The tumor suppressor p27Kip1 undergoes endo-lysosomal degradation through its
interaction with sorting nexin 6
José J. Fuster1,2,7, José M. González1,7, María D. Edo1,7, Rosa Viana1, Patricia Boya3, Javier
Cervera4, Marcel Verges5, José Rivera2, and Vicente Andrés1,2,6
1
Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy,
Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (IBVCSIC), 46010-Valencia, Spain.
2
Laboratory of Molecular and Genetic Cardiovascular Pathophysiology, Department of
Atherothrombosis and Cardiovascular Imaging, Spanish National Cardiovascular Research
Center (CNIC), 28029 Madrid, Spain
3
Development, Differentiation and Degeneration Laboratory, Department of Cellular and
Molecular Physiopathology, Centro de Investigaciones Biológicas, CSIC, 28040-Madrid,
Spain.
4
Molecular Recognition Laboratory and 5Laboratory of Epithelial Cell Biology, Centro de
Investigación Príncipe Felipe, 46012-Valencia, Spain.
6
Corresponding author.
CNIC, Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Telephone: +34-914531200
7
FAX: +34-914531265
email: vandres@cnic.es
Authors with equal contribution.
Short title: SNX6 targets p27 to endo-lysosomal degradation
1
ABSTRACT
A large body of evidence supports that proteasomal degradation of the growth suppressor p27Kip1
(p27) facilitates mammalian cell cycle progression. However, very few studies have addressed
the possibility of proteasome-independent mechanisms of p27 proteolysis. Here we provide
evidence for a novel pathway of p27 degradation via the lysosome that is mediated by its
interaction with the endosomal protein SNX6, a member of the sorting nexin family of vesiculartrafficking regulators. p27 and SNX6 interact in vitro and in vivo in mammalian cells, partially
colocalize in endosomes and are present in purified endosomal fractions. Gain- and loss-offunction studies revealed that SNX6 induces the endosomal accumulation of p27. Moreover, p27
is detected in lysosomes, and inhibition of lysosomal-dependent proteolysis impairs serummediated downregulation of p27 in a SNX6-dependent manner. To validate the localization of
p27 in these organelles, we analyzed several cell lines using two different anti-p27 antibodies,
several organelle-specific markers (e.g., EEA1, LAMP1, LAMP2, lysotracker), and
overexpression of fluorescent p27 and SNX6. Remarkably, silencing of SNX6 attenuates p27
downregulation in the G1 phase of the mitotic cell cycle and delays cell cycle progression. We
therefore conclude that, in addition to the proteasome-dependent pathway, SNX6-mediated
endo-lysosomal degradation of p27 also contributes to cell cycle progression in mammalian
cells.
Key words: p27, SNX6, endosome, lysosome, proteolysis
2
INTRODUCTION
The transition along the different phases of the mitotic cycle in eukaryotic cells is positively
regulated by the sequential activation of holoenzymes that comprise a catalytic cyclin-dependent
kinase (CDK) and a regulatory cyclin subunit, which are inhibited by their direct interaction with
specific CDK inhibitory proteins (1). p27 is a member of the Cip-Kip family of CDK inhibitory
proteins which plays a major role in the control of the mammalian cell cycle in different
pathophysiological conditions (1). In quiescent cells, p27 exhibits maximal translation and
stability and contributes to growth arrest via inhibition of cyclin-CDK complexes. Upon
mitogenic stimulation, the level of p27 protein decreases rapidly, thus allowing the activation of
cyclin E–CDK2 and cyclin A–CDK2 complexes and the subsequent transcriptional activation of
genes that are required for the G1/S transition and the initiation of DNA replication (1, 2).
Supporting its major role on cell cycle control, genetic ablation of p27 in the mouse augments
body size, causes multiple organomegalia coinciding with higher proliferative activity (3-5) and
aggravates the course of proliferative diseases, such as cancer (6) and atherosclerosis (7, 8).
Moreover, p27 regulates cell migration, apoptosis and autophagy (9-16).
Accelerated proteolysis of p27 is associated with poor prognosis in cancer patients (17, 18).
The proteolytic degradation of p27 at the proteasome, which has been recognized as a key
mechanism for the regulation of its expression and function, is thought to occur via two different
pathways that operate in the cytoplasm and nucleus (19). Degradation of p27 in early G1 occurs
at the cytoplasm via the ubiquitin ligase Kip1 ubiquitylation-promoting complex (KPC) (20),
whereas its degradation at the G1/S transition and in G2-phase prior to mitosis occurs in the
nucleus via the S-phase kinase-associated protein 2 (SKP2), the F-box protein component of the
SCFSKP2 ubiquitin ligase complex (21-25). The molecular mechanisms regulating the pathways
targeting p27 for proteasomal degradation are thoroughly reviewed elsewhere (19, 26).
3
Comparatively, very little is known about proteasome-independent mechanisms of p27
proteolysis, which include calpain- and caspase-mediated proteolysis of p27 in certain cell types
(27-31).
Since protein–protein interactions play a major role in the regulation of p27 stability and
subcellular localization (19, 26), in this study we used the yeast two-hybrid assay to identify
novel partners of p27 which might be involved in the regulation of its expression and/or
function. We find that p27 interacts with sorting nexin 6 (SNX6), a member of the SNX family
of mammalian proteins comprising more than 30 cytoplasmic and membrane-associated
polypeptides that contain a phosphoinositol-binding phox homology domain (32, 33). SNXs
regulate important aspects of intracellular protein trafficking, such as receptor internalization,
endosome-to-Golgi retrograde transport, endosomal recycling and prodegradative sorting (3337). However, the specific cellular functions of SNX6 remain largely undefined (see
Discussion). Our studies provide the first evidence that the tumor suppressor p27 undergoes
endo-lysosomal degradation through its interaction with SNX6.
4
MATERIALS AND METHODS
Plasmids. pBTM116-p27 was generated by subcloning into EcoRI/BamHI sites of pBTM116
(gift from P. Sanz, Spain) a PCR product encoding for murine p27 using as a template pcDNA3mp27 - (gift from B. Amati, Switzerland). pGEX-SNX6 (1-179) was generated by digestion with
BamHI/XhoI of pAD-Gal4-SNX6 (1-179) and subcloning into pGEX-4T-3 (Pharmacia). cDNAs
encoding wild-type (2-197) or deletion mutant (2-151, 2-88, 50-151) forms of murine p27 tagged
at their NH2 termini with 6xHIS were subcloned into BamHI/XhoI sites of pcDNA3 (Invitrogen).
pcDNA-HA-mp27(PA) (gift from J. Kato, Japan) is described in (38). pLAMP1-GFP (gift from
J. Lippincott-Schwartz, USA) is described in (39). pEYFP-p27, pEYFP-P27(PA), pECFP-SNX6
and pEYFP-SNX6 were generated by subcloning PCR-generated full-length cDNAs: p27 and
p27PA into pEYFP-C1 (Invitrogen) and SNX6 into pEYFP-N1 (Invitrogen) and pECFP-N1
(Invitrogen) using pEBB-HA-SNX6 as template (gift from W.T. Parks, USA, described in (40)).
RNA interference. For transient silencing of SNX6, we used a pcDNA 6.2-GW miR plasmid encoding
a mi-RNAi against SNX6 (Invitrogen). For stable silencing of SNX6, plasmids encoding shRNAs
against SNX6 were obtained using the pSilencer Express siRNA Expression Cassette Kit
according to manufacturer’s instructions (Ambion).
Yeast two-hybrid. The yeast strain TAT-7 (MATa ade2 his3 leu2 trp1 gal4 gal80 LYS2::lexAopHIS3 URA3::lexAop-lacZ) was cotransformed with with a p27 mutant version unable to bind
cyclin-dependent kinases (pBTM116-p27ck-) and a murine thymocyte cDNA library subcloned
in pADGal4-2.1 plasmid (gift from G. Gil, Spain). Transformants were selected in SC+2%
glucose plates lacking leucine, tryptophan and histidine and were subsequently screened for 5
galactosidase activity using a filter lift assay. Inserts from the isolated pAD-Gal4-2.1 plasmids
were
sequenced
using
5´-GAAGATACCCCACCAAACCC-3´
and
5´-
GCGGGTTTTTCAGTATCTA-3´ primers.
Cell culture. Cells were maintained in DMEM supplemented with 100 U/mL penicillin, 0.1
mg/mL streptomycin, 2 mM L-glutamine and either 10% fetal bovine serum (Hela, U2OS,
HepG2, and HEK-293 cells) or 10 % newborn calf serum (NIH-3T3 cells).
Anti-SNX6 antibody generation. After extraction of preimmune serum, three mice were
immunized with recombinant GST-SNX6 (1-179) isolated by SDS-PAGE, and serum containing
polyclonal
antibodies
was
collected
eight
weeks
after
immunization.
ELISA,
immunofluorescence and Western blot were used to select the mouse which produced the antiserum with the best signal-to-noise ratio (Fig.S1A,B, online supplement, and data not shown).
Spleen cells from this mouse were fused with SP2/0 myeloma cells. Screening of specific
antibody-secreting hybridomas was carried out by ELISA and Western blot and several selected
hybridoma clones were intraperitoneally inoculated in mice. Ascitic fluid was collected from
these mice and analyzed for its specificity for SNX6 by Western blot analysis and confocal
microscopy (Fig.S1C,D, online supplement, and data not shown).
GST pull-down assays. GST and GST-SNX6 recombinant proteins were purified using
glutathione-sepharose 4B (Amersham) according to the manufacturer´s instructions and were
eluted with 50 mM Tris-HCl (pH 8.0), 10 mM glutathione. Whole extracts (500 μg) of
transfected HEK-293 cells (overexpressing p27 wild-type or deletion mutants) were incubated
with recombinant proteins (3 μg) in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium
6
deoxycholate, 0.1% SDS, 50 mM TRIS pH 8.0) supplemented with protease inhibitor cocktail
(Complete, Roche) After overnight incubation at 4ºC, glutathione-sepharose 4B was added to a
final concentration of 10% and agitated at 4ºC for 1 h. Beads were collected by centrifugation
and washed three times with RIPA buffer. Pellets were air-dried, resuspended in 2x Laemmli´s
buffer, boiled for 5 min, and separated onto 12% SDS-PAGE.
Subcellular fractionation and immunoblots. Postnuclear supernatants were prepared as
described (41). Endosome fractions were isolated by centrifugation on a discontinuous sucrose
gradient essentially as described (42). Purified proteins and cell lysates were separated by SDSPAGE and transferred onto PVDF membranes (Immobilon-P, Millipore). Membranes were
blocked in 4% dry milk and incubated with primary antibodies. Secondary HRP-coupled antiisotype-specific antibodies (Santa Cruz Biotechnologies) were used for detection with enhanced
chemiluminescence reagent (ECL plus, Amersham). The monoclonal anti-SNX6 antibody 446A
was used for immunoblotting studies (see above). Commercial primary antibodies were acquired
from the following providers: anti-GST (sc-138), anti-lamin A/C (sc-7293), anti-ERK (sc-154),
anti-PCNA (sc-7907) and anti-tubulin (sc-8035) from Santa Cruz Biotechnologies; anti-HA (H9658) and anti-Actin (A-2066) from Sigma; anti-early endosome antigen 1 (EEA1) (ab14453)
and anti-LDH (Ab7639) from Abcam; anti-GFP (A6455) from Invitrogen; and anti-p27 (610242)
from BD Transduction Laboratories.
Confocal microscopy. Cells grown on glass coverslips were fixed for 15 min at room
temperature with 4% paraformaldehyde, permeabilized for 30 min with 0.5% Tween 20/PBS,
and blocked for 1 h at room temperature in 5% horse serum/PBS. An additional blocking step
was performed using an avidin-biotin blocking kit (Vector).
7
To visualize lysosomes, growing cells were incubated during 2h at 37ºC with lysotracker
red probe (DND-99, Invitrogen). After fixation, permeabilization and blockade, cells were
incubated overnight with antibodies anti-p27 (1/200, p27 (554), MBL), anti-SNX6 (1/25, clone
450A-R1 1st extraction), anti-EEA1-FITC (1/75, Becton Dickinson) or anti-lysosomalassociated membrane protein 2 (LAMP2) (1/75, Hybridoma Bank, University of Iowa, USA).
p27, SNX6 and LAMP2 were visualized with anti-rabbit or anti-mouse biotin/streptavidin-FITC.
For double p27-SNX6 immunofluorescence, cells were incubated overnight at 4ºC with
anti-p27 antibody (1/200, p27 (554), MBL, or p27 (C19), Santa Cruz), washed extensively with
PBS and incubated at room temperature during 1 h with anti-SNX6 antibody (1/25, clone 450A
R1 1st extraction). p27 and SNX6 were visualized with anti-rabbit biotin/streptavidin-FITC and
anti-mouse-TRITC, respectively.
For double SNX6-EEA1 immunofluorescence, cells were incubated overnight at 4ºC with
anti-SNX6 (1/25, clone 450A-R1), washed with PBS and incubated at room temperature during
1 h with anti-EEA1-FITC (1/75, Becton Dickinson). SNX6 was visualized with anti-mouse
biotin/streptavidin-Texas Red.
For double p27-LAMP1 or p27-LAMP2 immunofluorescence, cells were incubated
overnight at 4ºC with anti-p27 (1/200, p27 (554), MBL, or p27 (C19) Santa Cruz), washed with
PBS and incubated during 1h at room temperature with anti-LAMP1 or anti LAMP2 (1/75,
Hybridoma Bank, University of Iowa, USA). p27 was visualized with anti-rabbit
biotin/streptavidin-FITC and LAMP1 and 2 with species-specific TRITC-conjugated secondary
antibodies.
For triple immunofluorescence labelling, cells were incubated overnight at 4ºC with antiSNX6 antibody (1/25, clone 450A R1, 1st extraction), washed with PBS, and incubated during 1
h at room temperature first with anti-p27 antibody (1/200, MBL) and then with anti-EEA1-FITC
8
(1/75, BD Transduction Laboratories). SNX6 was visualized with anti-mouse-TRITC and p27
with anti-rabbit-biotin/streptavidin-Alexa633.
In double and triple immunofluorescence experiments, single staining of each protein was
performed to ensure non-overlapping fluorescence.
For YFP, CFP, SNX6-YFP, SNX6-CPF, CFP-p27 and YFP-p27 visualization, cells were
transfected with plasmids encoding for these proteins (4 g each plasmid) and studied 24 h after
transfection.
Images were acquired on a Leica TCS/SP2 confocal microscope with a 63X oil immersion
objective (NA 1.4). Confocal microscope settings were adjusted to produce the optimum signalto-noise ratio. The sequential mode was used in image acquisition in order to avoid any
interference from overlapping fluorescence. Fluorescence analysis was performed using
Metamorph software (Universal Imaging).
Fluorescence resonance energy transfer (FRET). The calcium phosphate method was used to
cotransfect U2OS cells with pECFP-SNX6 + pEYFP-p27, pECFP-SNX6 + pEYFP-p27PA or
pECFP-SNX6 + pEYFP as a negative control (4 g each plasmid). Images were acquired on a
Leica TCS/SP2 confocal microscope with a 63x oil immersion objective (NA 1.4). An argon
laser line of 458 nm was used to excite CFP (PMT window 465–505 nm) and a 514-nm line to
excite YFP (20% laser intensity for acquisition, and 65% for photobleaching) (PMT window
525–600 nm). FRET studies were performed in 4% paraformaldehyde-fixed cells using the
acceptor-photobleaching method as we have previously described (41, 43). Briefly, FRET is
calculated as the relative increase in donor fluorescence resulting from the reduction or
elimination of energy transfer when YFP is photobleached. The percentage of pixels exhibiting
9
increased CFP fluorescence intensity after photobleaching was quantified in the regions of
interest using the following equation:
FRET efficiency =(Cafter-Cbefore)/Cafter x100
where Cbefore and Cafter are the total fluorescence intensity (area x average intensity of
bright spots) of the CFP channel before and after photobleaching, respectively. Fluorescence
analysis was performed using Metamorph software (Universal Imaging).
Cell cycle analysis. Cultured cells were trypsinized, washed two times in PBS, and collected by
centrifugation for 5 min at 300g. After fixation in 80% ethanol for 1h at -20ºC, cells were
incubated for approximately 30 minutes with 50 μg/mL propidium iodide containing 0.25
mg/mL RNAse A. Labelled cells were analyzed in a FACSCanto flow cytometer (Becton
Dickinson) and DNA histograms were fitted into cell cycle distributions using the ModFit 3.0
software (Verity Software House).
10
RESULTS
p27 and SNX6 interact both in vitro and in vivo. By using the yeast two-hybrid system,
we identified SNX6 as a potential heterodimerization partner of p27 (data not shown). This
interaction was validated by GST pull-down assays using lysates of HEK-293 cells transiently
transfected with full-length p27 and various deletion mutants, which also revealed that the region
from amino acid 88 to 151 of p27 is essential for its interaction with GST-SNX6 (1-179)
(Fig.1A). Moreover, the interaction of p27 and SNX6 was abrogated by point mutations which
eliminated the proline-rich region of p27 located at amino acids 90-96 (HA-p27(PA), Fig.1B).
The possible interaction of p27 and SNX6 was also analyzed in vivo by FRET, which
allows the measurement of protein-protein interactions at a molecular resolution of 1-10 nm (44).
We used the acceptor photobleaching method, in which increments in the emission of the CFP
donor after photobleaching of the YFP acceptor indicate efficient FRET. U2OS cells were
cotransfected with a plasmid encoding SNX6-CFP and either YFP-p27, YFP-p27(PA) or YFP as
negative control. As shown in Fig.2A, YFP-p27 preferentially accumulated within the nucleus
but also exhibited some cytoplasmic localization, and SNX6-CFP showed a cytoplasmic
speckled pattern. FRET efficiency was analyzed in bright cytoplasmic spots where CFP-SNX6
and YFP-p27 proteins colocalized. In these regions, we found augmented SNX6-CFP emission
after photobleaching in cells overexpressing YFP-p27 but not in cells overexpressing YFP
(Fig.2B). Moreover, the interaction of YFP-p27 and SNX6-CFP was abrogated by point
mutations which eliminated the proline-rich region of p27 (YPF-p27(PA), Fig.2B). These
findings demonstrate the specific in vivo interaction between SNX6-CFP and YFP-p27 through
the poly-proline region of p27, in agreement with the GST pull-down experiments of Fig.1B.
11
p27 and SNX6 colocalize at the endosome in mammalian cells. We generated mouse
monoclonal anti-SNX6 antibodies which specifically recognized both recombinant HA-SNX6
and an endogenous protein of approximately 50 kDa with a speckled cytoplasmic pattern of
expression resembling the known SNX6 cellular distribution (Fig.S1, online supplement).
Moreover, the anti-SNX6 antibody revealed the colocalization of SNX6 with the early-endosome
marker EEA1 in HeLa (Fig.S2A, online supplement) and NIH-3T3 cells (data not shown), but
not with the lysosomal acidotropic probe lysotracker in HeLa (Fig.S2B, online supplement) and
COS7 cells (data not shown), in agreement with previous studies (32, 45). Colocalization of
lysotracker with lysosomal-associated membrane protein 2 (LAMP2) (Fig.S2C, online
supplement) and lack of overlap with EEA1 (Fig.S2D, online supplement) confirmed the
lysosomal accumulation of lysotracker. We found areas of colocalization between endogenous
p27, SNX6 and EEA1 in triple confocal immunofluorescence studies in NIH-3T3 and HeLa cells
using the anti-p27 (554) antibody (Fig.3A). Colocalization of p27 with EEA1 (Fig.3B) and
SNX6 (Fig.3C) in NIH-3T3 cells was also observed using a different anti-p27 antibody (anti-p27
(C19)). Moreover, Western blot analysis revealed the presence of p27 in endosome-enriched
fractions obtained from NIH-3T3 cells and mouse liver tissue (Fig.3D). These endosomal
fractions were enriched in SNX6 and EEA1 and exhibited undetectable levels of the nuclear
proteins PCNA (proliferating cell nuclear antigen) and lamin A/C and of the cytosolic protein
LDH (Fig.3D). Collectively, these expression studies strongly suggest that a fraction of
cytoplasmic p27 is localized in EEA1- and SNX6-immunoreactive endosomes.
SNX6 affects p27 subcellular distribution and protein levels. Since SNXs are major
regulators of intracellular protein trafficking (33), we investigated whether subcellular
localization of p27 was affected by overexpression of SNX6. Confocal microscopy studies
12
demonstrated increased accumulation of ectopically expressed YFP-p27 within the cytoplasm of
U2OS cells upon overexpression of SNX6-CFP compared to that of controls overexpressing
CFP, in which nuclear accumulation of YFP-p27 predominated (p<0.001, Fig.4A). Accordingly,
Western blot analysis revealed higher amount of both YFP-p27 and endogenous p27 proteins in
the cytoplasmic fraction (PNS: postnuclear supernatant) of cells expressing SNX6-CFP
compared to cells expressing CFP (Fig.4A), and increased level of endogenous p27 in endosomal
fractions of cells expressing SNX6-YFP compared to YFP (Fig.4B). In marked contrast, RNAimediated silencing of SNX6 expression in transiently-transfected U2OS cells (Fig.S3, online
supplement) diminished cytoplasmic localization of YFP-p27 and endogenous p27 (p<0.05, Fig.
4C). In addition, p27 endosomal localization was reduced in NIH-3T3 cells stably expressing an
RNAi against SNX6 (Fig.4D). Moreover, RNAi-mediated silencing of SNX6 increased the
amount of p27 in whole lysates (Fig.4C and 4D) and, conversely, adenovirus-mediated
overexpression of SNX6 led to diminished p27 expression level in HepG2 cells (Fig S4, online
supplement). Taken together, these results suggest that SNX6 plays a role in the regulation of
p27 protein levels.
SNX6 induces p27 lysosomal degradation in proliferating cells. Endocytosed material in
mammalian cells generally travels into early/sorting endosomes, which mature into late
endosomes and lysosomes (46). Since SNXs appear to be involved in these processes (47-54),
our finding that p27 interacts with SNX6 and partially localizes at the endosome prompted us to
hypothesize the existence of a lysosomal mechanism for p27 degradation. Supporting this view,
immunofluorescence confocal microscopy in different mammalian cell lines revealed areas of
colocalization between p27 and the lysosomal markers LAMP2 (Fig.5A,B; Fig.S5A, online
13
supplement), LAMP1 (Fig.S5B, online supplement) and lysotracker (Fig.S5C, online
supplement).
To elucidate the functional relevance of the potential degradation of p27 by the endo-lysosomal
pathway, we analyzed the effect of lysosomal inhibitors on the downregulation of p27 induced
by different stimuli. The reduction of p27 expression induced by UV irradiation of NIH-3T3
cells was unaffected by hydroxichloroquine, but was completely abolished by the proteasome
inhibitor MG132 (Fig.5C). Thus, the effects of UV irradiation on p27 protein levels appear to
depend on the proteasome, but not on the lysosomal pathway. On the contrary, mitogen-induced
downregulation of p27 in NIH-3T3 cells was attenuated by the lysosomal inhibitors
hydroxichloroquine or leupeptine/ammonium chloride (Fig.5D). The effect of lysosomal
inhibitors on p27 protein levels was specifically observed after 8 hours of serum stimulation,
when p27 levels in untreated cells decreased at least by 75%. This finding suggests that the
lysosomal pathway plays a role in p27 downregulation upon mitogenic stimulation. Supporting
this notion, we detected p27 inside swollen lysosomes in the presence of hydroxichloroquine
(Fig.S7, online supplement), particularly in serum-stimulated cells (p27 accumulated in 17.5 %
of LAMP2-immunorreactive vesicles in serum-stimulated cells, but only in 4.5 % of LAMP2immunorreactive vesicles in serum-starved cells) (Fig.S8, online supplement). Remarkably, as
shown in Fig.5E, SNX6 appears to be required for lysosomal degradation of p27 in serumstimulated cells, since SNX6 silencing attenuated p27 downregulation upon mitogen stimulation
and, importantly, abolished the effect of hydroxichloroquine, but not of MG132. Conversely,
SNX6 silencing did not affect p27 levels in the presence of hydroxichloroquine (Fig.5E), thus
suggesting that SNX6-mediated p27 downregulation depends on the lysosomal pathway. To
discard the possibility that these results were the consequence of a transcriptional mechanism,
we quantified p27 mRNA levels by real-time PCR. As shown in Fig.S6, neither RNAi-mediated
14
SNX6 silencing nor treatment with hydroxichloroquine or MG132 significantly affected p27
mRNA levels. Taken together, these results suggest that the endo-lysosomal proteolytic pathway
is the main mechanism mediating SNX6-dependent p27 downregulation and that SNX6 mediates
the lysosomal degradation of p27 upon serum stimulation.
SNX6 affects p27 downregulation and cell cycle progression upon mitogenic stimulation
The observation that SNX6 and the lysosomal pathway might contribute to p27 downregulation
after serum stimulation prompted us to investigate the potential role of this pathway in cell cycle
control. We first analyzed the effects of SNX6 silencing on the kinetics of p27 downregulation
throughout the entire cell cycle. Stable silencing of SNX6 in NIH-3T3 cells diminished seruminduced downregulation of p27 mainly during late G1 phase (approximately after 8-10 h of
serum stimulation) and at the G1/S transition (approximately after 12 h of serum stimulation),
but not in other phases of the cell cycle (Fig.6A,B). We therefore hypothesized that SNX6mediated downregulation of p27 plays an important role during the early stages of the cell cycle.
In support of this notion, SNX6 silencing delayed S-phase entry upon serum stimulation of
quiescent NIH-3T3 cells (Fig.6B). These findings suggest that SNX6-mediated lysosomal
degradation of p27 plays an important role in the control of cell proliferation. Consistent with
this, the lysosomal inhibitors hydroxichloroquine and leupeptine/ammonium chloride markedly
abrogated S-phase entry to a similar extent than the proteasome inhibitor MG132 (Fig. 6C).
Taken together, our findings strongly suggest that the interaction of p27 with SNX6 contributes
to cell cycle progression in mammalian cells.
15
DISCUSSION
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.
16
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-toGolgi 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
17
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 starvationsynchronized 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
18
elucidate the molecular mechanisms regulating this novel SNX6-dependent p27 proteolytic
pathway and its potential role in proliferative diseases such as atherosclerosis and cancer.
ACKNOWLEDGEMENTS
We thank M.A. Ibáñez for help with the preparation and analysis of endosomal fractions, M.J.
Andrés-Manzano for help preparing the figures, and colleagues who generously provided
reagents (B. Amati, Switzerland; J. Kato, Japan; J. Lippincott-Schwartz, USA; W.T. Parks, USA;
B. Vogelstein, USA; G. Gil, Spain; P. Sanz, Spain; M. Clague, UK). Work supported by grants
to VA from the Spanish Ministry of Science and Innovation (MICINN) and European Regional
Development Fund (grants SAF2004-3057 and SAF2007-62110), Instituto de Salud Carlos III
(ISCIII) (grant RD06/0014/0021, RECAVA), and Fundación Ramón Areces. J.M.G. was
supported by ISCIII, and J.J.F. by the CSIC-I3P predoctoral fellowship programme cosponsored
by the European Social Fund. The Centro Nacional de Investigaciones Cardiovasculares (CNIC)
is supported by the MICINN and the Pro-CNIC Foundation.
19
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Figure 1. The proline-rich domain of p27 mediates its interaction with SNX6. (A) Lysates
from HEK-293 cells transfected with wild-type and deletion mutants of p27 were incubated with
GST or GST-SNX6 (amino acids 1-179). Proteins present in the precipitated material were
analyzed by Western blot. (B) GST-SNX6 was incubated with lysates from HEK-293 cells
transfected with wild-type p27 or HA-p27(PA), a HA-tagged mutant carrying four proline-toalanine substitutions within the proline-rich region. Proteins were analyzed by Western blot in
both the input (1/10 of starting material) and precipitated fractions. NLS: Nuclear localization
signal; P-rich: Proline-rich region.
Figure 2. p27 interacts with SNX6 in vivo. FRET was determined using the acceptor
photobleaching method in U2OS cells cotransfected with SNX6-CFP and either YFP-p27, YPFp27(PA) or YFP. (A) The low magnification images show representative examples of SNX6CFP/YFP-p27 cotransfected cell before and after photobleaching. The SNX6-CFP fluorescence
in the two numbered squared regions in A is shown at higher magnification before and after
photobleaching. (B) Quantification of FRET efficiency in regions of interest (e.g. the squared
regions shown in panel A for SNX6-CFP/YFP-p27 wt) calculated as the percent of increment in
the total intensity of CFP fluorescence after photobleaching (averaged from 30-40 cells analyzed
in three independent experiments).
Figure 3. p27 and SNX6 partially colocalize at endosomes. Representative confocal
microscopy images of NIH-3T3 and HeLa cells showing points of colocalization in triple (A)
and double (B, C) immunofluorescence. Two different anti-p27 antibodies were used (p27 (554)
in A; p27 (C19) in B and C). The graphs represent fluorescence profiles along the dashed white
lines showing areas of colocalization. (D) Western blot analysis of whole lysates (WL) and
24
endosomal fractions (Endo) from NIH-3T3 cells and mouse liver. Expression of endosomal
(EEA1), nuclear (lamin A/C, PCNA) and cytosolic (LDH) markers was analyzed to assess the
purity of the subcellular fractions.
Figure 4. SNX6 gain-of and loss-of function affects cytoplasmic and endosomal localization
of recombinant and endogenous p27. (A) U2OS cells were cotransfected with plasmids
encoding YFP-p27 and either SNX6-CFP or CFP. Representative confocal microscopy images
are shown and the percentage of cells with cytoplasmic YFP-p27 is represented in the graph
(n=100-200 cells). Whole lysates and postnuclear supernatants (PNS) were analyzed by Western
blot using the indicated antibodies. (B) Western blot analysis of whole lysates and endosomal
fractions from U2OS cells singly transfected with either YFP or SNX6-YFP. (C) U2OS cells
were cotransfected with plasmids encoding CFP-p27 and either a control or a SNX6-targeting
RNAi. Representative confocal microscopy images are shown and CFP-p27 total intensity per
cytoplasmic area is plotted in the graph (mean ± SEM, n=100-150 cells). Whole lysates and PNS
were analyzed by Western blot. (D) Western blot analysis of the indicated subcellular fractions
from NIH-3T3 cells stably expressing either a control or a SNX6-targeting RNAi.
Figure 5. p27 partially colocalizes with the lysosomal protein LAMP2 and undergoes
SNX6-mediated lysosomal degradation (A, B) Representative confocal microscopy images of
NIH-3T3 cells showing cellular distribution of p27 (green) and the lysosomal marker LAMP2
(red) in double inmunofluorescence experiments using two anti-p27 antibodies: p27 (554) and
p27 (C19). The two squared regions in the low magnification pictures are enlarged. The graphs
represent fluorescence profiles along the dashed white lines showing areas of colocalization
(arrows). (C) NIH-3T3 cells were UV-irradiated (120 J/cm2) and analyzed by Western blot 8h
25
after irradiation in the absence (control) or the presence of 30 μg/mL HCQ or 10 M MG132.
p27 relative levels were normalized to tubulin as loading control and the average result of two
different experiments is shown (D) NIH-3T3 cells were starved during 48h and then stimulated
8h with 10% NCS in the absence or the presence of 30 μg/mL HCQ, 0.1 mM leupeptine/20 mM
NH4Cl (LEU) or 10 M MG132. p27 relative levels were normalized to actin as loading control
and the average result of two different experiments is shown. (E) NIH-3T3 cells stably
overexpressing control RNAi or RNAi against SNX6 were starved during 48h (Starved) and then
stimulated 8h with 10% NCS in the absence or the presence of 30 μg/mL HCQ, 0.1 mM
leupeptine/20 mM NH4Cl (LEU) or 10 M MG132. p27 relative levels were normalized to actin
as loading control and the average result of two different experiments is shown
Figure 6. SNX6 attenuates p27 downregulation in early stages of the cell cycle and delays Sphase entry. (A, B) NIH-3T3 cells stably expressing an RNAi targeting SNX6 or a control
RNAi were synchronized in G0 by 48h of serum deprivation and then restimulated with 10%
NCS for the indicated times. p27 protein levels were measured by Western Blot (A) and cell
cycle profile was analyzed by flow cytometry (B). The graph in A shows the relative content of
p27 (serum-starved cells=100%) determined by densitometric analysis (average from three
independent experiments). (C) NIH-3T3 cells were synchronized in G0 by 48h of serum
deprivation and then restimulated with 10% NBS for the indicated times in the absence or the
presence of 30 μg/mL HCQ, 0.1 mM leupeptine/20 mM NH4Cl (LEU) or 10 M MG132.
Figure 7. Pathways for the degradation of p27. Proteasomal degradation of p27 at the G1/Sphase transition and in G2 phase occurs in the nucleus via the SCFSKP2 ubiquitin ligase complex.
26
Cytoplasmic degradation of p27 in G1 phase can occur in the proteasome via the ubiquitin ligase
Kip1 ubiquitylation-promoting complex (KPC) and in the lysosome via its interaction with the
endosomal vesicular-trafficking regulator SNX6.
27