Podoplanin is a substrate of presenilin-1/γ-secretase

Abstract

Podoplanin (PDPN) is a mucin-like transmembrane glycoprotein that plays an important role in development and cancer. Here, we provide evidence that the intracellular domain (ICD) of podoplanin is released into the cytosol following a sequential proteolytic processing by a metalloprotease and -secretase. Western blotting and cell fractionation studies revealed that HEK293T and MDCK cells transfected with an eGFP-tagged podoplanin construct (PDPNeGFP,(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63) constitutively express two C-terminal fragments (CTFs): a ~3 3 kDa membrane-bound PCTF33, and a ~2 9 kDa cytosolic podoplanin ICD (PICD). While pharmacological inhibition of metalloproteases reduced the expression of PCTF33, treatment of cells with -secretase inhibitors resulted in enhanced PCTF33 levels. PCTF33 processing by -secretase depends on presenilin-1 (PS1) function: cells expressing a dominant negative form of PS1 (PS1 D385N), and mouse embryonic fibroblasts (MEFs) genetically deficient in PS1, but not in PS2, show higher levels of PCTF33 expression with respect to wild-type MEFs. Furthermore, transfection of PS1 deficient MEFs with wild-type PS1 (PS1 wt) decreased PCTF33 levels. N-terminal amino acid sequencing of the affinity purified PICD revealed that the -secretase cleavage site was located between valines 150 and 151, but these residues are not critical for proteolysis. We found that podoplanin CTFs are also generated in cells expressing podoplanin mutants harboring heterologous transmembrane regions. Taken together, these results indicate that podoplanin is a novel substrate for PS1/-secretase.

The Role of Podoplanin in the Biology of Differentiated Thyroid Cancers

“…Binding of podoplanin to these proteins through its cytoplasmic domain anchors PDPN to the actin cytoskeleton, what leads to the activation of small Rho GTPases and the induction of cell migration and invasion, as well as epithelilal-mesenchymal transition [21], [56], [69]. Moreover, as demonstrated recently, intracellular domain of podoplanin undergoes cleavage by γ-secretase, releasing a short intracellular domain into cytosol, which is suggested to play an important role in the podoplanin signaling and function [70]. Furthermore, a number of other proteins have been proposed to act together with podoplanin in controlling cell motility, migration and invasion, including molecules of the extracellular matrix [21], [56], [71], and matrix metalloproteinases (MMP-1, MMP-2, MMP-9, MMP-10), which are also thought to be involved in PDPN-dependent tumor progression [72], [73].…”

Plasma soluble podoplanin is a novel marker for the diagnosis of tumor occurrence and metastasis

“…PDPN is also expressed in many tumor cells, including squamous cell carcinoma, malignant mesothelioma, Kaposi's sarcoma, angiosarcoma, hemangioblastoma, testicular seminoma, dysgerminoma and brain tumors . PDPN is composed of 3 structural domains: a highly O ‐glycosylated extracellular domain, a hydrophobic transmembrane domain, and a short cytoplasmic domain . PDPN induces platelet aggregation through direct binding of its extracellular domain to C‐type lectin‐like receptor 2 (CLEC‐2) expressed on the platelet surface .…”

New Insights into the Role of Podoplanin in Epithelial–Mesenchymal Transition

“…In addition, we have described that the ectodomain of podoplanin is first cleaved by a metalloprotease and, thereafter, the membrane-bound C-terminal fragment is -secretase, between Val150 and Val151, in the cytoplasmic side of the TM domain, thus releasing the intracellular domain (Yurrita et al, 2014). …”