During Drosophila embryogenesis, both the cytoplasmic Abelson tyrosine kinase (Abl) and the membrane bound tyrosine phosphatase PTP69D are required for proper guidance of CNS and motor axons. Evidence is provided that PTP69D modulates signaling by Abl and its antagonist, Ena. An Abl loss-of function mutation dominantly suppresses most Ptp69D mutant phenotypes including larval/pupal lethality and CNS and motor axon defects, while increased Abl and decreased Ena expression dramatically increase the expressivity of Ptp69D axonal defects. In contrast, Ptp69D mutations do not affect Abl mutant phenotypes. These results support the hypothesis that PTP69D antagonizes the Abl/Ena genetic pathway, perhaps as an upstream regulator. It was also found that mutation of the gene encoding the cytoplasmic Src64B tyrosine kinase exacerbates Ptp69D phenotypes, suggesting that two different cytoplasmic tyrosine kinases, Abl and Src64B, modify PTP69D-mediated axon patterning in quite different ways (Song, 2008).

Two enzyme classes, tyrosine kinases and tyrosine phosphatases, dynamically maintain protein phosphotyrosine modifications that are critical for axon guidance. Studies that revealed physical interactions between members of these families have led to an investigation of the relationship between the membrane bound tyrosine phosphatase, PTP69D and cytoplasmic tyrosine kinases. Evidence is provided that PTP69D modulates signaling by the tyrosine kinase, Abl, and its substrate Ena. (1) Ptp69D mutant phenotypes, including adult lethality, embryonic CNS and ISNb motor axon defects, are significantly suppressed by loss of Abl function, and dramatically enhanced by gain of Abl function. (2) Ptp69D does not suppress Abl, suggesting that their interaction is asymmetric. (3) Ena, a strong suppressor and a downstream substrate of Abl, dominantly exacerbates the defects of Ptp69D.

Abl mutants display evident phenotypes such as adult lethality and ISNb arrest defects. Bi-directional suppression was expected between Ptp69D and Abl by analogy to Dlar mutations, which interact symmetrically with Abl for such phenotypes as midline crossing defects in the CNS. However, although Abl mutants modify Ptp69D phenotypes, no evidence was found of reciprocal suppression by introducing Ptp69D mutations into Abl embryos despite trying various combinations of alleles. While several models might explain this, one simple interpretation is that Abl is epistatic to Ptp69D, i.e., Ptp69D acts through Abl (Song, 2008).

What could be downstream targets of PTP69D and Abl? As a substrate of Abl and antagonistic genetic component of the Abl pathway, Ena is an excellent candidate, and indeed, ena mutations enhanced the lethality and axonal defects of Ptp69D mutants. Ena is known to play a role in cell motility, and likely supports F-actin assembly within cells by antagonizing capping protein at the barbed ends of actin and reducing filament branching. In Drosophila, Ena associates with the PTP69D D2 domain and is phosphorylated by Abl in vitro, and its specific cellular localization is regulated by Abl. The consistent pattern of interactions of Ptp69D with Abl and ena (suppressed by Abl mutations and enhanced by the Abl antagonist, ena) supports the idea that the Abl effector, Ena is also a key to signaling by PTP69D (Song, 2008).

The data define a functional relationship among PTP69D, Abl and Ena, but what could be their physical relationship? Extrapolating from genetic interactions to molecular mechanism is not straightforward, however, an attractive speculation that could provide one framework for further thinking is the idea that PTP69D, Abl and Ena may coexist in a complex, where PTP69D inhibits Abl, which in turn inhibits Ena. Such a model would be consistent with the available biochemical evidence, as well as with the genetic interactions observed. Many other models are equally possible, however, and a great deal of additional experimentation would be required to establish this hypothesis. For example, whether these three proteins co-immunoprecipitate has not been investigated, nor have it been demonstrated that they act simultaneously in the same cell. Moreover, although the kinase activity of Abl is required for its axonal function, it is thought that tyrosine phosphorylation of Ena is not the sole function of Abl. Axon guidance by Abl seems also, for example, to be associated with the action of small Rho family GTPases, particularly Rac, and any model for the mechanism of axon guidance by Abl and its partners will have to take these data into account (Song, 2008).

The patterns of genetic interactions of Ptp69D with Abl and ena described in this paper bear some similarities to those of Dlar. Does either RPTP substitute for each other? Previous studies demonstrated that Ptp69D and DLAR cooperate at growth cone choice points along one nerve, ISNb, while along another nerve, ISN, they do not act together. In the adult eye, moreover, a Dlar transgene rescues Ptp69D R7 axon phenotype, but not vice versa. Thus, the relationship between PTP69D and DLAR is complex and depends on cellular context (Song, 2008).

This analysis of PTP69D was extended by investigating the functional interaction of PTP69D with a Drosophila Src gene, Src64B. Mammalian Src protein (c-src) has been shown to regulate the stability and remodeling of actin structures. In Drosophila, Src64B has been shown to function in nervous system development in the embryo, the mushroom body of the adult brain and in the adult eye, making it a plausible candidate for interacting with PTP69D in axon guidance. Moreover, in mouse the RPTP CD45 functions to positively regulate SFK in T cells. Indeed, the data show that Ptp69D does interact with Src64B, but in a sense opposite to that with Abl: Ptp69D and Src64B interact synergistically rather than antagonistically (Song, 2008).

Hints as to a possible mechanism that could underlie the interaction of PTP69D with Src64B are suggested by experiments in mammals. The SFK Fyn binds to LAR and phosphorylates the LAR D2 domain. In turn, LAR dephosphorylates a C-terminal inhibitory motif of Fyn, increasing Fyn activity. The current data as well could potentially be explained by an analogous model whereby PTP69D derepresses Src activity by removing an inhibitory phosphate, though other models are clearly possible and more study is required to test this speculation. It is interesting that the biochemical association of LAR with Fyn in mammals is reminiscent of that observed for DLAR with Abl in Drosophila, but the biological consequences in the two cases are quite different, and in fact opposite: activation of Fyn activity, but suppression of Abl (Song, 2008).

Superficially, it seemed surprising that two cytoplasmic kinases had opposite interactions with PTP69D, antagonizing Abl but cooperating with Src64B. To test this further, the genetic interaction between Src64B and Abl was examined. Although Src64B^Δ17 did not show any significant effect on Abl lethality, it dramatically suppressed the ISNb stall defect of Abl mutants, further supporting the hypothesis that Src64B and Abl kinases may have opposing functions in axon guidance (Song, 2008).

In summary, the receptor protein tyrosine phosphatase PTP69D interacts both with the Abl-Ena tyrosine kinase pathway and with Src64B to control axon patterning in Drosophila. PTP69D antagonizes Abl, perhaps as an upstream regulator, but functions synergistically with Src64B, thus revealing previously unrecognized specificity in the action of these tyrosine kinase pathways (Song, 2008).