Abstract
Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs) containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1) domains, while the membrane-distal (D2) domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.
Highlights
The activity of Protein Tyrosine Phosphatases (PTPs) is critical for the regulation of signaling networks that govern cell growth, differentiation and communication
The tandem PTP domains of Phosphatase on Drosophila chromosome band 99A (PTP99A) (PTP99A D1D2) and Drosophila Leukocyte antigen Related (DLAR) (DLAR D1D2), the individual PTP domains (D1 and D2) as well as the active site mutants were expressed in E. coli and were purified using the protocols described earlier (Figure 1a, Figure S1a, b & c) [13]
Phosphatase activity measurements on tandem PTP domains of PTP99A and DLAR reveal that the PTPase activity in both PTP99A and DLAR was localized to the D1 domain, with no detectable activity in the D2 domain (Figure 1b, Figure 2a, b, c & d and Table S2)
Summary
The activity of Protein Tyrosine Phosphatases (PTPs) is critical for the regulation of signaling networks that govern cell growth, differentiation and communication. While the guidance decision of the ISN axons to navigate past their first branch point requires concerted activity of DLAR and PTP99A, the entry of the Segmental Nerve b (SNb) into the Ventrolateral Muscle field depends on the tightly modulated antagonistic actions of the two RPTPs [3,4,5]. These observations could be rationalized by a model wherein the synergistic action of DLAR and PTP99A relies on common substrates resulting in the transduction of identical downstream signals. We report experimental data and computational studies that suggest that interactions between the two PTP domains of these RPTPs play a significant role in the catalytic activity and substrate specificity of the proteins
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