PCSK9 is phosphorylated by a Golgi casein kinase-like kinase ex vivo and circulates as a phosphoprotein in humans Thilina Dewpura1,*, Angela Raymond1,*, Jose´ e Hamelin2, Nabil G. Seidah2, Majambu Mbikay1, Michel Chre´ tien1 and Janice Mayne1
1 Chronic Disease Program, Ottawa Health Research Institute, The Ottawa Hospital, Canada 2 Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Canada
Keywords cholesterol; hypercholesterolemia; kinase; PCSK9; phosphoprotein
Correspondence J. Mayne, Chronic Disease Program, Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, Ontario K1Y 4E9, Canada Fax: +1 613 761 4355 Tel: +1 613 798 5555, ext. 16084 E-mail: jmayne@ohri.ca
*These authors contributed equally to this article
(Received 4 March 2008, revised 23 April 2008, accepted 6 May 2008)
remains associated with it
doi:10.1111/j.1742-4658.2008.06495.x
cell
Proprotein convertase subtilisin ⁄ kexin 9 (PCSK9) is a secreted glycoprotein that regulates the degradation of the low-density lipoprotein receptor. Sin- gle nucleotide polymorphisms in its gene associate with both hypercholes- terolemia and hypocholesterolemia, and studies have shown a significant reduction in the risk of coronary heart disease for ‘loss-of-function’ PCSK9 carriers. Previously, we reported that proPCSK9 undergoes autocatalytic processing of its prodomain in the endoplasmic reticulum and that its inhibitory prosegment following secretion. Herein, we used a combination of mass spectrometry and radiolabeling to report that PCSK9 is phosphorylated at two sites: Ser47 in its propeptide and Ser688 in its C-terminal domain. Site-directed mutagenesis suggested that a Golgi casein kinase-like kinase is responsible for PCSK9 phosphory- lation, based on the consensus site, SXE ⁄ S(p). PCSK9 phosphorylation was cell-type specific and occurs physiologically because human plasma PCSK9 is phosphorylated. Interestingly, we show that the naturally occur- ring ‘loss-of-function’ variant PCSK9(R46L) exhibits significantly decreased propeptide phosphorylation in the Huh7 liver line by 34% (P < 0.0001). PCSK9(R46L) and the engineered, unphosphorylated vari- ant PCSK9(E49A) are cleaved following Ser47, suggesting that phosphory- lation protects the propeptide against proteolysis. Phosphorylation may therefore play an important regulatory role in PCSK9 function. These find- ings will be important for the future design of PCSK9 inhibitors.
Abbreviations GCK, Golgi casein kinase; LDLR, low density lipoprotein receptor; PCSK9, proprotein convertase subtilisin ⁄ kexin 9; SAP, shrimp alkaline phosphatase.
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Proprotein convertase subtilisn ⁄ kexin 9 (PCSK9) is a member of the mammalian PCSK family that, to date, includes eight other members: PCSK1 (PC1 ⁄ 3), PCSK2 (PC2), PCSK3 (PC4), PCSK5 (Furin), PCSK4 (PC5 ⁄ 6), PCSK6 (Pace4), PCSK7 (PC7) and PCSK8 (SKI-1 ⁄ S1P) [1]. Collectively, this family is responsible for the proteolytic maturation of secretory precursors to bioactive proteins and peptides including neuro- peptides, pro-hormones, cytokines, growth factors, receptors, cell-surface proteins and serum proteins [2,3]. Fitting with its role in cholesterol metabolism, PCSK9 is highly expressed in the liver and intestine, two tissues important in cholesterol homeostasis [4]. It is also found in circulation [5–7]. PCSK9, like its fam- ily members, is synthesized as a preproprotein contain- ing several defined motifs: a signal peptide domain for routing the PCSKs to the secretory pathway, a prodomain important for folding and acting as an
T. Dewpura et al.
PCSK9 circulates as a phosphoprotein in humans
at Ser47 the propeptide
endogenous inhibitor, a catalytic domain characteristic of serine proteases and a C-terminal Cys- and His-rich domain implicated in enzyme stability and protein– protein interaction [3]. We reported that PCSK9 is autocatalytically processed in the endoplasmic reticu- lum at the site FAQ152flSIP indicative of its consensus cleavage motif, travels to the Golgi where its sugar res- idues at the glycosylation site N533CS are matured and its propeptide is sulfated at Tyr38, and is secreted [4,5]. PCSK9 is unique among the PCSK family because it is secreted in association with its inhibitory propeptide.
of PCSK9-propeptide level
provide a promising therapy for treatment of hyper- cholesterolemia [7,11,24]. Toward this goal we decided to further analyze post-translational modifications of PCSK9. We had previously reported on molecular mass heterogeneity of the propeptide [4], showing that this was in part due to sulfation of Tyr38 [5]. Here we demonstrate that the heterogeneity is also due to phosphorylation of as assessed by MS analysis of PCSK9 immunoprecipi- tates in the presence and absence of shrimp alkaline phosphatase (SAP). Radiolabeling and site-directed mutagenesis also demonstrated the existence of a sec- ond major site of phosphorylation in the Cys- and His-rich domain at Ser688, very near its C-terminus. We demonstrate that these modifications are physio- logically relevant because: (a) they occur ex vivo in lines and in vivo in human plasma; human liver cell (b) in the case of the propeptide, phosphorylation is decreased in the naturally occurring ‘loss-of-function’ R46L and A53V PCSK9 variants; and (c) decreasing the phosphorylation increases subsequent proteolysis following Ser47, the site of phosphorylation. Site-directed mutagenesis of amino acids surrounding both the propeptide and C-terminal sites suggest that PCSK9 phosphorylation is carried out by a Golgi casein kinase (GCK)-like kinase.
Results
The secreted propeptide of PCSK9 is phosphorylated in a cell-type specific manner
Cell culture and animal models have established that the low-density lipoprotein receptor (LDLR) is one of the main downstream targets of PCSK9 [4,8–11]. Sup- porting this, several groups have reported that secreted PCSK9 can interact with and enter the endocytic recy- cling pathway with LDLR, affecting the equilibrium of LDLR recycling versus LDLR lysosomal-dependent degradation [6,12–15]. The ‘gain-of-function’ D374Y mutation in the catalytic domain of PCSK9 results in the most severe form of autosomal-dominant hyper- cholesterolemia [16,17]. Studies have shown that this variant binds the LDLR (within its epidermal growth factor-A domain) at the cell surface 25 times more effi- thereby shifting the ciently than wild-type PCSK9, equilibrium toward LDLR lysosomal-dependent degra- dation [12,15]. However the effect of other autosomal- dominant PCSK9 hypercholesterolemia-associated mutations, such as the PCSK9(S127R) on PCSK9- LDLR dependent degradation is less obvious because their binding equilibrium to the LDLR is only moder- ately increased [15,18]. Crystal structures have shown that this Ser127 residue does not interact directly with the LDLR [19].
for at
in this case,
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We examined the heterogeneity of the molecular mass of the propeptide of endogenous PCSK9 in the media of HepG2 cells by MS analyses of immunoprecipitates with immune (I) sera directed against PCSK9 or pre- immune (PI) sera (Fig. 1A,B, respectively). Figure 1A illustrates the two molecular forms of secreted PCSK9- propeptide, the peak at 13 834.6 Da is due to sulfation 2)Y38; calculated mass 13 835.5 Da with at Tyr38 (SO4 modification at pyroGlu31) [5], whereas the peak at 2)Y38 with an additional 13 915.5 Da is due to SO4 modification of (cid:2) 80 Da. Figure 1B shows a nonspe- cific peak interacting with the PI serum at 14 417 Da. To examine whether PCSK9-propeptide heterogeneity was due to phosphorylation, immunoprecipitates were incubated in the presence (Fig. 1C,D) of SAP. Follow- ing SAP incubation, PCSK9-propeptide heterogeneity was lost and a single peak corresponding to its sulfated molecular form was resolved at 13 834.5 Da (Fig. 1C), whereas the nonspecific peak was unaffected by this treatment (Fig. 1D). Heterogeneity of the propeptide Longitudinal population studies have shown signifi- cant reduction in the risk of coronary heart disease in [20,21]. Reduced ‘loss-of-function’ PCSK9 carriers plasma PCSK9 concentrations three least PCSK9 variants, R46L, Y142X and C679X, increase the amount of LDLR that is recycled, effectively reducing plasma LDL cholesterol [7,22]. As is the case with ‘gain-of-function’ PCSK9 variants, not all ‘loss- of-function’ variants can be attributed to a single mechanism, reduced plasma PCSK9. However these studies, along with the identification of two healthy PCSK9 ‘null’ individuals [7,23] have gen- erated much interest toward understanding the exact details of the mechanism(s) of PCSK9-dependent LDLR degradation, its site(s) of action, whether the effect is direct or indirect, and how different PCSK9 single nucleotide polymorphisms alter its function. It is believed that the design of PCSK9 inhibitors may
T. Dewpura et al.
PCSK9 circulates as a phosphoprotein in humans
HepG2 + PCSK9-V5
Endogenous PCSK9
7.5
A SO4
2–/PO4
2– = 55/45 ± 0.05
2–/PO4
2– = 46/54 ± 0.02
E SO4
15
13911.6 + H
13834.6 + H 13915.5 + H
5
10
14152.0 + H ns
13831.5 + H
2.5
5
0
0
Huh7 + PCSK9-V 5
B
14417.0 + H
Preimmune Control
7.5
2– =
15
F 13913.7 + H 2–/PO4 SO4 30/70 ± 0.04
5
y t i
10
y t i
ns
13833.7 + H
14155.4 + H
2.5
5
s n e t n
i
s n e t n
i
0
0
Endogenous PCSK9 + SAP
HEK293 + PCSK9-V5
C
G
7.5
15
SO4
2–/PO4
2– = 77/23 ± 0.002
13834.5 + H
l
l
13831.6 + H
k a e p e v i t a e R
5
10
k a e p e v i t a e R
13911.6 + H
5
2.5
14075.5 + H
0
0
D
14407.0 + H
Preimmune + SAP Control
CHOK1 + PCSK9-V5 13831.6 + H
15
7.5
2–/PO4
2– =
H SO4 100/0
10
5
2)),
5
2.5
14073.3 + H
13000
15000 16000
0 12000
0 12000
15000
16000
13000 14000 Mass/charge (m/z)
14000 Mass/charge (m/z)
Fig. 1. MS analysis of the PCSK9-propep- tide molecular mass heterogeneity. (A–D) TOF-MS analyses of the molecular forms of endogenously expressed PCSK9-propeptide immunoprecipitated from the media of HepG2 cells with either immune (anti- hPCSK9 IgG; A, C) or preimmune sera (B, D), and following dephosphorylation (C, D). (E–H) TOF-MS analyses of the molecular forms of the propeptide of V5-tagged PCSK9 immunoprecipitates from the media of transfected and overexpressing HepG2 (E), Huh7 (F), HEK293 (G) and CHOK1 (H) 2)) to cells. The ratio of the sulfated (SO4 sulfated and phosphorylated (PO4 calculated as the area under the peak as described in Experimental procedures, is shown ± SE. Analyses were conducted on at least three independent experiments. ns, nonspecific peak.
2)
and PCSK9-dependent
(SO4 ⁄ PO4
hPCSK9(WT)-V5 transfected with of PCSK9 in the absence of SAP indicated that not all secreted propeptide is phosphorylated (Fig.1A). This partial modification has been reported for a number of other secreted phosphoproteins such as insulin growth factor-binding proteins and osteopontin [25], and is unlike the complete modification reported for phos- secreted from mammary and salivary phoproteins glands [25]. Interestingly, MS analyses of the PCSK9- propeptide immunoprecipitated from HepG2 total cell lysates did not show intracellular phosphorylation of the PCSK9-propeptide (data not shown) suggesting that this phosphorylation occurs just prior to PCSK9 secretion, as we had previously documented for its sulfation at Tyr38 [4,5]. same Using the technique, we next
the HEK293 cell line
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the most commonly used to study PCSK9 biosynthe- sis LDLR degradation [5,10,26]. The ratio of secreted propeptide phosphory- lation, assessed by determining the ratio between unphosphorylated but sulfated propeptide and phos- 2)) as the phorylated propeptide signal area under the peak, was similar between transduced hPCSK9(WT)-V5 expressed in HepG2 cells (Fig. 1E; 46 ⁄ 54 ± 0.02, n=5) and that produced endogenously (Fig. 1A; 55 ⁄ 45 ± 0.05, p=0.16, n=7). However, the ratio of unphosphorylated but sulfated propeptide to phosphorylated propeptide from the media of Huh7 cells was 30 ⁄ 70 ± 0.04 suggesting that significantly more of the PCSK9-propeptide is secreted as a phosphoprotein from this cell line (Fig. 1F; p=0.003, n=5). By con- trast, expressing hPCSK9 (WT)-V5 secreted only 23 ± 0.2% (n=3) of PCSK9- propeptide in its phosphorylated form and signifi- cantly less than both liver cell lines; P < 0.0001 for both (Fig. 1G). No phosphorylation of PCSK9-pro- peptide was detected in the CHOK1 cell line (Fig. 1H, examined propeptide phosphorylation from the media of several cell lines transfected with the expression vector for a C-terminal V5-tagged wild-type hPCSK9 [hPCSK9 (WT)-V5], including the human liver cell lines HepG2 line the human embryonic kidney cell and Huh7, HEK293 and the Chinese hamster ovary cell line CHOK1 (Fig. 1E–H, respectively). These cell lines are
T. Dewpura et al.
PCSK9 circulates as a phosphoprotein in humans
Ser47 is the site of phosphorylation in the propeptide of PCSK9
in part,
and
2)
sulfated and
propeptide for (Fig. 2); when this
15
corresponding to SO4 To define the site of phosphorylation in the PCSK9- propeptide we immunoprecipitated hPCSK9(WT)-V5 from transfected HepG2 cells, treated half with SAP and then digested with trypsin (Fig. 2). MS analyses of the tryptic peptides incubated in the absence or pres- shifted by ence of SAP revealed a peptide that 79.5 Da, corresponding to pyroGlu31–Arg66 within the propeptide (Fig. 2A: observed 4172.9 Da versus calculated 4174.2 Da; and Fig. 2B: observed 4093.4 Da versus calculated 4094.2 Da, respectively). Ser47 within this peptide (Fig. 2C) exhibits a minimal consensus site (S47EED) for two kinases demonstrated to act on secretory proteins; GCK [consensus site SXE ⁄ S(P)] [29] and casein kinase II (consensus site S ⁄ TXXE ⁄ D) [30]. Phosphorylation of Ser47 was confirmed by site-direc- ted mutagenesis residue was mutated to Ala and the construct [hPCSK9(S47A)-V5] transduced into Huh7 cells, the propeptide was no showing a single peak of longer phosphorylated, 2) propeptide 13 817.3 Da, (calculated size 13 819.5 Da; Fig. 2D). n=3). These results demonstrate the cell-type specific- ity of phosphorylation of the PCSK9-propeptide, with ratio of unphosphorylated to phosphorylated the differing significantly among the cell lines examined. to cell-specific kinase This may be due, and ⁄ or phosphatase activities and ⁄ or differing levels therein. Although phosphorylated sulfated peptides < 8000 Da differ in their detection efficiency by MS this difference is lost when comparing like molecules that are > 8000 Da [27,28]. Therefore, our measure of the area under the peak for unphosphory- lated but sulfated PCSK9-propeptide (13 835.5 Da) to PCSK9-propeptide phosphorylated (13 915.5 Da) is a valid comparison. In addition, a minor but specific band is present in these spec- tra at (cid:2) 14 150 Da (Fig. 1E,F) and (cid:2) 14 070 Da that represents alternative signal pepti- (Fig. 1G,H) dase cleavage site following Ala28 (calculated mass 2)PO4 and SO4 14 159.8 Da 2) propeptide) instead of Ala30 14 079.8 Da for SO4 Indeed, signalp 3.0 server (a signal (Fig. 1E–H). peptide prediction program) predicted the primary sig- nal peptide site as ARA30flQE and a secondary signal peptide site as A28flRAQE.
PCSK9-V5 + trypsin
A
D
PCSK9(S47A)-V5 Δ Δ –16 Da
4093.4 + H
13817.3 + H
20
4 1 7 4172.9 + H
10
10
y t i s n e t n
y t i s n e t n
0
B
4093.4 + H
ns
PCSK9-V5 + trypsin + SAP
14045.9 + H
5
20
i k a e p e v i t a l e R
i k a e p e v i t a l e R
10
0
12000
13000
14000
16000
0 4000 4050 4100 4150 4200 4250
15000 Mass/charge (m/z)
Mass/charge (m/z)
C
Q31EDEDGDY[SO4
2–]EELVLALRS[PO4
2–]EEDGLAEAPEHGTTATFHR66
CAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHV
FHGLLPGFLVKMSGDLLEALKLPHVDYIEEDSSVFAQ152
Fig. 2. MS analysis of PCSK9-propeptide tryptic digests and phosphorylation site PCSK9-propeptide variant. (A, B) TOF-MS analyses of the tryptic peptides from the immunoprecipitates of the propeptide of V5-tagged PCSK9 from the media of transfected and overexpressing 2)Y38 and Huh7 cells in the absence (A) and presence (B) of SAP. (C) Amino acid sequence of the propeptide of PCSK9. pyroQ31, SO4 2)S47 are in bold. The phosphorylated tryptic peptide is highlighted by gray boxes in (A) and (B) and the corresponding amino acid PO4 sequence highlighted by a gray font in (C). (D) MS analyses of the molecular form of the propeptide variant (S47A) of V5-tagged PCSK9 immunoprecipitates from the media of transfected and overexpressing Huh7 cells. Analyses were conducted on at least three independent experiments. ns, nonspecific peak.
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PCSK9 circulates as a phosphoprotein in humans
Phosphorylation of Ser47 in the propeptide of PCSK9 is decreased by the naturally occurring R46L and A53V PCSK9 variants
addition, the level of phosphorylation of the R46L var- iant (46 ± 2%, n=6) was significantly less than the (58 ± 4%, n=5, p=0.01) A53V PCSK9 variant the n)1 basic (Fig. 3D). Therefore, replacement of residue Arg by Leu, decreases the rate of phosphoryla- tion at Ser47, indicating the importance of this residue in the consensus site or conformation recognition by its cognate kinase. Also, the reduced phosphorylation of the A53V variant in comparison with wild-type indi- cates that residues downstream of Ser47 may also impact its post-translational modification. In a previ- ous study, we observed that individuals heterozygous for the PCSK9(R46L) variant have reduced circulating PCSK9 compared with individuals carrying the normal PCSK9 alleles [22].
Site-directed mutagenesis shows consensus sequence site of GCK [SXE ⁄ S(p)] for propeptide phosphorylation
We next examined the effect of several hPCSK9-V5 variations on the phosphorylation of secreted propep- tide, namely the Y38F variation preventing sulfation of the prodomain, the common naturally occurring A53V variation that has no significant effect on plasma cholesterol levels and the R46L variation, a naturally occurring variant associated with hypocholes- terolemia [31] and reduced plasma PCSK9 [22]. As shown in Fig. 3A, mutating the site of propeptide sulf- ation [hPCSK9(Y38F)-V5] did not significantly affect PCSK9-propeptide phosphorylation in comparison with hPCSK9(WT)-V5 (Fig. 3D, p=0.67), as assessed by comparing the area under the peak for the unphos- phorylated versus phosphorylated signals. By contrast, levels of propeptide phosphorylation for both the R46L and the A53V PCSK9 variants were reduced by in 34% (p=0.0001) and 17% (p=0.04) respectively, In comparison with hPCSK9(WT)-V5 (Fig. 3B–D). To determine the consensus site of phosphorylation within the propeptide of PCSK9, immunoprecipitates
D
A
Y38F
15
13823.8+H
PCSK9(Y38F)-V5 +16 Da Δ Δ
UM –
2– =
10
UM/PO4 32/68 ±0.003
2–
PO4
13745.4+H 14065.0+H
5
0
R46L
B
15
PCSK9(R46L)-V5 –43 Da Δ Δ
2–
2– =54/46 ± 0.02
SO4
2–/PO4
SO4
y t i s n e t n
10
2–
PO4
13800.3 + H 13879.6 + H 14120.2 + H 14044.6 + H
5
ns
i k a e p e v i t a l e R
0
A53V
C
15
PCSK9(A53V)-V5 +28 Da
2–
SO4
2– = 42/58 ± 0.04
SO 4
2– /P O 4
10
2–
13948.2 + H
PO4
ns
13865.0 + H
5
14194.4 + H
2); gray bars) to sul- 2); black
0
12000 13000 14000 15000
WT
Mass/charge (m/z)
2–
SO4
2–
PO4
0
10 20 30 40 50 60 70 80
Percent molecular form
Fig. 3. MS analysis of immunoprecipitated PCSK9-propeptide from the media of trans- fected Huh7 cells overexpressing V5-tagged PCSK9 variants. (A–C) TOF-MS analyses of the propeptide of V5-tagged PCSK9 variants as labeled from the media of transfected and overexpressing Huh7 cells. For each variant the change in molecular mass due to the specific amino acid change is show as DDa. (D) A graphic representation of the data incorporating results from analyses of the propeptide of V5-tagged wild-type PCSK9. The ratio of unmodified (UM; white bar) or sulfated (SO4 fated and phosphorylated (PO4 bars), calculated as area under the peak as described in Experimental procedures, is shown ± SE. t-Tests were carried out to compare significant changes in phosphoryla- tion of the propeptide of PCSK9 between variants. Analyses were conducted on at least three independent experiments. ns, nonspecific; *P < 0.05; **P < 0.005; ***P < 0.0005.
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PCSK9 circulates as a phosphoprotein in humans
2) + PO4
2)-propeptide to PO4
and 1.0 ± 0.1 for HepG2 cells
consensus requires site of V5-tagged recombinant PCSK9 from transfected Huh7 cells were analyzed by MS (Fig. 4). Below each spectra is the observed and calculated (in parentheses) 2) and molecular masses for each mutant in its SO4 2) forms, as well as the major molecular SO4 form observed. Mutations E48A and E48D did not affect phosphorylation (Fig. 4C,D), nor did D50A and D50E (Fig. 4G,H). However mutations E49A and the propeptide E49D prevented phosphorylation of (Fig. 4E,F). The requirement of a Glu at n+2 (Fig. 4C) and the inability of Asp (Fig. 4D) to mimic its effect, is a strict requirement for phosphorylation by GCK, SXE ⁄ S(p) [29], and not casein kinase II whose n+3 E or D (SXXE ⁄ D).
hPCSK9(WT) (lanes 6 and 7, respectively). Again the phosphorylated form of PCSK9 and its propeptide were only detected extracellularly (data not shown). These results, and the MS analyses presented previ- ously, suggest that phosphorylation occurs just prior to PCSK9 secretion from the cell, or after secretion by an ectokinase [34]. Quantification of the ratio of 2)-PCSK9 is shown below PO4 each lane. The ratio of PCSK9-propeptide to mature PCSK9 phosphorylation for endogenous protein secreted into the media from Huh7 cells was 1.9 ± 0.1 (n=3) (n=3; p=0.006), reflecting the significantly higher level of phosphorylation of PCSK9-propeptide in Huh7 versus HepG2 shown earlier (Fig. 1). This difference was not due to changes in the amount of total PCSK9 relative to its propeptide as assessed by 35S-Met ⁄ Cys labeling of PCSK9 (data not shown). The minor, but specific band just above the major propeptide band represents the propeptide generated by the alternate signal pepti- dase cleavage following Ala28 instead of Ala30, as shown in the mass spectra previously (Figs 1E–H and 4A–H). (Fig. 4F, inset). This product was
differed the ratio of PO4 between We also noted that 2)-PCSK9
Interestingly, a cleaved PCSK9-propeptide product was detected in the media of Huh7 cells transfected with the E49A PCSK9 variant at 11738.2 Da (Fig. 4E, inset), due to cleavage following Ser47 and corre- sponded to 13% of the area under the peak for total propeptide 2026.1 Da (observed DDa Q31-Ser47 versus calculated 2044.1 Da), but not with the E49D variant also observed in immunoprecipitates from the media of cells expressing the R46L variant and corresponded to 5% of the area under the peak for total propeptide inset) (observed DDa Q31-Ser47 2000.1 Da (Fig. 4B, versus calculated 2001.0 Da). Phosphorylation is known to alter the stability of proteins and their resis- tance to proteolysis [32,33]. Our results suggest that Ser47 phosphorylation stabilizes the propeptide of PCSK9 by preventing its proteolysis. Also, cleavage of the propeptide of the R46L and E49A PCSK9 vari- ants, but not E49D PCSK9 variant, suggests that charge distribution around this site is also important for its stability. PCSK9 was in
PCSK9 is phosphorylated in its C-terminal domain
2)-propep- endogenous tide ⁄ PO4 PCSK9 (Fig. 5, lane 2; 1.0 ± 0.1) and hPCSK9(WT)- V5 (Fig. 5, lane 1; 3.2 ± 0.1) secreted from HepG2 cells. The attenuated phosphorylation of V5-tagged versus endogenous PCSK9 could be a consequence of: (a) saturation of the responsible kinase upon over- expression, or (b) the C-terminal V5-tag affecting the conformation of PCSK9 preventing kinase access- ibility. To test the first possibility we carried out sequential immunoprecipitations of V5-tagged PCSK9 followed by endogenous PCSK9 from transfected HepG2 cells (Fig. 5). Phosphorylation of endogenous identical secreted transfected lane 5) and untransfected (0.92 ± 0.1, n=3; Fig. 5, HepG2 cells (1.0 ± 0.1, n=3, p=0.39; Fig. 5, lane 2) so the responsible kinase was not saturated. To examine the second possibility, transfection of untag- ged hPCSK9(WT) into Huh7 cells did not affect C-terminal phosphorylation (1.7 ± 0.1, n=4; Fig. 5, lane 7) when compared with endogenous PCSK9 from the same cell line (1.9 ± 0.1, n=3, p=0.2; Fig. 5, lane 6). The same result was noted when this experiment line (data not was duplicated in the HepG2 cell shown).
and
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To further examine PCSK9 phosphorylation, we grew untransfected hPCSK9(WT)-V5- transfected expressing HepG2 cells in media containing 32P-ortho- phosphate, immunoprecipitated PCSK9 from cells and media, and analyzed samples by SDS ⁄ PAGE fraction- ation followed by phosphorimaging (Fig. 5). Lanes 1 immunoprecipitated and 2 and 3 and 4 represent PCSK9 from media and total cell lysates of HepG2, respectively. PCSK9 and its co-immunoprecipitating propeptide were secreted as phosphoproteins, whereas -prodomain were detected no phospho-PCSK9 or intracellularly. Radiolabeling and analyses were also conducted for untransfected and transfected Huh7 cells in this case S688QE] In addition, there is a commercially available anti- body whose epitope (C679RSRHLAQASQELQ692) was directed toward the C-terminus of PCSK9 and contained a potential consensus site of phosphoryla- [29]. This tion [SXE ⁄ S(P),
T. Dewpura et al.
PCSK9 circulates as a phosphoprotein in humans
2–
A
WT C
E48A E
D50A
E49A G
2–
2–
PO 4
SO 4
PO 4
2–
75
PO 4
11738.2+H
y t i s n e t n
2–
2–
2–
10
SO 4
10
10
50
SO4
SO4
10
ACS
ACS
5
ACS
5
5
5
ACS
0
0
0
0
25
11000 12500
11000 12500
11000 12500
11000 12500
i k a e p e v i t a l e R
0
1200 0
1400 0
1200 0
1400 0
1200 0
1400 0
1400 0 1200 0 Mass/charge (m/z)
2–
13827.9 Da (13835.5 Da)
13782.9 Da (13777.5 Da)
13775.1 Da (13777.5 Da)
13798.4 Da (13791.5 Da)
SO4
2–
13906.4 Da (13915.5 Da)
13863.9 Da (13857.5 Da)
2– (13857.5 Da)
13875.0 Da (13871.5 Da)
PO4
No PO 4
Form
phosphorylate d
phosphorylate d
sulfated
phosphorylate d
R46L
E49D
D50E
B
D
F
H
E48D 2–
2–
2–
PO 4
SO 4
2–
PO 4
75
SO 4
2–
PO 4
11782.8+H
y t i s n e t n
2–
2–
50
SO 4
ACS
10
10
10
SO 4
ACS
10
ACS
ACS
5
5
5
5
25
0
0
0
0
11000 12500
11000 12500
11000 12500
11000 12500
i k a e p e v i t a l e R
0
1200 0
1400 0
1200 0
1400 0
1200 0
1400 0
1200 0
1400 0
Mass/charge (m/z)
2–
13782.9 Da (13792.5 Da)
13822.2 Da (13821.5 Da)
13822.3 Da (13821.5 Da)
13853.3 Da (13849.5 Da)
SO4
2–
13864.0 Da (13872.5 Da)
13898.8 Da (13901.5 Da)
2– (13901 Da)
13919.0 Da (13929.5 Da)
PO4
No PO 4
phosphorylate d
Form
sulfated
sulfated
phosphorylate d
2)) and sulfated and phosphorylated (PO4
Fig. 4. MS analysis of the consensus site of PCSK9-propeptide phosphorylation from the media of transfected Huh7 cells overexpressing V5-tagged PCSK9 variants. (A–H) TOF-MS analyses of the propeptide of V5-tagged PCSK9 variants as labeled from the media of transfected and overexpressing Huh7 cells. For each variant the observed versus calculated (in brackets) molecular mass is shown below each panel for 2)) propeptide, as well as the major molecular form observed. Insets highlight the the sulfated (SO4 presence or absence of proteolysis fragments of the parent propeptide. Analyses were conducted on at least three independent experi- ments. ns, nonspecific. ACS, alternate signal peptidase cleavage site.
sera against PCSK9 (Fig. 6D) and human plasma show that its propeptide also circulates as a phospho- protein (Fig. 6D; observed mass 13 919 Da versus calculated 13 915.5 Da). Figure 6C shows nonspecific peaks that are immunoprecipitated with preimmune sera.
Phosphorylation of C-terminal PCSK9 was also dependent on GCK-like activity
FEBS Journal 275 (2008) 3480–3493 ª 2008 The Authors Journal compilation ª 2008 FEBS
3486
To confirm and determine the consensus site of phos- phorylation within the C-terminal of PCSK9, we cultured Huh7 cells, untransfected and transfected trans- antibody reacted with immunoprecipitates of fected V5-labeled PCSK9 (Fig. 6A, lane 1) but was unable to detect endogenous PCSK9 immunoprecipi- tates (Fig. 6A, lane 2). However, dephosphorylation of immunoprecipitated endogenous PCSK9 with SAP, restored antibody recognition (Fig. 6A, lanes 3 and 4). Of significance, this modification also occurs at Ser688 in vivo, as assessed by immunoblotting (using the same C-terminal PCSK9 antibody as above) of PCSK9 immunoprecipitates from human plasma in lanes 1 the absence and presence of SAP (Fig. 6B, and 2, respectively). MS analyses of immunoprecipi- tates using preimmune sera (Fig. 6C) and immune
T. Dewpura et al.
PCSK9 circulates as a phosphoprotein in humans
HepG2
Huh7
Media
Cells
Media
Media
3
1
6 UT
2 WT-V5 UT WT-V5
4 UT
5 PIP-WT
7 WT
proPCSK9
PCSK9
ACS prodomain
IP: V5 Ab
1.9 ± ± 0.1 1.7 ± 0.1
0.92 ± ± 0.1
3.2 ± ± 0.1 1.0 ± ± 0.1
IP: hPCSK9 Ab propeptide / PCSK9
Fig. 5. The prodomain and mature PCSK9 are secreted as phosphoproteins in vitro. HepG2 and Huh7 cells untransfected (lanes 2, 4 and 6) and transfected with the expression vector for either untagged (lane 7) or V5-tagged hPCSK9 (lanes 1, 3 and 5) were radiolabeled with 32P-orthophosphate as per Experimental procedures. Total cell lysates and media were immunoprecipitated with anti-hPCSK9 IgG or anti-V5 IgG ?accolade "acc1a"> and fractionated by SDS ⁄ PAGE for phosphorimaging as per Experimental procedures. Lane 5 represents the post-immunoprecipitation of endogenously labeled protein following a primary immunoprecipitation for overexpressed V5-tagged protein. The positions of PCSK9, propeptide and alternate propeptide signal peptidase cleavage product (ACS) are noted. Quantitation of the ratio of phos- phorylation for propeptide to PCSK9 is shown below each lane. Analyses were conducted on at least three independent experiments.
A
B
Human plasma
2
1
HepG2 media
4
2
1 WT–V5
3 UT UT UT
PCSK9-V5
PCSK9
PCSK9 IgG
10
–
–
–
Units SAP
1
10
Units SAP
IB: Anti- hPCSK9 C-terminal IgG
IB: Anti- hPCSK9 C-terminal IgG
PCSK9 propeptide from human plasma
13919.3 + H
Preimmune sera
C
D
3
3
PCSK9 Immune sera
ns:13754.4 + H
y t i s n e t n
y t i s n e t n
ns:13754.9 + H
2
2
ns:13894.6 + H
ns: 13604.7 + H
ns:13973.2 + H
1
1
i k a e p e v
l
l
0
0
i k a e p e v i t a e R
i t a e R
12000
13000
14000
15000
16000
12000
13000
14000
15000
16000
Mass/charge (m/z)
Fig. 6. The prodomain and mature PCSK9 are secreted as phosphoproteins in vivo. (A) Immunoprecipitation of overexpressed V5-labeled PCSK9 (lane 1) or endogenous PCSK9 (lanes 2–4) from the media of HepG2 cells followed by dephosphorylation of immunoprecipitates (lanes 3 and 4) and immunoblotting analyses with the anti- hPCSK9 C-terminal IgG (Imgenex). (B) Immunoprecipitation of PCSK9 from human plasma with the anti-hPCSK9 IgG followed by incubation in the absence (lane 1) or presence (lane 2) of SAP and immunoblot- ting analyses with the anti-(C-terminal hPCSK9) IgG (Imgenex). IgG, immuno- globulin band. (C, D) TOF-MS of the molecular forms of PCSK9-propeptide immunoprecipitated from human plasma with either preimmune sera (C) or immune (anti-hPCSK9 IgG; D). ns, nonspecific peaks.
for vectors untagged expression
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3487
with hPCSK9 mutants in media containing 32P-orthophosphate. We immunoprecipitated PCSK9 from these media, and analyzed itby SDS ⁄ PAGE fractionation followed by phosphorimaging. To assess total protein expression 35S-Met ⁄ Cys labeling was carried out (Fig. 7). There was (cid:2) 3.5 · more expression of both the S688A (3.5 · for PCSK9 and 3.2 · for its propeptide, respec- tively; lanes 1A and 1B) and E690A PCSK9 mutants (3.4 · for PCSK9 and 3.5 · for its propeptide, respec- tively; lanes 3A and 3B) when compared with endoge- nous levels of PCSK9 or its propeptide (both set as 1; lanes 2A and 2B). The mutation of either S688A (lanes 5B and 7B) or E690A (lanes 6B and 8B) in the C-terminal region of PCSK9 did not affect propeptide phosphorylation which was also (cid:2) 3 · more than
T. Dewpura et al.
PCSK9 circulates as a phosphoprotein in humans
Media from Huh7 cells
A
35S - Met/Cys labeled
32P - orthophosphate labeled
2
3
4
1
5
6
7
8
PCSK9
C 1
S688A 3.5
C 1
E690A 3.4
S688A 1.0
E690A 0.84
S688A E690A 0.83
0.71
1 Relative PCSK9
23 ± 0.2% in HEK293 cells and none in CHOK1 cells (Fig. 1). It also occurred very late in the secretory pathway or at the cell surface because no phosphory- lated PCSK9 was detected intracellularly by either MS analyses of immunoprecipitates or radiolabeling fol- lowed by immunoprecipitation and autoradiography, two very sensitive techniques.
B ACS propeptide
C
S688A
E690A
C
S688A E690AS688A E690A
1
3.5
3.1
2.9
2.8
1
2.2
3.2
Relative propeptide
Fig. 7. Site-directed mutagenesis of the C-terminal phosphorylation region of PCSK9. Huh7 cells untransfected (lanes 2 and 4; endoge- nous-C) and transfected with cDNAs encoding untagged PCSK9 C-terminal variants (lane 1 and 3 and 5–8 as labeled) were radiola- beled with either 35S-Met ⁄ Cys (lanes 1–3) or 32P-orthophosphate (lanes 4–8) as per Experimental procedures. Media was immuno- precipitated with anti-hPCSK9 IgG, fractionated by SDS ⁄ PAGE for phosphorimaging as per Experimental procedures. The positions of PCSK9, propeptide and alternate propeptide signal peptidase cleav- age product (ACS) are noted. Quantitation of the ratio of total pro- tein immunoprecipitated (setting untranfected endogenous-C as 1) is shown below each lane.
(CAKEP; human not
Serine phosphorylation occurred within the site RS47EED and was 100% dependant on Glu at the n+2 position (Fig. 1). This site is completely con- served among primates except for the tamarin monkey where an amino acid change occurs at n+3 (D50E), which should not affect propeptide phosphorylation based on our site-directed mutagenesis results (Fig. 4). There are two possible sites of prodomain phosphory- lation in the mouse and rat. The first site is conserved between human (RSEED), mouse and rat PCSK9 (both PSQED; supplementary Fig. S1A). Although, the n)1 and n+1 residues differ, they still conform to a consensus phosphorylation site for GCK (SXE). The second site is only conserved between mouse and rat supplementary (CSKEA), Fig. S1A). The prodomain of mouse PCSK9 is phos- phorylated (supplementary Fig. S1B,C) at PS50QED (supplementary Fig. S1D) and not at CSKEA (supple- mentary Fig. S1E).
endogenous PCSK9 (lane 4B). However, mutation of either S688A (lanes 5A and 7A) or E690A (lanes 6A and 8A) prevented phosphorylation at the C-terminus of PCSK9 because only background levels of phos- phorylation due to endogenous PCSK9 were measured (lane 4A). This can also be seen by comparing the ratio of propeptide ⁄ PCSK9 phosphorylation for wild- type untagged PCSK9 (1.9; Fig. 5, lane 6) with both of these untagged mutants (Fig. 7, lanes 5–8). The requirement for an E at n+2 suggests that, like for this phosphorylation is propeptide phosphorylation, carried out by a GCK-like kinase [consensus site SXE ⁄ S(p)].
Discussion
it
stabilizes
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3488
PCSK9 undergoes several post-translational modifica- tions; while in the ER it is glycosylated at a single N-linked site at amino acid 533 that is further matured in the Golgi increasing the molecular mass of secreted versus intracellular PCSK9 by (cid:2) 2200 Da [4]. We have also reported on sulfation of Tyr38 within the propep- tide of PCSK9. Sulfation occurred just prior to secre- tion from the trans-Golgi network because it was barely detected intracellularly [4,5]. In this study, we report that secreted PCSK9 is phosphorylated at Ser47 in its propeptide and at Ser688 in its Cys- and His-rich domain. Phosphorylation of the propeptide was cell- type specific with 70 ± 4% phosphorylation in Huh7 cells, cells, 54 ± 2% in HepG2 followed by Phosphorylation is an important post-translational modification shown to affect several parameters inclu- ding: (a) stability and turnover by interfering with or promoting proteolysis [32,33], (b) activating or inacti- vating enzymes [35], (c) subcellular localization and transport [36,37], and (d) protein–protein interactions and ⁄ or protein conformation [33,38,39]. What is the function of propeptide phosphorylation in PCSK9? Biophysical studies of the structure of PCSK9 have shown that its propeptide region is solvent exposed, and crystal structure studies of PCSK9 have failed in this region due to lack of electron density [15,19,40,41] and therefore descriptions of the prodomain of PCSK9 begin downstream of the site of phosphorylation (Ser47) at Thr61 [15,19]. Neither study predicts direct interaction of the PCSK9-propeptide with the LDLR is epidermal growth factor-A domain; however, interesting to note that several documented ‘loss-of- function’ PCSK9 variants such as the R46L [31,42,43] occur within this domain, suggesting a regulatory func- tion for this region. We provide evidence here that phosphorylation at Ser47, as well as charge distri- bution within this propeptide region, it against proteolysis following this site of post-transla- tional modification (Fig. 4). Recently, Kwon et al. [19] recombinant propeptide D53-PCSK9 reported that the than sevenfold affinity for exhibited greater
T. Dewpura et al.
PCSK9 circulates as a phosphoprotein in humans
extracellular, epidermal growth factor-A domain of LDLR in comparison with wild-type PCSK9, support- ing our results that the N-terminal region of the pro- peptide of PCSK9 may modulate or stabilize its interaction with LDLR, either directly or indirectly.
respectively), it
We also report that PCSK9 is phosphorylated in its Cys- and His-rich domain, five amino acids from its C-terminus at Ser688, within the sequence QAS688- QELQ (Figs 5 and 6). Like the N-terminal propeptide region of PCSK9, its C-terminal region (from amino acids 683–692) has not been characterized by existing crystal structure studies [15], and although this site is not conserved in the mouse or rat (KASWVQ and KASWVHQ, is 100% conserved among 12 of the 14 primate species [44]. Because the C-terminus of PCSK9 is solvent-exposed, it may be involved in interactions with other PCSK9 domains (e.g. the propeptide) or other peptides ⁄ proteins. Phos- phorylation status may be an important mode of regulating such interactions. We previously reported that heterozygous carriers of the PCSK9 R46L variant have less circulating PCSK9 than those carrying normal alleles for PCSK9 [22]. In this report we show that the propeptide region of this variant is subject to proteolysis in the Huh7 cell line. Does this occur in vivo and does it effectively shorten the half-life of PCSK9 resulting in the ‘loss-of-function’ phenotype documented for carriers of these variants, or as hypothesized above, could the reduction in propep- tide phosphorylation decrease its affinity for the LDLR? If phosphorylation regulates PCSK9 activity, under- standing the physiological stimuli that affect it, as well as mapping any additional sites of phosphorylation will be important in further understanding of its cell biology, and will improve PCSK9 drug-design strate- gies, having important implications in the future treat- ment of hypercholesterolemic individuals. To begin to address the importance of PCSK9 and its propeptide phosphorylation we examine the effects of these phosp- homutants (in both hypo- and hyperphosphorylated states) on PCSK9-dependent LDLR degradation.
Experimental procedures
Constructs and antibodies
The cDNA of human PCSK9 was cloned into the pIRES2- enhanced green fluorescent protein with or without a C-ter- minal V5 tag as described previously [2]. Mutations were introduced by site-directed mutagenesis as described [49]. Anti-hPCSK9 IgG, used for the immunoprecipitation of endogenous or untagged recombinant PCSK9 was raised in rabbits by cDNA vaccination with the mammalian expres- sion vector pcDNA3 into which the cDNA for human PCSK9 had been inserted [50]. Animal protocol for antibody production was approved by the institutional Animal Care Committee. The mouse anti-V5 IgG used for immunopre- cipitation of V5-tagged recombinant PCSK9 was from Invi- trogen (Burlington, Canada) and the goat anti-(C-terminal PCSK9) IgG used for immunoblotting from Imgenex (San Diego, CA, USA). Secondary anti-mouse and anti- (rabbit HRP) IgG were from Amersham (Piscataway, NJ, USA) and the secondary anti-(goat HRP) IgG was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
the HEK293 and CHO cell is, We also demonstrated that the addition of the C-ter- minal V5-tag greatly diminished phosphorylation at Ser688 (Fig. 5). Many binding, co-localization and crystal structure studies for PCSK9 and LDLR have been carried out using tagged and therefore hypo- phosphorylated PCSK9 and ⁄ or employing cell lines in which propeptide phosphorylation is diminished or absent lines, (that respectively, Fig. 1). PCSK9 is co-regulated at the transcriptional
Cell culture, transfection and sample collection
HepG2, Huh7, HEK293 and CHOK1 cells were grown at 37 (cid:2)C in Dulbecco’s modified Eagle’s medium + 10% FBS + gentamycin (28 lgÆmL)1). Cells (3 · 105) were trans- fected with a plasmid expression vector for human PCSK9 (hPCSK9; 1.5 lg) as described using Lipofectamine 2000 (Invitrogen) in a 1 : 1 ratio to cDNA [2]. Spent media from untransfected and transfected cells were collected in the
level with LDLR and many studies have asked the physio- logical relevance of a co-directional regulation of two proteins with opposing functions. Recent studies have shown that PCSK9 catalytic activity is not required for LDLR degradation and suggest that it instead binds to LDLR directly, re-routing it to the lysosome [45,46]. Most studies have focused on the physiological consequences of the ‘gain-of-function’ D374Y PCSK9 mutation that causes severe hypercholesterolemia. This variant binds 6-25 · more strongly to the LDLR than wild-type PCSK9 at pH 7.5 and 8-25 · more strongly at pH 5.3 found in the late endosomes [15,47]. Few studies have addressed how wild-type PCSK9 might alter its affinity toward the LDLR under normal physi- ology. Could phosphorylation of PCSK9 and ⁄ or its propeptide affect PCSK9 ⁄ LDLR binding and provide an acute mechanism(s) to regulate its ‘activity’ in circu- lation and ⁄ or upon appropriate stimulation? Not all
FEBS Journal 275 (2008) 3480–3493 ª 2008 The Authors Journal compilation ª 2008 FEBS
3489
tissues or cell-types respond equally to PCSK9 [48]. We have shown that PCSK9 phosphory- lation is cell-type specific (Fig. 1), so tissue-specific kinases and ⁄ or phosphatases may provide an addi- tional trophic level of regulation.
T. Dewpura et al.
PCSK9 circulates as a phosphoprotein in humans
presence of a general protease inhibitor cocktail (Roche, Laval, Canada) and 200 lm sodium orthovanadate (a phos- phatase inhibitor; Sigma-Aldrich, Oakville, Canada) and centrifuged at 13 000 g for 3 min to remove suspended cells and debris. Cells were lysed in 1 · RIPA buffer (50 mm Tris pH 7.6, 150 mm NaCl, 1% v ⁄ v NP-40, 0.5% w ⁄ v deoxycho- late, 0.1% w ⁄ v SDS) in the presence of inhibitors, as above. Lysates were rotated at 4 (cid:2)C for 30 min, centrifuged at 13 000 g for 3 min and supernatants collected. Protein con- centrations in total cell lysates were determined by the Brad- ford dye-binding method using Bio-Rad’s Protein Assay Kit (Bio-Rad, Mississauga, Canada).
that captured PCSK9 as previously described, except following immunoprecipitation, the antibody ⁄ antigen com- plex was eluted from the protein A beads by incubation in 2 · 150 lL 0.1 m glycine (pH 2.8) for 10 min at room tem- perature. Supernatants were collected, combined and neu- tralized with 30 lL 1 m Tris ⁄ HCl (pH 9.0), concentrated 20· with an Amicon Ultra YM10 Centricon (Millipore Corp., Temecula, CA, USA) and retentates equilibrated in 0.1% trifluoroacetic acid. Ten microliters of the sample was applied to a Gold ProteinChip Array (Ciphergen Biosys- tems Inc., Fremont, CA, USA) and air-dried. One microli- ter of saturated 3,5-dimethoxy-4-hydroxycinnamic acid in 50% (v ⁄ v) acetonitrile + 0.5% (v ⁄ v) trifluoroacetic acid was added and samples analysed by TOF-MS in a Cipher- gen Protein Biology System II. Analyses represent an aver- age of 100 shots and masses were externally calibrated with All-in-1 Protein Standards (Ciphergen Biosystems Inc.). All data were normalized for total ion current and peak areas calculated using the indirect method (with a bracket height of 0.4 and width expansion factor of 2) contained within Ciphergen’s proteinchip 3.1 software.
at
4 (cid:2)C.
Immunoprecipitation, immunoblotting and radiolabeling
Enzymatic dephosphorylation was carried out by incu- bating immunoprecipitates in the presence of 10 units (except where indicated) of SAP (Fermentas, Burlington, Canada) in the provided reaction buffer system for 30 min at 37 (cid:2)C with agitation.
Dephosphorylation
Immunoprecipitations were carried out in 1 · Tris-buffered saline + 0.1% Tween-20 with anti-hPCSK9 IgG (dilution 1 : 500), preimmune sera (dilution 1 : 500) or anti-V5 IgG (1 : 500) and 30 lL of protein A agarose (Sigma-Aldrich) overnight Immunoprecipitates were washed 4 · with 1 mL Tris-buffered saline + 0.1% Tween-20 and fractionated through a 12% polyacrylamide gel. Proteins were electroblotted onto nitrocellulose and immunoblotted following a standard protocol. The primary anti-(C-termi- nal PCSK9) and anti-V5 IgG were used at 1 : 2000 dilu- tions and the secondary antibodies at 1 : 5000 dilutions. Immunoblots were revealed by chemiluminescence using Western Lightening Plus (Perkin-Elmer, Woodbridge, Can- ada) on Kodak X-OMAT film (VWR International, Mon- treal, Canada). The signal was quantified by densitometry using Syngene’s Chemigenius 2XE imager and genetool software (VWR International).
Trypsin digestion was carried out by incubating immuno- precipitates in the presence of 6 ngÆlL)1 trypsin (Roche) in 25 mm NH4HCO3 and 1% (v ⁄ v) acetonitrile overnight at 37 (cid:2)C with agitation.
Trypsin digestion
All results are expressed as mean ± standard error (SE), except where indicated. Data were analyzed using graph- pad prism 5.0 statistical software with significance defined as P < 0.05.
Statistical analyses
Acknowledgement
Untransfected and transfected HepG2 and Huh7 cells were grown to confluence as above. Prior to radiolabeling cells were incubated for 4 h in serum-free Dulbecco’s modified Eagle’s medium without sodium phosphate (Invitrogen) or Met ⁄ Cys-free Dulbecco’s modified Eagle’s medium (Invitro- gen) and then incubated for 16 h in the same media in the presence of either 250 lCi 32P-orthophosphate or 250 lCi 35S-Met ⁄ Cys. Media and total cell lysates were harvested and immunoprecipitated as described above. Samples were frac- tionated through a 12% SDS ⁄ PAGE. Following electropho- resis, gels were dried and visualized by phosphorimaging using a Typhoon Imager. Signals were quantified using imagequant 5.2 software using the integer integration method when comparing samples within a lane and, for sam- ples between lanes, by volume quantitation as recommended.
supported by CIHR team grant This work was (CTP 82946).
MS analyses
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