Two tyrosine kinases, Src64 and Tec29, regulate the growth of actin rich-ring canals in the Drosophila ovary. Src64 directs the localization of Tec29 to ring canals, but the mechanism underlying this process has remained unknown. This study shows that Tec29 localizes to ring canals via its Src homology 3 (SH3) and Src homology 2 (SH2) domains. Tec29 activity is required for its own ring canal localization, suggesting that a phosphotyrosine ligand for the SH2 domain is generated by Tec29 itself. Src64 regulates this process by phosphorylating Y677 within the kinase domain of Tec29, an event required for Tec29 activation. The pleckstrin homology (PH) domain of Tec29 has dual functions in mediating Src64 regulation. In the absence of Src64, the PH domain prevents Tec29 ring canal localization. In the presence of Src64, it enhances membrane targeting of Tec29 by a PI(3,4,5)P3-mediated mechanism. In the absence of its PH domain, Tec29 constitutively localizes to ring canals, but still requires Src64 for full activation (Lu, 2005).
Thus SH3 and SH2 protein-protein interaction domains of Tec29 are necessary and sufficient for ring canal localization. Localization of a truncated protein that contains these two domains (DeltaKinase), however, is dependent on endogenous Tec29 activity. A likely reason for this result is that endogenous Tec29 activity can generate ring canal binding sites for the SH2 domain of DeltaKinase. The fact that Tec29 activity is directly correlated with phosphotyrosine contents on ring canals is consistent with this model. Alternatively, endogenous Tec29 may phosphorylate DeltaKinase, thus allowing it to bind to an SH2-domain-containing protein on the ring canal. A tyrosine residue within the SH3 domain of Btk has been shown to be a major autophosphorylation site (Rawlings, 1996). When the corresponding tyrosine is mutated in Tec29, however, the mutant protein localizes to ring canals and fully rescues all defects associated with Tec29 mutants, indicating that this residue is not important for the function of Tec29 on ring canals. In addition, no phosphotyrosine content was detected within the DeltaKinase protein by immunoblotting with an antiphosphotyrosine antibody. Therefore, the most likely scenario is that Tec29 phosphorylates a ring canal protein, thus generating binding sites for its own SH2 domain (Lu, 2005).
Since Src64 protein is localized to nurse cell cortical membrane, as well as ring canals, it is interesting to consider why localization of Tec29, a process regulated by Src64, is directed to ring canals. One possible explanation is that Src64 activates Tec29 everywhere on the membrane, but the substrate (and therefore binding partner) of Tec29 is only present on ring canals. Alternatively, Tec29's substrate may be present on membranes and ring canals, but Src64 may be activated only at ring canals to ensure local activation of Tec29. A third possibility is that activation of Src64 and Tec29's substrates are both restricted to ring canals. The data that overexpressed type 1 Tec29 localizes to ring canals in the absence of Src64 are consistent with Tec29 substrates being on ring canals only. Therefore a model is proposed for how type 2 Tec29 may localize to ring canals. Interactions between the PH domain and PIP3 can target type 2 Tec29 to the cortical membrane, thus allowing it to be phosphorylated and activated by Src64. Once activated, the Tec29 protein that is localized to the membrane region adjacent to ring canals might access and phosphorylate substrate proteins, and bind to them via its SH3 and SH2 domains. In addition, ring-canal-localized Tec29 can phosphorylate additional substrate proteins, thus generating ring canal binding sites and directly recruit undocked Tec29 protein from the cytoplasm through a positive feedback mechanism (Lu, 2005).
The results suggest that Tec29's binding partner on the ring canal is one of its substrates. This substrate may be a scaffolding protein, on which a signaling complex can assemble, or it may be an effector that is directly involved in the formation and rearrangement of actin networks on ring canals. Several proteins that interact with either the SH3 (hnRNP-K, Vav, WASp, etc.) or the SH2 domain (BLNK, BRDG-1, SLP-76) of other TFKs have been identified. Although none of these proteins has been shown to be a direct substrate of TFKs, their Drosophila homologs remain attractive candidates for downstream effectors of Tec29. For example, the SCAR protein, which has functions similar to WASp in promoting actin polymerization, has been shown to localize to actin-rich structures during Drosophila development. SCAR mutant ring canals have abnormal morphology and exhibit growth defects. Analysis of the phosphorylation of these and other candidate proteins, including Kelch, in response to Tec29, their mutant phenotypes in the ovary and the functional consequences of their potential association with Tec29, may lead to the identification of Tec29's ring canal binding partner (Lu, 2005).
TFKs are the only group of nonreceptor protein tyrosine kinases that contain PH domains. The existence of Tec29 splicing variants that differ in this domain provide an opportunity to analyze the in vivo significance of its function and how it affects Tec29 localization. The results show that the PH domain can accentuate Src64 regulation of Tec29 by inhibiting Src64-independent ring canal localization of Tec29. Interestingly, a recent study has shown that truncation of the PH domain increases the basal activity of Btk in vitro, but eliminates PIP3-dependent regulation (Saito, 2001). This suggests that an inhibitory function by the PH domain may be more universal among other TFKs, perhaps to confer a more stringent regulatory mechanism by activators such as SFKs. However, these results also raised questions as to the biological significance of type 1 Tec29. The fact that type 1 Tec29 lacks a PH domain dictates that it can be regulated by Src64 but not PIP3. This may be important in other tissues and developmental processes, where temporal and spatial coordination of multiple signaling pathways may be critical. Interestingly, Tec29 has been shown to be involved in dorsal closure and male genital formation (Baba, 1999a; Tateno, 2000). Type 1 or type 2 Tec29-specific expression in the CNS has also been described (Baba, 1999a). Further experiments will be necessary to address the physiological importance of type 1 and type 2 Tec29 proteins in these tissues (Lu, 2005).
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