanism by which SH2-B and APS multimers contribute to autophosphorylation of TrkA should provide insight into NGF signaling in developing neurons.