Abstract
The receptor-linked protein tyrosine phosphatases (RPTPs) are key regulators of cell-cell communication through the control of cellular phosphotyrosine levels. Most human RPTPs possess an extracellular receptor domain and tandem intracellular phosphatase domains: comprising an active membrane proximal (D1) domain and an inactive distal (D2) pseudophosphatase domain. Here we demonstrate that PTPRU is unique amongst the RPTPs in possessing two pseudophosphatase domains. The PTPRU-D1 displays no detectable catalytic activity against a range of phosphorylated substrates and we show that this is due to multiple structural rearrangements that destabilise the active site pocket and block the catalytic cysteine. Upon oxidation, this cysteine forms an intramolecular disulphide bond with a vicinal “backdoor” cysteine, a process thought to reversibly inactivate related phosphatases. Importantly, despite the absence of catalytic activity, PTPRU binds substrates of related phosphatases strongly suggesting that this pseudophosphatase functions in tyrosine phosphorylation by competing with active phosphatases for the binding of substrates.
Highlights
The receptor-linked protein tyrosine phosphatases (RPTPs) are key regulators of cell-cell communication through the control of cellular phosphotyrosine levels
We were unable to detect activity for PTPRU-D1 against pNPP, even at high enzyme concentrations or when using an extended assay duration (Fig. 1d), whereas PTPRK-D1 activity was readily detectable in the low nanomolar range (Fig. 1d)
We show that PTPRU does not exhibit detectable phosphatase activity against a range of substrates or in pTyr dephosphorylation assays with cell lysates
Summary
The receptor-linked protein tyrosine phosphatases (RPTPs) are key regulators of cell-cell communication through the control of cellular phosphotyrosine levels. The PTPRU-D1 displays no detectable catalytic activity against a range of phosphorylated substrates and we show that this is due to multiple structural rearrangements that destabilise the active site pocket and block the catalytic cysteine. Upon oxidation, this cysteine forms an intramolecular disulphide bond with a vicinal “backdoor” cysteine, a process thought to reversibly inactivate related phosphatases. The human classical protein tyrosine phosphatases (PTPs) are key signalling regulators that work with kinases to finetune cellular levels of phosphotyrosine (pTyr), impacting on multiple cellular pathways including metabolism, differentiation, and adhesion[1,2,3]. Despite the importance of catalysis for the function of many PTPs, there are numerous reports of non-catalytic functions[8,9,10]
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