Protein tyrosine phosphatase 69D

Protein Interactions

Genetic analysis of growth cone guidance choice points in Drosophila has identified neuronal receptor protein tyrosine phosphatases (RPTPs) as key determinants of axon pathfinding behavior. The Drosophila Abl tyrosine kinase functions in the intersegmental nerve b (ISNb) motor choice point pathway as an antagonist of the RPTP Dlar. The function of Abl in this pathway is dependent on an intact catalytic domain. The Abl phosphoprotein substrate Enabled (Ena) is required for choice point navigation. Both Abl and Ena proteins associate with the Dlar cytoplasmic domain and serve as substrates for Dlar in vitro, suggesting that they play a direct role in the Dlar pathway. These data suggest that Dlar, Abl, and Ena define a phosphorylation state-dependent switch that controls growth cone behavior by transmitting signals at the cell surface to the actin cytoskeleton (Wills, 1999).

In addition to the Dlar D2 domain, Drosophila Abl can weakly phosphorylate the D2 domain of another receptor tyrosine kinase, Ptp69D; this is interesting, since Ptp69D is tyrosine phosphorylated in S2 cells. The physical interactions between Abl and Dlar support a model whereby both proteins function in the same signaling pathway. Furthermore, the phosphorylation of the D2 domain in vitro raises the intriguing possibility that d-Abl activity regulates Dlar function in vivo. The genetic relationship between Abl and Dlar and the requirement of Ena function for ISNb target entry suggest that Ena might act in the Dlar signaling pathway. To test this model, it was asked whether Ena associates with the cytoplasmic domain of Dlar. Endogenous Ena protein associates with a Dlar full-length cytoplasmic domain (GST-Dlar D1-D2) or with D2 alone but not comparably with wild-type D1. Since Abl is known to associate with Ena, and since binding between Abl and Dlar has been demonstrated, it is possible that Ena binding to Dlar requires Abl or additional proteins. Purified Ena has been shown to bind to the Dlar cytoplasmic domain. In both extract and recombinant protein binding assays, Ena shows only weak association with DPTP10D. However, Ena binds effectively to the D2 domain of Ptp69D. The preferential binding of Ena to the D2 domains of Dlar and Ptp69D, as compared with the D1 domains of the same RPTPs, suggests that these interactions are specific. The parallel between Dlar and Ptp69D binding is interesting, given the published observation that Ptp69D is required for ISNb guidance and can partially substitute for Dlar in vivo. Furthermore, the nature and penetrance of ISNb defects in ena mutants suggest that Ena may function downstream of multiple inputs (Wills, 1999).

Previous studies have demonstrated that a number of mammalian RPTPs (including LAR, RPTPµ, and RPTPsigma) are cleaved within their extracellular domains. Since the extracellular domain can be shed from the cell surface, the extracellular and intracellular domains of these proteins may function separately. A model for Ptp69D function in which ligand binding regulates the activity of the intracellular domain requires that the protein or a fraction thereof is not cleaved, or alternatively, if cleaved, the two fragments must remain associated. A test was performed to see whether Ptp69D is proteolytically cleaved and whether the extracellular and intracellular domains associate with each other. A monoclonal antibody directed toward an N-terminal extracellular epitope of Ptp69D recognizes a single band of about 110 kDa on Western blots of extracts prepared from third instar eye-brain complexes. Since this is substantially smaller than the size predicted based on its amino acid composition (about 180 kDa), it suggested that Ptp69D could be proteolytically processed. This was examined further in S2 cell lines and in eye-brain complexes from transgenic animals expressing Ptp69D with a C-terminal Myc-epitope tag. In transfected S2 cells, both the N- and C-terminal-directed antibodies recognize a common band of about 200 kDa, which is proposed to correspond to the full-length glycosylated form of the protein. While the N-terminal-directed antibody recognizes the 110 kDa species previously observed in extracts of eye-brain complexes, the C-terminal antibody recognizes a band of about 90 kDa. Examination of the Ptp69D sequence reveals a basic residue-rich sequence, KLRDKR, in the MPR that may serve as a proteolytic cleavage site to generate these two fragments. Since the 200 kDa band is not observed in extracts of eye-brain complexes, proteolytic cleavage in the developing animal is efficient (Garrity, 1999).

Since the Ptp69D protein is cleaved, the ability of the two fragments to associate in transfected S2 cells was assessed. The C-terminal fragment of Ptp69D was immunoprecipitated from S2 cells expressing the C-terminal epitope-tagged Ptp69D using anti-Myc antibody. The N-terminal fragment was also found in the immunoprecipitate, indicating that some of the cleaved fragments remain associated with each other. Although these results do not exclude a model in which the two fragments function separately, they are consistent with a model in which they function together in a complex to regulate targeting (Garrity, 1999).

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