The tumor suppressor p27Kip1 undergoes endo-lysosomal degradation through its interaction with sorting nexin 6

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The proteolytic degradation of p27 contributes significantly to cell proliferation in physiological and pathological conditions. It is generally accepted that p27 degradation occurs mainly through the proteasomal pathway (19, 26). To the best of our knowledge, here we provide the first evidence for the existence of an endo-lysosomal pathway for the proteolytic degradation of p27 triggered by its interaction with the vesicular-trafficking regulator SNX6, which contributes to p27 downregulation in the early stages of the mitotic cell cycle. The following findings support this notion: 1) p27 and SNX6 interact both in vitro (yeast-two hybrid and GST-pull down assays) (Fig.1) and in vivo (FRET) (Fig.2); 2) p27 and SNX6 partially colocalize at the endosome (Fig.3) and SNX6 gain-of- and loss-of-function increases and reduces, respectively, the cytoplasmic and endosomal localization of p27 (Fig.4); 3) a fraction of p27 localizes to the lysosome, its mitogen-induced degradation is attenuated by lysosomal inhibitors and SNX6 silencing (Fig.5); and 4) SNX6 loss-of-function delays cell-cycle progression (Fig.6). In order to validate the unanticipated localization of p27 in endosomes and lysosomes, we have utilized different controls: 1) our studies were carried out in several cell lines, thus suggesting that the observed subcellular localization of p27 is a general feature of mammalian cells; 2) two different anti-p27 antibodies were employed, both of which revealed endosomal and lysosomal localization of p27 using several organelle-specific markers (e.g., EEA1, lysotracker, LAMP1, LAMP2); 3) the coexpression of p27 and SNX6 in endosomes observed by immunofluorescence assays was confirmed by Western blot analysis of endosome-enriched subcellular fractions; and 4) colocalization of p27 and SNX6 was confirmed in cells expressing fluorescently–labeled p27 and SNX6 proteins.

SNXs comprise a family of more than 30 mammalian proteins involved in intracellular protein trafficking and prodegradative sorting of proteins (33-35, 37, 55). SNX6 is a component of the retromer (45), the multimeric protein complex which mediates retrograde endosome-to-Golgi transport of proteins (55). SNX6 also interacts with the TGF-β family of receptor Ser-Thr kinases and negatively regulates TGF-β signalling (40) possibly due to receptor degradation. Moreover, SNX6 has been recently shown to interact with the G-protein coupled receptor kinase-2 interacting protein 1 and to accelerate degradation of the epidermal growth factor receptor (54), thus highlighting its prodegradative role. In this study, we identified SNX6 as a novel interaction partner of p27 in yeast two-hybrid assays. GST-pull down assays confirm the association of p27 and SNX6 and demonstrate that p27/SNX6 heterodimerization requires the proline-rich motif of p27 located at amino acids 90-96, which also mediates its interaction with Grb2 (38, 56). Supporting the in vivo occurrence of an interaction between p27 and SNX6, we found that both proteins colocalize in endosomes, as demonstrated by immunofluorescence experiments and Western blot analysis of subcellular fractions. Moreover, our FRET studies provide direct evidence that p27 and SNX6 interact in vivo. This interaction, however, is likely to occur in a very short temporal window and/or may require a particular cellular environment which is lost upon cell lysis (perhaps the endosome), since we have not been able to coimmunoprecipitate the endogenous p27 and SNX6 proteins under various experimental conditions.

Proteins that undergo lysosomal-dependent degradation can be first detected in early endosomes, which sequentially mature into late endosomes and lysosomes through a mechanism regulated in part by SNXs (46). Thus, the observation that SNX6 and p27 partially colocalize at the endosome and that SNX6 affects the amount of endosomal p27 prompted us to investigate the possible existence of an endo-lysosomal pathway for p27 degradation mediated by its interaction with SNX6. Supporting this notion, we observed colocalization of p27 with several lysosomal markers (e.g. lysotracker, LAMP1, LAMP2) and reduced mitogen-dependent downregulation of p27 upon treatment with pharmacological inhibitors of lysosomal proteolysis. Moreover, SNX6 loss-of-function attenuates the lysosomal pathway of p27 degradation, without affecting its mRNA levels. Thus, in addition to the well-established proteasome-dependent pathway, p27 may also undergo lysosomal proteolysis. We propose the model depicted in Fig.7 whereby the interaction of p27 with SNX6 promotes its translocation to early endosomes, which eventually mature into lysosomes where p27 is degraded. This model is consistent with the observation that SNX6 and p27 colocalize in endosomes, but only p27 is detected in the lysosomal compartment. Moreover, this hypothesis is in agreement with previous findings suggesting a direct role of SNX6 in EGFR targeting to endo-lysosomal degradation (54).

SNX6-mediated endo-lysosomal degradation of p27 seems to play a role in the regulation of cell cycle progression, since SNX6 silencing reduces p27 downregulation during G1 phase and at the G1/S-phase transition and delays S-phase entry upon serum stimulation of starvation-synchronized NIH-3T3 cells. These findings indicate that the interaction of p27 with SNX6 promotes cell proliferation and identify SNX6 as a putative cell cycle regulatory protein. Indeed, it has been reported that ectopically-expressed SNX6 partially rescues growth of embryonic stem cells in the absence of leukemia inhibitory factor (LIF) (57).

In summary, we conclude that p27 can undergo endo-lysosomal degradation through its interaction with the vesicular-trafficking regulator SNX6. This novel pathway of p27 degradation may cooperate with the well-known degradation of p27 via the proteasome, thus allowing the rapid downregulation of p27 that occurs in early stages of the mitotic cell cycle. Additional examples of proteins for which both lysosomal and proteasomal proteolysis have been reported include Fos (58), Jun (59) and Deltex (60). Future studies are warranted to elucidate the molecular mechanisms regulating this novel SNX6-dependent p27 proteolytic pathway and its potential role in proliferative diseases such as atherosclerosis and cancer.