Abstract
Receptor-type protein tyrosine phosphatase α (RPTPα) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. The domain structure of RPTPs comprises an extracellular region, a transmembrane helix, and two tandem intracellular catalytic domains referred to as D1 and D2. The D2 domain of RPTPs is believed to mostly play a regulatory function; however, no regulatory model has been established for RPTPα-D2 or other RPTP-D2 domains. Here, we solved the 1.8 Å resolution crystal structure of the cytoplasmic region of RPTPα, encompassing D1 and D2, trapped in a conformation that revealed a possible mechanism through which D2 can allosterically inhibit D1 activity. Using a D2-truncation RPTPα variant and mutational analysis of the D1/D2 interfaces, we show that D2 inhibits RPTPα phosphatase activity and identified a 405PFTP408 motif in D1 that mediates the inhibitory effect of D2. Expression of the gain-of-function F406A/T407A RPTPα variant in HEK293T cells enhanced SRC activation, supporting the relevance of our proposed D2-mediated regulation mechanism in cell signaling. There is emerging interest in the development of allosteric inhibitors of RPTPs but a scarcity of validated allosteric sites for RPTPs. The results of our study not only shed light on the regulatory role of RPTP-D2 domains, but also provide a potentially useful tool for the discovery of chemical probes targeting RPTPα and other RPTPs.
Highlights
Receptor-type protein tyrosine phosphatase ␣ (RPTP␣) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis
Because residues at the D1/D2 interface and linker region are highly conserved between receptor PTPs (RPTPs)␣ and RPTP⑀ (Fig. 1F), we speculated that the observed differences might reflect a degree of interdomain flexibility constrained as distinct rigid conformations by the crystal packing
Despite D2’s modest direct contribution to the enzymatic activity, the observation that, in the crystal structure of RPTP␣, residues in D2 come in close contact and restrict the conformational freedom of the crucial PXXP motif suggests that D2 might be able to directly regulate the activity of D1
Summary
Receptor-type protein tyrosine phosphatase ␣ (RPTP␣) is an important positive regulator of SRC kinase activation and a known promoter of cancer growth, fibrosis, and arthritis. Because residues at the D1/D2 interface and linker region are highly conserved between RPTP␣ and RPTP⑀ (Fig. 1F), we speculated that the observed differences might reflect a degree of interdomain flexibility constrained as distinct rigid conformations by the crystal packing.
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