title: 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 γ-secretase. Western blotting and cell fractionation studies revealed that HEK293T and MDCK cells transfected with an eGFP-tagged podoplanin construct (PDPNeGFP, 50-63kDa) constitutively express two C-terminal fragments (CTFs): a ∼33kDa membrane-bound PCTF33, and a ∼29kDa cytosolic podoplanin ICD (PICD). While pharmacological inhibition of metalloproteases reduced the expression of PCTF33, treatment of cells with γ-secretase inhibitors resulted in enhanced PCTF33 levels. PCTF33 processing by γ-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 γ-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/γ-secretase.
authors.affiliation: Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain.
authors.forename: Maria M
authors.initials: MM
authors.lastname: Yurrita
authors.affiliation: Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Fernández-Muñoz
authors.affiliation: Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain.
authors.forename: Gaelle
authors.initials: G
authors.lastname: Del Castillo
authors.affiliation: Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain.
authors.forename: Ester
authors.initials: E
authors.lastname: Martín-Villar
authors.affiliation: Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain.
authors.forename: Jaime
authors.initials: J
authors.lastname: Renart
authors.affiliation: Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain. Electronic address: mquintanilla@iib.uam.es.
authors.forename: Miguel
authors.initials: M
authors.lastname: Quintanilla
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D004285:Dogs; D057809:HEK293 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D053764:Presenilin-1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000098853:Podoplanin
chemical_list: D053829:Amyloid Precursor Protein Secretases; D008562:Membrane Glycoproteins; D053764:Presenilin-1; C117468:PDPN protein, human; D000098853:Podoplanin
keywords: C-terminal fragment; CT; CTF; EC; ERM; ICD; MEF; Metalloprotease; PDPN; PS; Podoplanin; Proteolytic processing; RIP; TM; cytoplasmic; extracellular; ezrin radixin moesin; fl-PDPN; full-length PDPN; intracellular domain; mouse embryonic fibroblast; podoplanin; presenilin; regulated intramembrane proteolysis; transmembrane; γ-Secretase