Download Mucolipin 1 channel activity is regulated by protein kinase A

Biochem. J. (2008) 410, 417–425 (Printed in Great Britain)
417
doi:10.1042/BJ20070713
Mucolipin 1 channel activity is regulated by protein kinase A-mediated
phosphorylation
Silvia VERGARAJAUREGUI*, Ross OBERDICK†, Kirill KISELYOV† and Rosa PUERTOLLANO*1
*Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, U.S.A., and †Department of Biological Sciences,
University of Pittsburgh, Pittsburgh, Pennsylvania 15260, U.S.A.
Mucolipins constitute a family of cation channels with homology
with the transient receptor potential family. Mutations in
MCOLN1 (mucolipin 1) have been linked to mucolipidosis
type IV, a recessive lysosomal storage disease characterized
by severe neurological and ophthalmologic abnormalities. At
present, little is known about the mechanisms that regulate
MCOLN1 activity. In the present paper, we addressed
whether MCOLN1 activity is regulated by phosphorylation. We
identified two PKA (protein kinase A) consensus motifs in the Cterminal tail of MCOLN1, containing Ser557 and Ser559 . Ser557 was
the principal phosphorylation site, as mutation of this residue
to alanine caused a greater than 75 % reduction in the total
levels of phosphorylated MCOLN1 C-terminal tail. Activation of
PKA with forskolin promoted MCOLN1 phosphorylation, both
in vitro and in vivo. In contrast, addition of the PKA inhibitor
H89 abolished MCOLN1 phosphorylation. We also found that
PKA-mediated phosphorylation regulates MCOLN1 channel
activity. Forskolin treatment decreased MCOLN1 channel activity, whereas treatment with H89 increased MCOLN1 channel
activity. The stimulatory effect of H89 on MCOLN1 function
was not observed when Ser557 and Ser559 were mutated to alanine
residues, indicating that these two residues are essential for PKAmediated negative regulation of MCOLN1. This paper presents
the first example of regulation of a member of the mucolipin
family by phosphorylation.
INTRODUCTION
N- and C-terminal tails oriented within the cytosol. The region
of homology with the other members of the TRP family consists of transmembrane domains 3–6 (amino acids 331–521),
which includes the pore located between transmembrane
segments 5 and 6 (amino acids 496–521). The selectivity of the
MCOLN1 channel remains controversial, as different studies have
suggested that the channel is permeable to Ca2+ [16]; Ca2+ , K+
and Na+ [17]; and H+ [18]. In addition, MCOLN1 activity can be
modulated by changes in pH and Ca2+ concentration [17,19].
The lysosomal defects observed in MLIV patients have led to
the suggestion that MCOLN1 might play a role in the biogenesis
of lysosomes [20]. This idea was supported by the observation that
knockout of cup-5, the orthologue of MCOLN1 in Caenorhabditis
elegans, resulted in formation of enlarged hybrid organelles
that contained both late-endosomal and lysosomal markers [21].
Recently, several groups have suggested additional roles for
MCOLN1 in different cellular processes, including regulation of
lysosomal acidification [18], autophagy of mitochondria [22] and
lysosomal secretion [23].
One way to gain information on the function of a protein
is to analyse its distribution and trafficking into the cell. For
example, consistent with its role in lysosomal function, MCOLN1
contains specific sorting signals that mediate its transport to
lysosomes [24–26]. These sorting signals include two di-leucine
motifs located at the N- and C-terminal tails that mediate
direct interaction with clathrin adaptors [24]. Post-translational
modifications also play an important role in the regulation of
protein function. We have shown previously that the C-terminal
tail of MCOLN1 is palmitoylated, and that palmitoylation
MLIV (mucolipidosis type IV) is an autosomal recessive disease
characterized by mental and psychomotor retardation, diminished
muscle tone or hypotonia, achlorhydria and visual problems,
including corneal clouding, retinal degeneration, sensitivity to
light and strabismus [1–3]. This disorder is found at a relatively
high frequency among Ashkenazi Jews, with 1:93 of the
population estimated to be a genetic carrier [4]. Analysis of
fibroblasts from MLIV patients by electron microscopy revealed
the presence of enlarged vacuolar structures that accumulate
mucopolysaccharides, phospholipids and gangliosides [5–8].
These enlarged vacuoles are present not only in fibroblasts, but
also in many different cell types, indicating a general impairment
of lysosomal function in MLIV patients. However, in contrast
with other lysosomal storage diseases, lysosomal hydrolases do
seem to be functional and correctly transported to lysosomes in
MLIV, suggesting that the observed defect in lysosomal function
may be the result of alterations in trafficking along the lysosomal
pathway [9,10].
Mutations in MCOLN1 (mucolipin 1) have been shown to be
the cause of MLIV [11–14]. MCOLN1 is an ion channel with
homology with the TRP (transient receptor potential) channel
superfamily. The TRPs constitute a large family of cation channels
that are expressed in most cell types and function as cellular
sensors of various internal and external stimuli, including changes
in temperature and osmolarity, light, pain, pheromones, odorants
and membrane stretch (reviewed in [15]). The predicted topology
of MCOLN1 is of six transmembrane-spanning domains, with the
Key words: lysosome, mucolipin 1 (MCOLN1), mucolipidosis
IV (MLIV), protein kinase A (PKA), transient receptor potential
(TRP).
Abbreviations used: BIS, bisindolylmaleimide I; CaMKII, Ca2+ /calmodulin-dependent protein kinase II; CTail, C-terminal tail; DMEM, Dulbecco’s modified
Eagle’s medium; DTT, dithiothreitol; ER, endoplasmic reticulum; FSK, forskolin; GFP, green fluorescent protein; GST, glutathione transferase; HEK-293,
human embryonic kidney; LAMP, lysosome-associated membrane protein; MCOLN1, mucolipin 1; MLIV, mucolipidosis type IV; NRK cell, normal rat kidney
cell; NTail, N-terminal tail; PKA, protein kinase A; PKC, protein kinase C; TGN, trans -Golgi network; TRP, transient receptor potential; TRPC, TRP canonical;
TRPM, TRP melastatin; TRPV; TRP vanilloid; YFP, yellow fluorescent protein.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
418
S. Vergarajauregui and others
promotes efficient internalization of MCOLN1 from the plasma
membrane [24].
Phosphorylation is another modification that critically regulates
protein activity. The activity of many TRP channels is regulated by phosphorylation and dephosphorylation events. A
comprehensive overview of the regulation of TRP channels,
including phosphorylation and interaction with auxiliary proteins
is reviewed in [26a,26b]. TRP phosphorylation may occur on
serine, threonine and tyrosine residues, and is mediated by
different kinases, including PKC (protein kinase C), PKA (protein
kinase A), Src, Fyn, PKG (protein kinase G) and CaMKII
(Ca2+ /calmodulin-dependent protein kinase II) (reviewed in [27]).
In this present paper, we addressed whether the activity
of MCOLN1 is regulated by phosphorylation. We report the
identification of two consensus PKA phosphorylation sites in
the C-terminal tail of MCOLN1 at Ser557 and Ser559 . Activation
of PKA by FSK (forskolin) induced MCOLN1 phosphorylation,
both in vitro and in vivo, and negatively regulated MCOLN1
channel activity in vivo. Conversely, treatment with the PKA
inhibitor H89 inhibited phosphorylation of the MCOLN1 Cterminal tail and caused an increase in MCOLN1 activity.
Therefore our results show for the first time that PKA-mediated
phosphorylation regulates MCOLN1 channel activity.
EXPERIMENTAL
Antibodies and reagents
Rabbit polyclonal anti-GFP (green fluorescent protein) antibody
was purchased from MBL International (Woburn, MA, U.S.A.)
and mouse monoclonal anti-FLAG M2 antibody from Sigma.
Mouse monoclonal anti-GFP (ab1218) and rabbit anti-LAMP1
(lysosome-associated membrane protein 1) (ab24170) antibodies
were from Abcam. Rabbit polyclonal anti-(MCOLN1 CTail) was
raised against the C-terminal tail of MCOLN1 (amino acids 518–
580) fused to GST. The specificity of the antibody was verified
by recognition of transfected proteins and by the absence of a
specific signal in fibroblasts from MLIV patients. Phosphatase
inhibitor cocktails 1 and 2, genistein, FSK and protease inhibitor
cocktail were obtained from Sigma. Serine/threonine kinase
inhibitor set [containing BIS (bisindolylmaleimide I), H89, KN93, ML-7 and staurosporine], Akt inhibitor IV and the purified
catalytic fragment of PKA were supplied by Calbiochem. [γ 32
P]ATP (3000 Ci/mmol) was obtained from GE Healthcare.
[32 P]Pi (orthophosphoric acid, 8500–9120 Ci/mmol) was obtained
from PerkinElmer.
Plasmids
Full length MCOLN1–GFP, MCOLN1 L15A/L16A/L577A/
L578A–GFP and GFP–MCOLN1-CTail (where CTail is Cterminal tail) were cloned as described previously [24]. The
N-terminal (amino acids 1–66) and the C-terminal cytosolic
tail (amino acids 518–580) of MCOLN1 were cloned into the
EcoRI and SalI sites of pGEX-5X (Pharmacia Biotech). To
generate MCOLN1–FLAG, full length MCOLN1 was cloned
into the EcoRI and SalI sites of pCMV-Tag 4B (Stratagene).
Mutations of residues in the cytosolic tails or the first extracellular
loop of MCOLN1 were introduced using the QuikChange sitedirected mutagenesis kit following the manufacturer’s instructions
(Stratagene). The incorporation of site-directed mutations was
confirmed by subsequent DNA sequencing.
Cell lines and transfections
HeLa and NRK cells (normal rat kidney cells) were maintained in DMEM (Dulbecco’s modified Eagle’s medium,
c The Authors Journal compilation c 2008 Biochemical Society
Life Technologies) supplemented with 100 units/ml penicillin
(Gibco), 100 µg/ml streptomycin (Gibco) and 10 % (w/v)
FBS (fetal bovine serum). Transfection of HeLa and NRK
cells was performed using FuGENETM 6 (Roche) according to
the manufacturer’s recommendations. In transient transfection
assays, analyses were conducted 24 h post-transfection.
In vitro phosphorylation
GST (glutathione transferase) fusion proteins were expressed
in bacteria and affinity purified using standard methods as
described previously [28]. Purified GST fusion proteins (10 µg)
bound to glutathione–Sepharose (20 µl volume) were washed
once in lysate kinase buffer [20 mM Tris (pH 7.5), 0.5 mM
DTT (dithiothreitol) and 10 mM MgCl2 ], and then incubated
with either 1 µl postnuclear supernatant (14 µg total protein)
from HeLa cells or with 1000 units of the purified catalytic
subunit of PKA (Calbiochem) for 20 min at 30 ◦C in lysate
kinase buffer containing 200 µM ATP and [γ -32 P]ATP (final
specific radioactivity of 500 µCi/µmol). The reactions were
stopped by washing with cold PBS containing 15 mM EDTA.
Phosphorylated proteins were eluted by the addition of 25 µl
Laemmli buffer before separation by electrophoresis on 4–
20 % gradient polyacrylamide gels. Gels were fixed and stained
with Coomassie Brilliant Blue, and then dried and subjected
to autoradiography. Preparation of the postnuclear supernatant
was performed as described previously [28]. Briefly, cells were
harvested using trypsin, resuspended in 10 mM Hepes/KOH
(pH 7.4), 320 mM sucrose, 10 mM EDTA, 5 mM EGTA and
1 mM DTT, supplemented with protease inhibitors, and disrupted
by sonication (15 pulses, intermediate power setting). Nuclei and
cell debris were removed by centrifugation at 1300 g for 5 min at
4 ◦C, and the supernatant stored at − 70 ◦C until required. For
experiments using inhibitors, the postnuclear supernatant was
pre-incubated with the indicated inhibitor for 30 min at room
temperature (23 ◦C) prior to the phosphorylation reaction.
Metabolic labelling and immunoprecipitation
HeLa cells transfected with GFP–MCOLN1-CTail, GFP–
MCOLN1-CTail S557A/S559A, MCOLN1–FLAG or MCOLN1
S557A/S559A–FLAG for 24 h were incubated with serum-free
DMEM lacking phosphate for 4 h at 37 ◦C, and then radiolabelled
for 3 h in the same medium in the presence of 0.15 mCi/ml [32 P]Pi.
For kinase stimulation, 100 µM FSK was added by rapid mixing
during the last 30 min of radiolabelling. After radiolabelling, the
cells were washed with ice-cold PBS and lysed in phosphorylation
buffer [20 mM Tris/HCl (pH 7.4), 400 mM NaCl, 1 mM EDTA,
1 mM MgCl2 and 1 mM CaCl2 ] supplmented with 2 % (v/v)
Triton X-100 and protease and serine/threonine phosphatase
inhibitors. The lysate was pre-cleared by centrifugation at 16 000 g
for 10 min at 4 ◦C and incubated for 4 h at 4 ◦C with 5 µl
monoclonal anti-GFP antibody or 3 µl anti-FLAG antibody,
followed by Protein G–Sepharose (20 µl of a 50 % slurry;
Amersham Biosciences) for 4 h at 4 ◦C. Immunoprecipitates were
collected, washed three times with phosphorylation buffer with
1 % Triton X-100, once with phosphorylation buffer with 0.1 %
SDS and once with PBS. The washed immunoprecipitates were
then eluted using Laemmli sample buffer, analysed by SDS/PAGE
(4–20 % gradient gels) under reducing conditions, and transferred
on to nitrocellulose. The nitrocellulose membrane was exposed
to Kodak X-AR film for 1 day to visualize radiolabelled proteins
and then subjected to Western blotting using polyclonal anti-GFP
(1:1000 dilution) and anti-(MCOLN1 CTail) (1:1000 dilution)
antibodies, which were incubated overnight at 4 ◦C.
PKA-mediated phosphorylation of MCOLN1
419
Electrophysiology
HEK-293 (human embryonic kidney) cells were transfected
with GFP–MCOLN1 constructs or with YFP (yellow fluorescent
protein)- and HA-tagged wild-type human MCOLN1 and
maintained in DMEM. Cells were seeded 24–48 h post transfection on to coverslips and used for experiments. The wholecell mode of patch–clamp technique was executed as described
previously [18,29]. The pipette solution contained 150 mM
caesium aspartate, 2 mM MgCl2 , 5 mM sodium ATP (pH 7.2) and
10 mM Hepes, with 0.1 µmol Ca2+ buffered at pCa 8 with 2 mM
EGTA or 10 mM BAPTA [1,2-bis-(o-aminophenoxy)ethaneN,N,N ,N -tetra-acetic acid]. The extracellular solution contained
10 mM Hepes, 150 mM NaCl (or sodium gluconate), 1 mM
CaCl2 , 1 mM MgCl2 and 5 mM KCl (pH 7.6). Drugs were applied
by bath perfusion.
Pipette resistance was in the 2–7 M range and seal resistance
was always greater than 1 G. Shortly after establishing the seal
and breaking into the cells, cells were challenged with 20 mV
voltage steps (250 ms long) between − 100 and + 100 mV, and the
resulting current was recorded using a EPC-10 amplifier (HEKA
Instruments, Lambrecht, Germany). Currents were recorded
using the Patchmaster program (HEKA Instruments). The liquid
junction potential was neutralized and currents were digitized at
1 kHz, filtered using a low-pass digital Bessel filter set at 300 Hz,
and were otherwise not conditioned. Offline analysis was
performed using the Origin 7 program (OriginLabs, Northampton,
MA, U.S.A.).
RESULTS
The C-terminal tail of MCOLN1 is phosphorylated by
serine/threonine protein kinases
To better understand the molecular mechanisms that regulate
MCOLN1 activity, we analysed whether the N- and C-terminal
tails of MCOLN1 are modified by phosphorylation. The soluble
N-terminal tail [GST–MCOLN1-NTail (where NTail is the Nterminal tail), residues 1–66] and the C-terminal tail (GST–
MCOLN1-CTail, residues 518–580) proteins were synthesized
in bacteria as GST-tagged fusion proteins. Following purification,
these fusion proteins were incubated with HeLa cell lysates and
subjected to in vitro kinase assays. After 20 min, the reactions
were terminated by adding PBS containing EDTA, the products were separated by SDS/PAGE, and the gel was dried
and exposed to X-ray film to detect 32 P incorporation. The
samples were also stained with Coomassie Brilliant Blue to
verify that equal amounts of protein were loaded in each lane.
As seen in Figure 1(A), GST–MCOLN1-CTail was efficiently
phosphorylated in vitro by protein kinase activities present in
HeLa cell lysates. The N-terminal tail of MCOLN1 was also
phosphorylated, although to a much lower extent than the Cterminal tail. Therefore we concentrated on the characterization
and relevance of phosphorylation of the MCOLN1 C-terminal
tail.
To determine whether phosphorylation of the GST–MCOLN1
C-terminal tail occurs on tyrosine or serine/threonine residues,
we performed in vitro phosphorylation assays in the presence of
specific inhibitors. Incubation with staurosporine, a broad-range
serine/threonine kinase inhibitor, almost completely abolished
GST–MCOLN1-CTail phosphorylation, whereas the addition
of serine/threonine phosphatase inhibitors slightly increased
the signal (Figure 1B). In contrast, incubation with tyrosine
phosphatase inhibitors or with genistein, an inhibitor of tyrosine
phosphorylation, did not have any effect on the phosphorylation
Figure 1
In vitro phosphorylation of the C-terminal tail of MCOLN1
(A) Phosphorylation of MCOLN1 N- and C-terminal tail fusion proteins. Fusion proteins
containing GST fused to wild-type MCOLN1 N-terminal tail (GST–MCOLN1-NTail) or C-terminal
tail (GST–MCOLN1-CTail) were incubated in vitro with [γ -32 P]ATP and postnuclear HeLa cell
supernatant for 20 min at 30 ◦C. The proteins were then separated by electrophoresis, stained
with Coomassie Brilliant Blue (TOTAL, lower panel) and subjected to autoradiography (32 P,
upper panel). (B) Treatment with staurosporine causes a reduction in MCOLN1 phosphorylation.
GST–MCOLN1-CTail was incubated as stated in (A) with postnuclear supernatant from HeLA
cells that had been pre-incubated for 30 min at room temperature with the broad spectrum
serine/threonine kinase inhibitor staurosporine (100 nM), the tyrosine kinase inhibitor genistein
(100 µM), a serine/threonine phosphatase inhibitor (Ser Pase inh; phosphatase inhibitor cocktail
1, 1:100 dilution) or with a tyrosine phosphatase inhibitor (Tyr Pase inh; phosphatase inhibitor
cocktail 2, 1:100 dilution). Samples were separated by electrophoresis, stained with Coomassie
Brilliant Blue (TOTAL, lower panel) and subjected to autoradiography (32 P, upper panel). Staining
with Coomassie Brilliant Blue showed similar levels of fusion protein in each lane.
of the MCOLN1 C-terminal tail (Figure 1B). Therefore our results
suggest that a serine/threonine kinase present in HeLa cell lysates
phosphorylates the C-terminal tail of MCOLN1.
Ser557 and Ser559 are two phosphorylation sites in MCOLN1
C-terminal tail
We then conducted a sequence search using the NetPhosK 2.0
program, which predicts potential phosphorylation sites at serine,
threonine and tyrosine residues [30]. We found six residues
that are potential candidate substrates for serine/threoninedependent phosphorylation in the MCOLN1 C-terminal tail
sequence: Thr523 , Ser547 , Ser550 , Ser557 , Ser559 and Ser572 . To identify
the residues that participate in MCOLN1 phosphorylation,
several mutant proteins were generated (Figure 2A). When all
the above detailed serine residues were mutated to alanine
residues (S547A/S550A/S557A/S559A/S572A mutant), there
was virtually no incorporation of 32 P into GST–MCOLN1-CTail,
indicating that phosphorylation occurs at serine residues. In
agreement with this observation, the T523A mutant exhibited
phosphorylation levels equivalent to those of the wild-type
MCOLN1 C-terminal tail. Moreover, mutation of Ser574 , Ser550 ,
and Ser572 to alanine (S572A and S547A/S550A/S572A mutants)
did not affect MCOLN1 C-terminal tail phosphorylation. In
contrast, the proteins which were mutated on Ser557 and
Ser559 (S547A/S550A/S557A/S559A/S572A and S557A/S559A/
S572A mutants) were not phosphorylated (Figure 2B).
To address the individual contribution of Ser557 and Ser559
in the C-terminal tail of MCOLN1, we mutated these residues
separately. When the S557A mutant was used as a substrate, there
was an >75 % reduction in the extent of in vitro phosphorylation
of GST–MCOLN1-CTail. In contrast, there was only a 25 %
difference in protein phosphorylation levels between the wildtype GST–MCOLN1-CTail and the S559A mutant. Finally, the
S557A/S559A double mutant was not significant phosphorylated
c The Authors Journal compilation c 2008 Biochemical Society
420
Figure 2
S. Vergarajauregui and others
MCOLN1 is phosphorylated on Ser557 and Ser559
(A) The primary structure of the C-terminal tail of wild-type MCOLN1 (WT), with the putative phosphorylated residues in bold identified using the NetPhosK 2.0 Program, and the different
mutants carrying alanine replacements at the indicated residues. 5S/A, S547A/S550A/S557A/S559A/S572A; S547,550,572 /AAA, S547A/S550A/S572A; S557,559,572 /AAA, S557A/S559A/S572A; S572 /A,
S572A; T523 /A, T523A. (B) In vitro phosphorylation of wild-type GST–MCOLN1-CTail (WT) or GST–MCOLN1-CTail T523A (T523 /A), S547A/S550A/S557A/S559A/S572A (5S/A), S572A (S572 /A),
S557A/S559A/S572A (S557,559,572 /AAA) or S547A/S550A/S572 (S547,550,572 /AAA) mutants. Samples were separated by electrophoresis, stained with Coomassie Brilliant Blue (TOTAL, lower panel)
and subjected to autoradiography (32 P, upper panel). Staining with Coomassie Brilliant Blue showed similar levels of fusion protein in each lane. (C) In vitro phosphorylation was performed using
HeLa cell extracts to phosphorylate wild-type GST–MCOLN1-CTail (WT) or GST–MCOLN1-CTail S557A (S557 /A), S559A (S559 /A) or S557A/S559A (S557,559 /AA) mutants. Samples were separated
by electrophoresis, stained with Coomassie Brilliant Blue (TOTAL, lower panel) and subjected to autoradiography (32 P, upper panel). Staining with Coomassie Brilliant Blue showed similar levels of
fusion protein in each lane. The right-hand panel shows quantification of phosphorylation of the GST–MCOLN1-CTail S557A (S557/A), S559A (S559/A) or S557A/S559A (S557,559/AA) mutants,
expressed as a percentage of wild-type GST–MCOLN1-CTail (WT) phosphorylation (y -axis). Results are means +
− S.D. (n = 3).
(Figure 2C). Taken together, the results indicate that Ser557 is the
major in vitro phosphorylation site in the MCOLN1 C-terminal
tail, whereas Ser559 is phosphorylated to a much lesser extent.
PKA induces phosphorylation of the MCOLN1 C-terminal tail
To determine the type of serine/threonine protein kinase
responsible for phosphorylation of MCOLN1, we examined the
effects of various serine/threonine protein kinase inhibitors. Using
our in vitro kinase assay, we found that addition of H89 to HeLa
cell lysates effectively inhibited phosphorylation at concentrations
at which this inhibitor is known to inhibit PKA [31]. In contrast,
there was no effect on phosphorylation with other inhibitors,
including BIS, KN-93, Akt inhibitor IV or ML-7, which are known
to inhibit PKC, CaMKII, Akt and MLCK (myosin light-chain
kinase) respectively (Figure 3A).
c The Authors Journal compilation c 2008 Biochemical Society
Because H89 inhibition of MCOLN1 phosphorylation is
indicative of PKA activity, we sought to determine whether
MCOLN1 is phosphorylated after treatment of cells with FSK,
a PKA activator. As expected, phosphorylation of the C-terminal
tail of MCOLN1 was significantly increased after FSK treatment.
Densitometric measurements indicated an >3-fold increase in
phosphorylation after incubation with FSK (Figure 3B).
Finally, we tested the ability of GST–MCOLN1-CTail to serve
as a substrate for purified PKA. As shown in Figure 3(C),
GST–MCOLN1-CTail was substantially phosphorylated after
incubation with purified PKA. Once again, mutation of Ser557
to an alanine residue resulted in a 90 % reduction in PKAmediated phosphorylation of GST–MCOLN1-CTail, whereas
phosphorylation of the S557A/S559A double mutant was not
detectable. In conclusion, our results demonstrate that MCOLN1
is directly phosphorylated by PKA.
PKA-mediated phosphorylation of MCOLN1
421
were maintained in serum-free DMEM for 4 h prior to treatment
with FSK. Subsequent metabolic labelling with [32 P]Pi and
immunoprecipitation of HeLa cells expressing GFP–MCOLN1CTail showed that incubation with FSK induced an >3-fold
increase in the levels of phosphorylation of this chimaera, whereas
it had no effect on the S557A/S559A double mutant (Figure 4A).
Finally, we expressed FLAG-tagged full length MCOLN1
(MCOLN1–FLAG) in HeLa cells. We have shown previously that
the presence of epitopes at the C-terminal tail of MCOLN1 does
not alter the function or cellular distribution of the protein, as these
chimaeras are targeted to lysosomes and induce accumulation of
late endosome/lysosome hybrid organelles [24]. As demonstrated
in Figure 4(B), analysis of [32 P]Pi-labelled HeLa cells revealed
that the level of phosphorylation of full length MCOLN1 was
increased after treatment with FSK and decreased by H89. In
contrast, MCOLN1 S557A/S559A was not phosphorylated
in response to FSK. Therefore we conclude that full length
MCOLN1 is phosphorylated in vivo by PKA on Ser557 and Ser559 .
Effect of PKA-mediated phosphorylation on MCOLN1 distribution
Figure 3
Phosphorylation of GST–MCOLN1-CTail by PKA
(A) In vitro phosphorylation of GST–MCOLN1-CTail was performed using HeLa extracts
pre-incubated with either 1 µM BIS (PKC inhibitor), 1 µM H89 (H-89; PKA inhibitor), 1 µM
KN-93 (CaMKII inhibitor), 5 µM Akt inhibitor IV (AKT inh IV; protein kinase B inhibitor),
1 µM ML-7 [MLCK (myosin light-chain kinase) inhibitor] or 100 nM staurosporine (Staurosp;
broad-range serine/threonine kinase inhibitor). Samples were separated by electrophoresis,
stained with Coomassie Brilliant Blue to determine total protein levels (TOTAL, lower panel) and
subjected to autoradiography (32 P, upper panel). (B) GST–MCOLN1-CTail phosphorylation
by HeLa cell extracts (14 µg total protein) treated with increasing concentrations of the
PKA inhibitor H89 (0.1, 1 and 10 µM), or with 10 µM of the PKA activator FSK. Samples
were separated by electrophoresis (left-hand panel), stained with Coomassie Brilliant Blue
to determine total protein levels (TOTAL, lower panel) and subjected to autoradiography
(32 P, upper panel). Quantification of GST–MCOLN1-CTail phosphorylation, expressed as
fold activation of samples without treatment, is shown in the right-hand panel. Results are
means +
− S.D. (n = 3) (C) Phosphorylation of GST–MCOLN1-CTail by purified PKA. GST,
wild-type GST–MCOLN1-CTail (WT) or GST–MCOLN1-CTail S557A (S557 /A), S559A (S559 /A) or
S557A/S559A (S557,559 /AA) mutants were incubated with [γ -32 P]ATP and the catalytic fragment
of PKA for 20 min at 30 ◦C. Samples were separated by electrophoresis (left-hand panel),
stained with Coomassie Brilliant Blue to determine total protein levels (TOTAL, lower panel) and
subjected to autoradiography (32 P, upper panel).Wild-type GST–MCOLN1-CTail shows robust
phosphorylation, and mutation of Ser557 largely reduced this phosphorylation, whereas the
double mutant eliminated phosphorylation.
We have described previously that MCOLN1 reaches lysosomes
through either a direct [from TGN (trans-Golgi network) to
early endosomes and subsequently to lysosomes] or an indirect
(from TGN to plasma membrane, followed by internalization to
endosomes and transport to lysosomes) pathway [24]. To assess
whether phosphorylation of the MCOLN1 C-terminal tail has an
effect on the trafficking of the protein, we analysed the cellular
distribution of two MCOLN1 mutants in which Ser557 and Ser559
were mutated to alanine residues (MCOLN1 S557A/S559A–
GFP) or to an aspartic acid residue (MCOLN1 S557D–GFP)
to prevent or mimic phosphorylation respectively. Both mutants
extensively co-localized with the lysosomal marker LAMP1,
indicating that phosphorylation of Ser557 and Ser559 did not
affect trafficking of MCOLN1 to lysosomes (see Supplementary
Figure 1 at http://www.BiochemJ.org/bj/410/bj4100417add.htm).
We also queried whether phosphorylation occurs once
MCOLN1 reaches lysosomes, or in a pre-lysosomal compartment.
To address this question, we used a plasma-membrane-retained
MCOLN1 mutant. Generation of this mutant has been described
previously [24] and was obtained by mutating two di-leucine motifs located in the N- and C-terminal tails of MCOLN1 (MCOLN1
L15A/L16A/L577A/L578A). As seen in Supplementary Figure 2 (http://www.BiochemJ.org/bj/410/bj4100417add.htm),
MCOLN1 L15A/L16A/L577A/L578A was phosphorylated
in vivo, and the level of phosphorylation was increased after
treatment with FSK. Moreover, another MCOLN1 mutant that
contains mutations at the first extracellular loop (MCOLN1
V195A/D196A/P197A/P198A) and is retained at the ER
(endoplasmic reticulum) was also phosphorylated in vivo after
incubation with FSK (see Supplementary Figure 2). Therefore
these results suggest that MCOLN1 phosphorylation may occur
in pre-lysosomal compartments.
PKA activation and inhibition affects MCOLN1 activity
FSK induces phosphorylation of MCOLN1 in vivo
Although our biochemical experiments showed that PKA incorporated 32 P into MCOLN1, the conditions used for in vitro
phosphorylation may not reflect the conditions found in the
cellular environment. In addition, treatment of proteins with
detergent for solubilization may expose residues that are
typically not available for phosphorylation in vivo. Therefore
we investigated whether a chimaera consisting of the C-terminal
tail of MCOLN1 fused to GFP (GFP–MCOLN1-CTail) was also
phosphorylated in vivo. To reduce basal phosphorylation, cells
In order to test the effect of MCOLN1 phosphorylation on
its activity, we analysed changes in amplitudes of MCOLN1dependent currents associated with inhibition or activation of
PKA. Full length recombinant human MCOLN1 was transfected
into HEK-293 cells, a model that has been shown previously to
deposit some of the recombinant overexpressed MCOLN1 into
the plasma membrane [18,29]. To facilitate MCOLN1 detection, we also used the plasma-membrane-retained MCOLN1
mutant MCOLN1 L15A/L16A/L577A/L578A–GFP. In these
experiments, the pipette solution contained 150 mM caesium, and
c The Authors Journal compilation c 2008 Biochemical Society
422
Figure 4
S. Vergarajauregui and others
In vivo phosphorylation of MCOLN1
(A) HeLa cells transfected with wild-type GFP–MCOLN1-CTail (WT) or with a GFP–MCOLN1-CTail S557A/S559A double mutant (S557,559 /AA) were starved for 4 h in DMEM and metabolically
labelled with [32 P]Pi for 3 h. FSK (100 µM) was added to the medium during the last 30 min of labelling. Cells were then lysed, immunoprecipitated with a monoclonal anti-GFP antibody, subjected
to SDS/PAGE and analysed either by Western blotting (WB) using a polyclonal anti-GFP antibody (lower panel) or by autoradiography (32 P, upper panel). Quantification of GFP–MCOLN1-CTail
in vivo phosphorylation is expressed as fold activation of wild-type GFP–MCOLN1 CTail in the absence of FSK (right-hand panel). Results are means +
− S.D. (n = 3). (B) Phosphorylation of full
length MCOLN1–FLAG. HeLa cells transfected with full length MCOLN1–FLAG (MCOLN1 WT) or MCOLN1 S557A/S559A–FLAG (MCOLN1 S557,559 /AA) were metabolically labelled in the presence
of [32 P]Pi for 3 h at 37 ◦C, and 100 µM FSK or 1 µM H89 was added for the last 30 min of labelling. Cells were then lysed, immunoprecipitated with a monoclonal anti-FLAG antibody, subjected
to SDS/PAGE and analysed either by Western blotting using a polyclonal anti-(MCOLN1 CTail) antibody (WB, lower panel) or by autoradiography (32 P, upper panel). The arrows correspond to the
theoretical molecular mass of the monomeric and dimeric forms of the full length MCOLN1–FLAG fusion protein. Higher bands may correspond to oligomers of MCOLN1–FLAG. Quantification of
full length MCOLN1–FLAG in vivo phosphorylation is expressed as fold activation of MCOLN1–FLAG in the absence of FSK (right-hand panel). Results are means +
− S.D. (n = 3).
the outside medium contained 150 mM sodium as the main charge
carrier. These conditions were used to preserve continuity with
our previous publications [18,29]. Whole-cell current recordings
in HEK-293 cells showed that expression of plasma-membraneretained MCOLN1 resulted in characteristically outwardly
rectifying currents, which are very similar to those of wildtype MCOLN1 localized to the plasma membrane under
overexpression conditions (Figure 5), and to those reported
previously in the plasma membrane under overexpression
conditions [18,29] or in artificial lipid bilayers using cell-free
synthesized protein [17,19]. Control YFP only transfected cells
did not show such activity (Figure 5). The similarities include
monovalent cation permeability and strong outward rectification.
Similar to previously published reports [17,19], MCOLN1
rectification did not depend on the permeating cation species
and was unaffected by omission of either Ca2+ or Mg2+ from
the extracellular and intracellular solutions (results not shown).
Rectification is a characteristic feature of many TRP channels and
it is usually attributed to the dependence of the open probability
of the channel on membrane potential. This exact phenomenon
has been directly shown for MCOLN1: its open probability,
but not the single channel current amplitude, is significantly
decreased at negative membrane potentials [17,19]. The
currents recorded using the membrane-retained construct were
somewhat less variable than those detected with wild-type recombinant MCOLN1. Among other factors, fusion of wild-type
c The Authors Journal compilation c 2008 Biochemical Society
MCOLN1 containing lysosomes with the plasma membrane or
endocytosis of MCOLN1 may contribute to such variability, and
thus we opted to use the plasma-membrane-targeted constructs
alongside wild-type MCOLN1 to test the effect of FSK and H89
on MCOLN1 activity.
Figure 6 shows examples of such experiments. In all experiments, the cells were allowed to stabilize for 6 to 9 min after the
start of the experiment, during which time the basal MCOLN1
activity was recorded every 3 min. After application of the
drugs, the current was recorded every 3 min for 15–24 min.
Figures 6(A) and 6(B) (upper panels) show that cell treatment
with 10 µM H89 induces an increase in amplitudes of both
wild-type and membrane-retained MCOLN1. The increase was
gradual, and in most cases lasted beyond the timeframe of our
experiments. In Figure 6(E), we summarize the effect of H89 on
MCOLN1 activity. Amplitudes of MCOLN1-dependent currents
recorded 15 min after H89 application were normalized to
current amplitudes recorded before H89 application, which were
considered to be 100 % in each separate experiment. Most of
the control cells did not respond to H89 application; in the
few cases that responded to H89, the current amplitude was
significantly lower than that measured in MCOLN1-expressing
cells (Figures 6D and 6E).
Figures 6(A) and 6(B) (lower panels) show that treatment
with 50 µM FSK inhibited both membrane-retained and wildtype MCOLN1 currents. As with H89, the effect was gradual
PKA-mediated phosphorylation of MCOLN1
423
Figure 5 Monovalent cation permeability through wild-type and
membrane-targeted MCOLN1
HEK-293 cells were transfected with MCOLN1 L15A/L16A/L577A/L578A–GFP
(MCOLN1-GFP-L15 L/AA-L577 L/AA) or co-transfected with wild-type MCOLN1–HA (WT
MCOLN1-HA) and YFP. Transfection with YFP only was performed as a control. Whole-cell
currents were recorded with 150 mM caesium aspartate in the pipette solution and 150 mM
NaCl in the extracellular solution. Similar outward rectification and reversal potentials of
the monovalent currents were mediated by wild-type HA-tagged MCOLN1 and the plasma
membrane-retained MCOLN1 L15A/L16A/L577A/L578A–GFP mutant.
and fully developed within 15 min following application of the
drug. Neither H89 nor FSK seemed to have a noticeable effect
on the S557A/S559A double mutant (MCOLN1 S557A/S559A–
GFP) (Figures 6C and 6E). On the basis of these results, we conclude that inhibition of PKA and MCOLN1 dephosphorylation increases the activity of MCOLN1, whereas MCOLN1 phosphorylation inhibits the activity of MCOLN1.
DISCUSSION
In this present paper, we have shown that the activity of MCOLN1
is regulated by phosphorylation. We found that PKA activation by
FSK induced MCOLN1 phosphorylation, both in vitro and in vivo,
and reduced MCOLN1 channel activity. In contrast, inhibition of
PKA by treatment with H89 dramatically increased MCOLN1
activity. Mutagenesis analysis identified two PKA consensus sites
in the C-terminal of MCOLN1 that mediate PKA phosphorylation
of MCOLN1. Ser557 seems to be the major phosphorylation site for
MCOLN1, as mutation of this residue to alanine caused an >75 %
reduction in the levels of phosphorylation on the MCOLN1 Cterminal tail. Alternatively, Ser559 was shown to be a relatively
minor phosphorylation site that accounts for approx. 25 % of total
phosphorylation of the C-terminal tail. As expected, activation of
the MCOLN1 S557A/S559A double mutant was not observed in
response to H89 treatment, indicating that these residues mediate
PKA-induced regulation of MCOLN1.
Numerous studies have suggested an important role for protein
kinases in the regulation of TRP channels. In most cases,
phosphorylation leads to an increase in the activity of the channel.
For example, phosphorylation of TRPM4 (TRP melastatin 4)
by PKC increases the activity of the channel [32], whereas Src
phosphorylation enhances the activity of TRPM7 [33]. Within
the TRPV (TRP vanilloid) family, it has been demonstrated that
PKA, PKC and CaMKII can activate TRPV1 [34]. In addition,
the channel activity of TRPC3 (TRP canonical 3) and TRPC6 is
increased after phosphorylation by Src and Fyn respectively [35].
However, there are also examples that show that phosphorylation
inactivates some TRP channels. For example, the activity of
TRPC4, TRPC5 and TRPC6 may be inhibited by PKC-mediated
phosphorylation [36].
Figure 6
The effect of PKA activation and inhibition on MCOLN1 activity
(A–D) Examples of whole-cell current recordings obtained from cells incubated with FSK
(50 µM) (A, B) or H89 (10 µM) (A–D) after co-transfection with YFP and MCOLN1 wild-type
(WT MCOLN1) (A), MCOLN1 L15A/L16A/L577A/L578A–GFP (MCOLN1-GFP-L15 L/AA-L577 L/
AA) (B), MCOLN1 S557A/S559A–GFP (MCOLN1-GFP-S557,559 /AA) (C), and from YFP-only
transfected cells treated with 10 µM H89 (control) (D). In (A) and (B), the time and current
amplitude scales are shared between the upper and lower panels. Larger control currents were
selected for FSK experiments to emphasize the effect. The pipette solution contained 150 mM
caesium aspartate and the extracellular solution contained 150 mM NaCl as the main charge
carrier. The current traces illustrate channel activity before and 15 min after application of the
drugs. (E) A summary of the effects of FSK and H89 on MCOLN1 channel activity. To statistically
analyse the effect of the drugs, currents at +100 mV were recorded 15 min after stimulation and
expressed as a percentage of the current before stimulation, which was taken as 100 %. Results
are means +
− S.D. (n = 4–6), in which MCOLN1-mediated currents were clearly detectable.
All the differences are statistically significant, with the exception of the effect of H89 on the
MCOLN1 S557A/S559A–GFP (MCOLN1-GFP-S557,559 /AA) mutant, which did not demonstrate
a statistically significant change.
The mechanism by which phosphorylation regulates MCOLN1
is unclear. Some ion channels regulated by phosphorylation
contain specific regulatory domains, the best known example of
which is the R domain of CFTR (cystic fibrosis transmembrane
conductance regulator) (reviewed in [37]). No such domains are
evident in the region of MCOLN1 that we identified as containing
the MCOLN1 phosphorylation site. The fact that the MCOLN1
phosphorylation domain is located far from the pore region makes
it unlikely that phosphorylation directly affects ion permeation
through the MCOLN1 pore. It is possible that phosphorylation
induces a conformational change in the MCOLN1 C-terminal,
which changes its interaction with other proteins, such as
c The Authors Journal compilation c 2008 Biochemical Society
424
S. Vergarajauregui and others
the MCOLN1 subunits, or perhaps with other TRP channels.
It should be noted that several TRP channels are known to
homomultimerize and heteromultimerize, and recent results from
Montell and colleagues show that MCOLN1 is no exception [38].
MCOLN1 homomultimerization seems to be very dynamic as,
for example, pH changes were shown to reorganize MCOLN1
conductance substates [17,19] and induce structural changes
consistent with the disorganization of MCOLN1 homomultimeric
complexes. Heteromultimerization of TRP channels have been
shown to dramatically affect the selectivity and activity of
TRP channels [39]. Therefore it is possible that MCOLN1
phosphorylation may affect the interaction of MCOLN1 subunits,
or perhaps influence its interaction with other TRP channels.
In many cases, the activation of TRP channels is accompanied
by changes in their cellular distribution such as translocation
from internal vesicles to the plasma membrane (reviewed in
[40]). However, mutation of Ser557 and Ser559 did not change
the distribution of MCOLN1, indicating that PKA-mediated
phosphorylation does not play a role in the regulation of MCOLN1
trafficking to lysosomes.
One attractive possibility is that phosphorylation at the Nterminal regulates MCOLN1 trafficking. Interestingly, sequence
searches performed using the NetPhosK 2.0 program predicted
a potential PKA phosphorylation site at Ser10 . This residue is
especially interesting as a result of its proximity to the acidic dileucine motif (S10 ETERLL16 ) that regulates direct trafficking of
MCOLN1 from the TGN to late endosomes through interactions
with clathrin adaptors [24]. However, mutation of Ser10 to an
alanine residue did not affect the levels of phosphorylation of
the N-terminal tail or distribution of the full length protein,
suggesting that this residue does not regulate MCOLN1 trafficking
(see Supplementary Figures 3 and 4 at http://www.BiochemJ.org/
bj/410/bj4100417add.htm).
PKA plays an important regulatory role in several anterograde
traffic pathways, including transportation between the ER and the
Golgi, intra-Golgi transport and budding of constitutive vesicles
from the TGN to the plasma membrane [41,42]. In addition, PKA
has also been implicated in retrograde transport from endosomes
to the Golgi [43] and from the Golgi to the ER [44]. Our results
suggest that PKA could also be involved in the regulation of lysosomal function through its ability to modulate the activity of
MCOLN1.
One of the characteristics of patients with MLIV is the presence
of achlorhydria with elevated plasma gastrin levels [3,45], as
well as the accumulation of enlarged vacuoles in the parietal
cells of the gastric mucosa [46]. Treatment of animals with
omeprazole, an inhibitor of the H+ /K+ –ATPase, causes similar
abnormalities to those observed in MLIV patients in parietal
cells, suggesting that the absence of MCOLN1 could affect the
activity or distribution of the H+ /K+ –ATPase [47]. Interestingly,
acid secretion into the lumen of the stomach requires translocation
of the H+ /K+ –ATPase from vacuoles to the plasma membrane, and
this process is dependent on PKA [48]. Lysosomal exocytosis
has also been shown to be impaired in MLIV fibroblasts [22]
and, at the same time, PKA is known to play a critical role in
regulated exocytosis [49]. Therefore lysosomal exocytosis and
acid secretion are examples of processes that may be dependent
on both MCOLN1 and PKA. Further studies will help to determine
the role of PKA-mediated modulation of MCOLN1 in those
processes listed above and other traffic events.
We thank the NIH (National Institutes of Health) Fellows Editorial Board for their editorial
assistance prior to acceptance of this manuscript. This project was supported by funding
from the Intramural Research Program of the NHLBI (National Heart, Lung, and Blood
Institute) to R. P., and by funding from the Mucolipidosis 4 Foundation to K. K.
c The Authors Journal compilation c 2008 Biochemical Society
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Received 29 May 2007; accepted 7 November 2007
Published as BJ Immediate Publication 7 November 2007, doi:10.1042/BJ20070713
c The Authors Journal compilation c 2008 Biochemical Society