Fearnley, G. W. | Search bioRxiv

bioRxiv2021

Calcium-binding protein S100A6 interaction with VEGF receptors integrates signaling and trafficking pathways

The mammalian endothelium which lines all blood vessels responds to soluble factors which control vascular development and sprouting. Endothelial cells bind to vascular endothelial growth factor A via two different receptor tyrosine kinases (VEGFR1, VEGFR2) which regulate such cellular responses. The integration of VEGFR signal transduction and membrane trafficking is not well understood. Here, we used a yeast-based membrane protein screen to identify VEGFR-interacting factor(s) which modulate endothelial cell function. By screening a human endothelial cDNA library, we identified a calcium-binding protein, S100A6, which can interact with either VEGFR. We found that S100A6 binds in a calcium-dependent manner to either VEGFR1 or VEGFR2. S100A6 binding was mapped to the VEGFR2 tyrosine kinase domain. Depletion of S100A6 impacts on VEGF-A-regulated signaling through the canonical mitogen-activated protein kinase (MAPK) pathway. Furthermore, S100A6 depletion caused contrasting effects on biosynthetic VEGFR delivery to the plasma membrane. Co-distribution of S100A6 and VEGFRs on tubular profiles suggest the presence of transport carriers that facilitate VEGFR trafficking. We propose a mechanism whereby S100A6 acts as a calcium-regulated switch which facilitates biosynthetic VEGFR trafficking from the TGN-to-plasma membrane. VEGFR-S100A6 interactions thus enable integration of signaling and trafficking pathways in controlling the endothelial response to VEGF-A.

cell biology↗

bioRxiv2019

The receptor PTPRU is a redox sensitive pseudophosphatase

The dynamic regulation of protein tyrosine phosphorylation is a critical feature of intercellular communication and is regulated by the actions of kinases and phosphatases. The receptor-linked protein tyrosine phosphatases (RPTPs) are key signaling molecules that possess an extracellular domain and intracellular phosphatase domains. Most human RPTPs have tandem intracellular tyrosine phosphatase domains: a catalytically active membrane proximal (D1) domain; and a membrane distal (D2) inactive \"pseudophosphatase\" domain. The receptor PTPRU plays a role in development, multiple cancers and has been implicated in the dephosphorylation of cell adhesion proteins. However, PTPRU has a non-canonical D1 domain containing several sequence variations in key catalytic loops that suggest it may function using a mechanism distinct from related RPTPs. Here, we demonstrate through biochemical and structural studies that PTPRU is unique amongst the RPTPs in possessing two pseudophosphatase domains. We show that PTPRU-D1 displays no detectable catalytic activity against a range of phosphorylated substrates and determine that this is due to substantial disorder in the substrate-binding pocket as well as rearrangement of the catalytic loop such that the active site cysteine is occluded. We also show that this cysteine can form an intramolecular disulfide bond with a vicinal \"backdoor\" cysteine. Further, we demonstrate that the PTPRU D2 domain can recruit substrates of related RPTPs suggesting that this pseudophosphatase functions by competing with active phosphatases for the binding of substrates involved in cell adhesion. Therefore, PTPRU is a bona-fide pseudophosphatase and its functional role in cell signaling is via a non-catalytic mechanism.\n\nSIGNIFICANCE STATEMENTProtein tyrosine phosphorylation is a key post-translational modification required for cellular communication that is dynamically regulated by the activities of tyrosine kinases and phosphatases. Receptor tyrosine phosphatases (RPTPs) possess an extracellular receptor domain and intracellular phosphatase domains. We show that PTPRU is a non-canonical RPTP devoid of catalytic activity and demonstrate that this is due to multiple structural rearrangements. Despite this, PTPRU retains the capacity to bind the substrates of related phosphatases suggesting that the non-catalytic function of this pseudophosphatase is to compete with active phosphatases for the binding of substrates. Such pseudoenzymes represent an exciting and growing area of research with implications as key regulators of signaling networks.

biochemistry↗