Pyruvate kinase type M2 promotes tumor cell exocytosis via phosphorylating synaptosome-associated protein-23

Tumor cells secret large amount of microvesicles (MVs) which are involved in remodeling tumor-stromal interactions to promote malignancy. The regulatory mechanisms governing the active exocytosis in tumor cells, however, remain incompletely understood. We show here that tumor cell exocytosis is tightly controlled by aerobic glycolysis (Warburg effect) and pyruvate kinase type M2 ( PKM2), which is commonly upregulated and phosphorylated in tumor cells, plays critical roles in promoting exocytosis. Upon stimulation by EGF, more phosphorylated PKM2 switches to dimer form and is recruited to exosomes where it associates with synaptosome-associated protein (SNAP)-23 (SNAP-23). The association of PKM2 and SNAP-23 in secreted exosomes is confirmed by cross immunoprecipitation and Western blot analysis. Dimerized PKM2 enhances the exosome release via phosphorylating SNAP-23. Direct phosphorylation assay and mass spectrometry identifies SNAP-23 phosphorylation at Ser 95 by PKM2. Ectopic expression of non-phosphorylated SNAP-23 mutant (Ser 95 →Ala 95 ) significantly reduces PKM2-mediated exocytosis whereas phosphomimetic SNAP-23 mutants (Ser 95 →Ele 95 ) partially rescue the impaired exocytosis in tumor cells with PKM2 knockdown. Our findings reveal a previously unrecognized non-metabolic function of PKM2 in promoting tumor cell exocytosis.