Exosomes re-suspended in PBS at a concentration of 5?g of protein per ml were further diluted 100- to 500-fold to achieve between 20 and 100 objects per frame

Exosomes re-suspended in PBS at a concentration of 5?g of protein per ml were further diluted 100- to 500-fold to achieve between 20 and 100 objects per frame. Direct phosphorylation assay and mass spectrometry confirm that PKM2 phosphorylates SNAP-23 at Ser95. Ectopic expression of non-phosphorylated SNAP-23 mutant (Ser95Ala95) significantly reduces PKM2-mediated exosomes release whereas expression of selective phosphomimetic SNAP-23 mutants (Ser95Glu95 but not Ser20Glu20) rescues the impaired exosomes release induced by PKM2 knockdown. Our findings reveal a non-metabolic function of PKM2, an enzyme associated with tumour cell reliance on aerobic glycolysis, in promoting tumour cell exosome release. As a mechanism to communicate with the microenvironment, tumour cells actively release large quantity of extracellular vesicles (EVs), including exosomes, microvesicles (MVs) or microparticles, and apoptotic body. These tumour-released EVs, which are abundant in the body fluids of patients with malignancy, play a critical role in promoting tumour growth and progression1,2. For example, NCI-H460 tumour cells actively release MVs made up of EMMPRIN, a transmembrane glycoprotein highly expressed by tumour cells, MV-encapsulated EMMPRIN that facilitates tumour invasion and metastasis via stimulating matrix metalloproteinase expression in fibroblasts3. Tumour cell exosomes also deliver active Wnt proteins to regulate target cell -catenin-dependent gene expression4. Malignancy cell-derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation phosphorylation assay was performed using both the recombinant SNAP-23 (rSNAP-23) and the recombinant PKM2 (rPKM2) purified from nuclear extracts of SW620 cells21. Since PKM2 uses PEP instead of ATP as a phosphate donor to phosphorylate ADP in the glycolysis, we replaced ATP by PEP in the reaction. After incubation under numerous conditions at room heat for 1?h, the reaction mixtures were then subjected to SDS-PAGE or Phos-tag SDS-PAGE analysis detection of SNAP-23 phosphorylation. As shown in Fig. 6a, WB analysis demonstrated that this rSNAP-23 was phosphorylated by the rPKM2 in the presence of PEP, confirming that PKM2 acts as a protein kinase to remove the phosphate group from PEP and puts the phosphate on SNAP-23. Open in a separate window Physique 6 Direct phosphorylation of recombinant SNAP-23 (rSNAP-23) at Ser95 by recombinant PKM2 (rPKM2).(a) Direct phosphorylation of rSNAP-23 by rPKM2. The rSNAP-23 was incubated with or without PEP, rPKM2 or PEP plus rPKM2 at room heat for 1?h. The reaction mixtures were then subjected to SDS-PAGE or Phos-tag SDS-PAGE analysis. SNAP-23 was detected Dimethyl biphenyl-4,4′-dicarboxylate by anti-SNAP-23 antibody in WB analysis. (b) Phosphorylated SNAP-23 by rPKM2 analysed by mass spectrometry (MS). Note that MS analysis of tryptic fragment Dimethyl biphenyl-4,4′-dicarboxylate of rSNAP-23 treated with PEP/rPKM2 Rabbit polyclonal to SR B1 matches to the peptide 92NFESGK97 of SNAP-23, suggesting that SNAP-23 Ser95 was phosphorylated. To identify the phosphorylation site on SNAP-23 used by PKM2, we further performed mass spectrometry (MS) analysis of purified recombinant SNAP-23 after phosphorylation assay (http://proteomecentral.proteomexchange.org, accession code: PXD005204). After fragmentation using trypsin, MS analysis recognized a phosphorylated fragment matched to the peptide 92NFESGK97, suggesting that Ser95 was phosphorylated (Fig. 6b). The theoretical mass-to-charge ratio of ions with Ser95 phosphorylation (Y+ ions) and Ser95 dephosphorylation (Y+-P ions) are outlined in Fig. 6b. There were five ions detected and marked in reddish. To further examine the role of phosphorylation of SNAP-23 by PKM2 in mediating tumour cell exosome release, we built three plasmids expressing SNAP-23 mutants. The Ser95 of wild-type (WT) SNAP-23 was changed with Glu95 (SNAP-23 (Ser95Glu95)), whose carbolyic acid side chain shall imitate the result of phosphorylation. In contrast, to render a dephosphorylated condition constitutively, we changed Ser95 of WT SNAP-23 with Ala95 (SNAP-23 (Ser95Ala95)). To make sure that serine phosphorylation by PKM2 may be the important factor (instead of phosphorylation of various other residue) allowing the part of SNAP-23 in exosome exocytosis, we also mutated Ser20 of SNAP-23 to Glu20 (SNAP-23 (Ser20Glu20)). Furthermore to producing three mutated variations of SNAP-23 DNA, we also produced siRNA-resistant constructs for Dimethyl biphenyl-4,4′-dicarboxylate every of our three mutated SNAP-23 plasmids. As demonstrated in Figs 3 and 7a nucleotides inside the.