Leuven, Leuven-Heverlee, Flanders, Belgium The Saccharomyces cerevisiae type 2A protein phosphatase PP2A Pph22p differs from the catalytic subunits of PP2A PP2Ac present in mammals, plant
Trang 1The Saccharomyces cerevisiae type 2A protein phosphatase Pph22p
is biochemically different from mammalian PP2A
Piotr Zabrocki1, Wojciech Swiatek1, Ewa Sugajska1, Johan M Thevelein2, Stefaan Wera2
and Stanislaw Zolnierowicz1,*
1
Cell and Molecular Signaling Laboratory, Intercollegiate Faculty of Biotechnology UG-MUG, Gdansk, Poland;2Laboratorium voor Moleculaire Celbiologie, K.U Leuven, Leuven-Heverlee, Flanders, Belgium
The Saccharomyces cerevisiae type 2A protein phosphatase
(PP2A) Pph22p differs from the catalytic subunits of PP2A
(PP2Ac) present in mammals, plants and
Schizosaccharom-yces pombeby a unique N-terminal extension of
approxi-mately 70 amino acids We have overexpressed S cerevisiae
Pph22p and its N-terminal deletion mutant DN-Pph22p in
the GS115 strain of Pichia pastoris and purified these
enzymes to apparent homogeneity Similar to other
heterologous systems used to overexpress PP2Ac, a low yield
of an active enzyme was obtained The recombinant
enzymes designed with an 8· His-tag at their N-terminus
were purified by ion-exchange chromatography on
DEAE-Sephacel and affinity chromatography on Ni2+
-nitrilotri-acetic acid agarose Comparison of biochemical properties
of purified Pph22p and DN-Pph22p with purified human
8· His PP2Ac identified similarities and differences
between these two enzymes Both enzymes displayed similar
specific activities with 32P-labelled phosphorylase a as
substrate Furthermore, selected inhibitors and metal ions
affected their activities to the same extend In contrast to the mammalian catalytic subunit PP2Ac, but similar to the dimeric form of mammalian PP2A, Pph22p, but not DN-Pph22p, interacted strongly with protamine Also with regard to the effects of protamine and polylysine on phos-phatase activity Pph22p, but not DN-Pph22p, behaved similarly to the PP2Ac–PR65 dimer, indicating a regulatory role for the N-terminal extension of Pph22p The N-terminal extension appears also responsible for interactions with phospholipids Additionally Pph22p has different redox properties than PP2Ac; in contrast to human PP2Ac it cannot be reactivated by reducing agents These properties make the S cerevisiae Pph22p phosphatase a unique enzyme among all type 2A protein phosphatases studied so far Keywords: Saccharomyces cerevisiae; protein phosphatase Pph22p; protein phosphatase 2A; heterologous expression, Pichia pastoris
Reversible protein phosphorylation catalysed by protein
kinases and phosphoprotein phosphatases is a major
mechanism utilized by eukaryotic organisms to regulate
various cellular processes [1] Protein kinases are apparently
derived from one primordial gene In contrast, protein
phosphatases are encoded by at least three unrelated gene
families Based on primary and tertiary structure
similarit-ies, protein phosphatases are currently classified into PPP,
Mg2+-dependent PPM (both PPP and PPM are specific
against phosphoserine/phosphothreonine residues) and
PTP (phosphotyrosine residues-specific) families [2,3] The
PTP family comprises also dual-specificity phosphatases
that are able to dephosphorylate all three phospho-residues [4] Mammalian type 2A protein phosphatase (PP2A), a member of the PPP family, displays a broad substrate specificity in vitro However, its in vivo substrate selectivity, enzymatic activity and subcellular localization are regulated
by the association with regulatory subunits [5,6] Thus, two different dimeric forms of PP2A are formed by the association of the catalytic subunit (PP2Ac) with PR65/A scaffolding subunit or a4 protein In addition, association of
a third variable subunit derived from the unrelated protein families PR55/B, PR61/B¢ or PR72/B¢¢ to the PR65/A– PP2Ac dimer results in the formation of trimeric PP2A [6]
In vivo substrates of PP2A in mammalian cells comprise protein kinases and transcription factors [7] However, the identity of many physiological substrates of PP2A still remains elusive
In budding yeast Saccharomyces cerevisiae protein kin-ases and protein phosphatkin-ases regulate cell growth, cell cycle progression, bud formation and morphogenesis as well as nutrient- and pheromone-induced signalling [8] The num-ber of protein kinases in yeast (119) is approximately four times higher than the number of protein phosphatases (31) [9] However, by association of a single catalytic subunit with different regulatory subunits, protein phosphatases can form several functional holoenzymes and thus match the complexity of protein kinases [2,3,5–7] All above listed families of protein phosphatases are encoded by the
S cerevisiae genome and represented by 12 (PPP),
Correspondence to S Wera, Laboratorium voor Moleculaire
Celbiologie, K.U Leuven, Kasteelpark Arenberg 31,
B-3001 Leuven-Heverlee, Flanders-Belgium.
Fax: + 32 16 32 19 79, Tel.: + 32 16 32 15 00,
E-mail: stefaan.wera@bio.kuleuven.ac.be
Abbreviations: PP2A, protein phosphatase type 2A; PP2Ac, the
catalytic subunit of PP2A; Pph21/22p, PP2Ac from Saccharomyces
cerevisiae; PR65/A, the structural subunit of PP2A; KM71
and GS115, strains of Pichia pastoris; GSSG, glutathione disulfide;
GSH, reduced glutathione.
Enzyme: protein phosphatase 2A (EC 3.1.3.16).
*Note: deceased on 13 February 2001.
(Received 31 January 2002, revised 15 April 2002,
accepted 29 April 2002)
Trang 26 (PPM) and 13 (PTP) members [8,9] In budding yeast
Schizosaccharomyes pombe, PP2A is encoded by PPH21
and PPH22 [10] Both Pph21p and Pph22p are involved in
actin cytoskeleton reorganization, bud morphogenesis and
cell cycle progression from G2to M [11–13] Pph21p and
Pph22p are highly similar (87%) and apparently perform
overlapping functions Deletion of both PP2A catalytic
subunit genes in budding yeast results in very slow growth
Additional deletion of the PP2A-related PPH3 gene is lethal
[11,14] Four polypeptides, encoded by CDC55, TPD3,
RTS1 and TAP42, form complexes with PP2A catalytic
subunits in yeast [12,15–17] Cdc55p, Tpd3p, Rts1p and
Tap42p correspond, respectively, to mammalian PR55/B,
PR65/A, PR61/B¢ and a4 The corresponding genes are not
essential but their mutation results in specific phenotypes
Moreover, two genes (RRD1 and RRD2) encoding
homo-logues of mammalian phosphotyrosine phosphatase
activa-tor (PTPA), a protein isolated from mammalian tissue
based on its ability to stimulate PP2A activity against
phosphotyrosine residues, are present in the budding yeast
genome [18,19]
All catalytic subunits of PP2A from various species are
subject to diverse regulatory control mechanisms
Carbo-xymethylation of Leu309 (Leu377 of S cerevisiae)
influen-ces PP2A activity of PP2A and is a signal for exchanging
variable regulatory B family subunits [20–22] (reviewed in
[23]) Phosphorylation of Tyr307is dependent on insulin,
epidermal growth factor, interleukin-1, tumour necrosis
factor a (and some other pathways) and inactivation of
phosphatase activity (reviewed in [24]) However no data
are available concerning phosphorylation of Tyr375 in
S cerevisiae PP2Ac is also phosphorylated on a threonine
residue, but the role and site(s) of phosphorylation is
unknown [25] PP2A interacts with second messenger
C2-ceramide and phospholipids, which stimulate its activity
(reviewed in [23,24,26]) Moreover, PP2A can potentially be
regulated by changes in the redox state of the catalytic
subunit [27,28]
Both Pph21p and Pph22p differ from the catalytic
subunits of PP2A (PP2Ac) of mammals, plants and
Schizosaccharomyces pombe by the presence of a unique
N-terminal extension of approximately 70 amino acids In
order to assess the impact of this N-terminal extension on
enzymatic properties of PP2A we expressed 8· His-tagged
Pph22p and a mutant of Pph22p lacking the N-terminal
extension (DN-Pph22p) in the yeast Pichia pastoris, purified
the phosphatases to apparent homogeneity and compared
their biochemical properties to that of purified 8·
His-tagged human PP2Ac expressed in Pichia
M A T E R I A L S A N D M E T H O D S
Host strains, media and buffers
The strain GS115 (his4, AOX1, AOX2) of P pastoris was
used for the overexpression of S cerevisiae Pph22p,
DN-Pph22p and N-terminus of Pph22p (first 77 amino
acids) Human PP2Aca and PR65a/Aa were overexpressed
and purified using KM71 (his4, aox1, AOX2) as described
previously [29] All strains were grown, transformed, and
analyzed according to the manufacturer’s (Invitrogen)
instructions Escherichia coli strains DH5a and Top 10F¢
were used for all plasmid constructions and propagations
The following media were used to grow P pastoris: RDB-agar: 1Msorbitol, 2% glucose, 1.34% yeast nitrogen base without amino acids, 4· 10)5% biotin, 2% agar; MD medium: 1.34% yeast nitrogen base without amino acids,
4· 10)5% biotin, 2% glucose; MM medium: 1.34% yeast nitrogen base without amino acids, 4· 10)5% biotin, 0.5% methanol; YPD: 1% yeast extract, 2% peptone, 2% glucose
pH 5.8 adjusted with HCl; MGY medium: 1.34% yeast nitrogen base without amino acids, 4· 10)5% biotin, 1% glycerol The following buffers were applied to purify recombinant Pph22p and DN-Pph22p: SCED buffer: 1 M
sorbitol, 10 mM sodium citrate pH 7.5, 10 mM EDTA,
10 mM dithiothreitol; breaking buffer: 50 mM Tris/HCl
pH 7.5, 1 mM EDTA, 0.1% 2-mercaptoethanol, 10 mM
NaCl, 5% glycerol, 10 mMphenylmethanesulfonyl fluoride and 20 mM benzamidine; buffer A: 20 mM Tris/HCl
pH 7.5, 170 mM NaCl (150 mMfor DN-Pph22p purifica-tion), 0.1 mM EDTA, 0.1% 2-mercaptoethanol, 5% gly-cerol, 1 mM phenylmethanesulfonyl fluoride and 2 mM
benzamidine; buffer B: 20 mM Tris/HCl pH 7.5, 450 mM
NaCl, 30 mM imidazole, 5% glycerol and 0.01% Triton X-100; buffer C: 20 mM Tris/HCl pH 7.5, 20% glycerol (± 0.5 mMdithiothreitol)
Molecular cloning of the Pph22p expression constructs Genomic DNA of S cerevisiae strain W303 was obtained
by the ammonium acetate method [30] and used as template
to amplify the PPH22 open reading frame with Pfu DNA polymerase (Stratagene) using a standard protocol The following primers were used: sense (1), 5¢-CGGGATCC ACCATGCATCATCATCATCATCATCATCATGATA TGGAAATTGATGACCCTATG-3¢ (BamHI site under-lined, 8· His-tag bold) and antisense (2), 5¢-CGGAA TTCTTATAAGAAATAATCCGGTGTCTTC-3¢ (EcoRI site underlined) For cloning of DN-Pph22p (Pph22p without first 77 amino acids) and the N-terminus of Pph22p (only the first 77 amino acids) we used: sense primer: 5¢-CGGGATCCACCATGCATCATCATCATCATCAT CATCATCTTGACCAATGGATTGAGCATTTG-3¢ (BamHI site underlined, 8· His-tag bold) and antisense: 5¢-CGGAATTCTTACTGATTTATATTTGTATTGGT CAG-3¢ (EcoRI site underlined) The PCR products were digested with EcoRI and BamHI and purified by agarose gel electrophoresis using the Geneclean III kit (BIO101) The isolated fragments were first subcloned into pBluescript, and the resulting plasmid amplified in Escherichia coli Subsequently, the Pph22p-encoding fragments were sub-cloned into the pPIC3.5K vector (Invitrogen) All plasmids used were sequenced with vector- and cDNA-specific primers
Homologous recombination in KM71 and GS115 strains
ofPichia pastoris Ten micrograms of plasmid DNA produced in E coli DH5a strain, was either used without restriction enzyme digestion or linearized with either SalI, NotI or BglII in the case of pPIC3.5K-PPH22 and SalI in the case of pPIC3.5K-DN-PPH22 and pPIC3.5K-Nterm (N-terminus
of Pph22p) or not digested, and transformed by the spheroplast method into KM71 and GS115 strains of
P pastoris Transformed yeast cells were plated on
Trang 3RDB-agar plates and transformants were transferred to
plates with either glucose (MD) or methanol (MM) medium
as a carbon source Transformants that displayed the ability
to grow on both carbon sources were selected for further
evaluation The presence of cDNA encoding Pph22p,
DN-Pph22p and N-terminus of DN-Pph22p integrated into the yeast
genome was confirmed by PCR analysis applying sense and
antisense oligonucleotides to amplify the PPH22 gene
(sequences listed above) Transformants obtained using
undigested plasmid DNA in KM71 strain and those
obtained after linearization of plasmid with both SalI and
NotI in GS115 in case Pph22p were used for further
evaluation In case DN-Pph22p and N-terminus of Pph22p
transformants obtained from both kind of DNA
(undigest-ed and digest(undigest-ed with the SalI) were us(undigest-ed for further
experiments In order to select transformants with the
highest copy number of PPH22 genes and mutants genes
inserted into the Pichia genome, yeast colonies were
transferred to YPD-agar plates or YPD-agar plates
con-taining G418 (Calbiochem) added at 2 and 4 mgÆmL)1 The
fastest growing colonies were selected from YPD-agar
plates containing 4 mgÆmL)1G418 and those were selected
for mini-scale expression studies
Mini-scale expression of Pph22p and DN-Pph22p
inP pastoris
Recombinants obtained in the KM71 strain (His+MutS,
slow methanol utilization) or in the GS115 strain (either
His+Mut+or His+MutS, fast and slow methanol
utiliza-tion, respectively) were grown for 24 h in 10 mL of MGY to
reach a D600between 2 and 6 Yeast cells were centrifuged
and resuspended in MM medium using 0.2 volume of
starting culture volume for His+MutSor adjusting D600to 1
for His+Mut+ Methanol-induced cultures were grown at
30C in an Aquatron AI 15 incubator (Infors HT) with
shaking set up to 280 r.p.m Methanol was added to 0.5%
(v/v) every 24 h and the induction was carried out for
9 days To determine the optimal time for protein
expres-sion, aliquots of the cultures were removed at 24-h intervals
and analyzed for the presence of the heterologous protein
by SDS/PAGE with Coomassie staining, immunodetection
with Tetra-His antibodies (Qiagen) and phosphatase
activity measurements Recombinant GS115 (His+Mut+)
obtained after transformation of yeast with the NotI
linearized plasmid displaying the highest level of Pph22p
expression, was used for further experiments In case of
DN-Pph22p and N-terminus for further experiments
trans-formants GS115 (His+Mut+) obtained with plasmids
linearized with the SalI were used
Midi-scale expression of recombinant proteins
inP pastoris
For the midi-scale expression of proteins the selected GS115
(His+Mut+) strain was cultured in a 100-mL baffled flask
in 25 mL of MGY medium at 30C with shaking at
205 r.p.m This yeast preculture reached an D600 5 after
24 h; then 5 mL of the preculture was used to inoculate five
portions of 1 L each of MGY medium and grown in 3 L
flasks to D600between 2 and 6 Cells were harvested, washed
and resuspended in 1 L of MM medium to induce
overexpression of heterologous proteins These cultures
were grown for 24 h at 30C with shaking set at 205 r.p.m After centrifugation at 2000 g for 5 min at room tempera-ture the cell pellet was washed with ice-cold water and stored at)80 C
Purification of recombinant Pph22p and DN-Pph22p Methanol-stimulated GS115-PPH22 (or GS115-DN-PPH22) cells (approximately 50 g) were resuspended in
100 mL of SCED buffer supplemented with 150 mg (84.7UÆmg)1) of yeast lyticase from Arthrobacter luteus (ICN) and incubated at 30C for 90 min to achieve spheroplast formation The spheroplasts were harvested by centrifugation at 750 g for 10 min at 25C and resus-pended in 50 mL of ice-cold breaking buffer Acid-washed glass beads (size 450–500 lm) were added (1 : 1, v/v) and the mixture was vortexed (MS-1 minishaker, IKA) 10 times for 1 min each with 1-min intervals for cooling on ice The lysates were cleared by centrifugation at 30 000 g for 30 min at 4C Cell-free supernatants were combined and fractionated with ammonium sulfate added to obtain 45% saturation The precipitated protein was collected by centrifugation, dissolved in 20 mL of buffer A and dialysed against buffer A The dialysate was loaded at a flow rate of
15 mLÆh)1 onto a DEAE-Sephacel column (2· 10 cm) equilibrated previously with buffer A The column was washed with 20 column volumes of buffer A and phosphatase activity was eluted with a linear gradient from 170 mMto 500 mMNaCl (in the case of DN-Pph22p from 150 to 700 mM NaCl) in buffer A collecting 3-mL fractions The fractions containing phosphatase activity were combined, dialysed against buffer B and loaded onto
a Ni2+-nitrilotriacetate agarose (Qiagen) column (2· 3 cm) The column was washed with buffer B and protein eluted with a linear gradient of imidazole from 30
to 200 mM, collecting 2-mL fractions The fractions were analysed by immunodetection with Tetra-His antibodies and phosphatase activity assays Combined fractions corresponding to the peak of Pph22p activity were dialysed against buffer C with or without dithiothreitol and stored
in small aliquots at )80 C Protein concentration was determined by the Bradford method using bovine serum albumin as standard
Antibodies and immunodetection
To detect recombinant His-tagged proteins monoclonal mice IgG1 Tetra-His antibodies (Qiagen) were applied as primary antibodies followed by goat anti-mouse horse radish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology Inc.) The colour reaction was devel-oped in the presence of reduced form of NAD plus either nitro blue tetrazolium (Sigma) or 4-chloro-1-naphthol (Sig-ma) When goat anti-mouse alkaline phosphatase-coupled secondary antibodies were applied the colour reaction was developed in the presence of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (ICN)
Phosphatase activity assays Protein phosphatase activity was measured with32P-labelled phosphorylase a (10 lM) as substrate as described pre-viously [31] When indicated, protamine (33 lgÆmL)1) and
Trang 4ammonium sulfate (16 mM), were included in the assay
buffer The recombinant PR65/A subunit was preincubated
with PP2Ac, Pph22p or DN-Pph22p in 20 mM Tris/HCl
pH 7.4, 50 mMNaCl, 0.1 mMEDTA and 0.1%
2-mercapto-ethanol for 10 min at 30C before the reaction was
started with32P-labelled phosphorylase a To measure the
effect of pH on PP2A activity the buffer containing 20 mM
sodium acetate/acetic acid, 20 mM imidazole/HCl and
20 mMTris/HCl covering pH from 5.0 to 10.0 was applied
One unit of phosphatase activity corresponds to 1 lmol of
32Pireleased from32P-labelled phosphorylase a per min at
30C
For activity assays with lipids, reactions were carried out
described previously [26] with minor changes Lipids and
phosphatases were incubated on ice for 30 min, prior to the
phosphatase activity assay Reactions were carried out for
15–30 min at 30C The assay was terminated by addition
of 0.1 mL 1 mMKH2PO4in 1MH2SO4and 0.3 mL 2%
ammonium molybdate After 10 min of incubation a
toluene/isobutyl alcohol mixture (1 : 1) was added, vortexed
for 10 s and centrifuged for 10 min Free 32Pi was
determined from the radioactivity recovered in the organic
phase 32P-Labelled phosphorylase a hydrolysis did not
exceed 20% of total phosphorylase a in all samples All
illustrated data represent the mean of at least two
independent experiments
Preparation of liposomes
Phospholipids were solubilized in chloroform and after
evaporation of chloroform were resuspended in 50 mMTris
pH 7.4, 0.1 mMEDTA, 0.1% 2-mercaptoethanol buffer by
sonication under argon Sonication was carried out in an
ice-bath for 10 min with breaks (24 kHz) (BioMetra
Ultrasonicator) Before use, liposomes were kept for 2 h
on ice to allow association of lipids
Determination of the influence of disulfides on yeast
and mammalian recombinant PP2Ac
Pph22p, PP2Ac and DN-Pph22p were incubated with
20 mM dithiothreitol overnight at 4C Mixtures were
dialyzed extensively against buffer containing 50 mMTris,
pH 7.4, 0.1 mMEDTA and 20% glycerol Determination
of the influence of glutathione disulfide (GSSG) and
reduced glutathione (GSH) was carried out by mixing this
redox agent with the purified phosphatase and incubation at
30C for 30 min The phosphatase assay was initialized by
adding 32P-labelled phosphorylase a The reaction buffer
contained 20 mM Tris, pH 7.4, 0.1 mM EDTA and 10%
glycerol
Reactivation of PP2Ac and Pph22p activity
Reactivation was carried out as described previously [27]
with minor changes PP2Ac, Pph22p and DN-Pph22p were
inactivated by incubation with 20 mMGSSG overnight at
4C Mixtures were dialyzed extensively against buffer
contained 50 mM Tris, pH 7.4, 0.1 mM EDTA and 20%
glycerol
Aliquots of the inactivated enzymes were mixed with
dithiothreitol or 2-mercaptoethanol at various
concentra-tions Phosphatase activity in samples was determined after
a 10-min incubation at 30C by addition of 32P-labelled phosphorylase a Reactions were carried out for 30 min under standard conditions
R E S U L T S A N D D I S C U S S I O N
Comparison of PP2A catalytic subunits fromS cerevisiae and other species
S cerevisiaeprotein phosphatases encoded by PPH21 and PPH22 are homologues of mammalian PP2Ac Pph21p consists of 369 amino acids and Pph22p of 377 amino acids Both enzymes are hence larger than PP2Ac from mammals, plants and S pombe which are composed of 306–322 amino acids This difference in size results from the presence of an acidic stretch of approximately 70 amino acids (pI 3.78 and 4.07for Pph21p and Pph22p, respectively) at the N-termini
of Pph21/Pph22p (Fig 1) The role of this N-terminal extension present in budding yeast PP2Ac is currently unknown One may speculate that these regions are responsible for targeting Pph21p and Pph22p to intracellu-lar compartments or to specific substrates, or fulfil a special regulatory function Interestingly, the N-terminal regions of Pph21p and Pph22p are quite divergent showing only 49.4% amino-acid sequence identity (the first N-terminal 42 amino acids of Pph22p display only 33.3% identity to the corresponding region in Pph21p) whereas the overall identity between enzymes equals 87% This might indicate that the N-termini of Pph21p and Pph22p may have distinct functions In order to determine whether the N-terminal extension present in Pph22p influences its biochemical properties we decided to overexpress this phosphatase and its deletion mutant without 77 N-terminal amino acids in
P pastoris, purify these enzymes to apparent homogeneity and compare their enzymatic properties to those of human PP2Ac
Purification ofS cerevisiae Pph22p expressed
inP pastoris
We determined the growth curves of control GS115, recombinant GS115-PPH22, and GS115-DN-PPH22 strains in minimal medium containing methanol (data not shown) A lag period of approximately 100 h was observed
in the case of the GS115-PPH22 and GS-115-DN-PPH22 strains cultured starting from a D600of 0.05, but not in the wild-type control After this period the recombinant strains resumed growth and eventually reached a D600similar to that of the wild-type strain Protein levels in both strains were similar, but phosphatase activity in GS115-DN-PPH22 lysates was lower than in lysates of GS115-PPH22; it is likely that more DN-Pph22p was in the insoluble state and this might also explain why the yield of purification was lower in case of DN-Pph22p Cultures in the stationary phase (high D600) showed less pronounced differences between the strains, but even under these conditions the strain overexpressing Pph22p grew somewhat slower Phosphorylase phosphatase activity was measured in cell-free extracts of all strains and its dependence on growth phase (reflected in D600value) was analysed At stationary phase (high D600), both Pph22p and DN-Pph22p proteins were maximally overexpressed 24 h after methanol induc-tion; amounts of active phosphatase decreased after this
Trang 5time, as confirmed also by Western blotting analysis (data
not shown)
The long lag period in the growth of the GS115-PPH22
strain observed after transferring the cells to
methanol-containing medium is similar to that described for the strain
overexpressing human PP2Ac [29] and might reflect effects
of higher phosphatase activity on yeast growth or on the cell
cycle
Figure 2 illustrates the purification of Pph22p and
DN-Pph22p from P pastoris cells using ammonium sulfate
fractionation, DEAE-Sephacel and Ni2+-nitrilotriacetic
acid agarose, as described in the Materials and methods section The final preparation, stained with Coomassie Brilliant Blue, appeared to be homogeneous Purity was confirmed by gel filtration (data not shown) Pph22p and DN-Pph22p proteins were purified with a yield of active protein of 80 and 60 lgÆL)1of P pastoris culture, respect-ively, in intracellular overexpression
The inclusion of Triton X-100 (0.01%) in the buffers used for chromatography on Ni2+-nitrilotriacetic acid agarose greatly enhanced recovery of active phosphatase from this column Similarly to mammalian PP2Ac, Pph22p and DN-Pph22p migrated as a doublet of two proteins Pph22p migrated on SDS/PAGE with a molecular mass of 52–53 kDa, different from its calculated molecular mass
of 44 kDa DN-Pph22p migrated on SDS/PAGE at its theoretical molecular mass of 37kDa The specific activity
of the final Pph22p and DN-Pph22p appropriate prepara-tions was 1.3 and 1.8 lmolÆmin)1Æmg protein)1using phos-phorylase a as substrate The specific activity of DN-Pph22p
is similar to the 1.7 lmolÆmin)1Æmg protein)1obtained for recombinant human PP2Ac [29], but the value for Pph22p is lower indicating an inhibitory effect of the N-terminus Characterization of purified Pph22p and DN-Pph22p PP2Ac was initially described as a metal-ion-independent protein phosphatase [32] In agreement with this, none of metal ions tested increased significantly the activity of Pph22p, DN-Pph22p or PP2Ac (Table 1) In contrast, several metal ions applied at a concentration of 1 mM
(Co2+, Ni2+, Fe2+, Fe3+ and Zn2+) inhibited Pph22p and PP2Ac activities with phosphorylase a as a substrate In order to exclude the latter effects being substrate dependent
we confirmed the data from Table 1 using kemptide as substrate (not shown) It remains to be determined whether the inhibitory effect of these high concentrations of metal ions reflect an interaction with SH groups exposed on the enzyme surface or formation of complexes with amino-acid
Pph22_S.cerevisiae
ppa1_S.pombe
PP2Ac/beta_rabbit
PP2Ac/alfa_H.sapiens
ppa2_pombe
PP2Ac_2_A.thaliana
Pph3_S.cerevisiae
Pph22_S.cerevisiae
ppa1_S.pombe
PP2Ac/beta_rabbit
PP2Ac/alfa_H.sapiens
ppa2_pombe
PP2Ac_2_A.thaliana
Pph3_S.cerevisiae
Pph22_S.cerevisiae
ppa1_S.pombe
PP2Ac/beta_rabbit
PP2Ac/alfa_H.sapiens
ppa2_pombe
PP2Ac_2_A.thaliana
Pph3_S.cerevisiae
Fig 1 Alignment of PP2A from S cerevisiae and other organisms Sequence alignment of PP2A catalytic subunits from S cerevisae (Pph21p, Pph22p, Pph3p), S pombe (ppa1), rabbit, Homo sapiens and Arabidopsis thaliana Conserved residues are coloured The N-terminal extension is only found in the S cerevisiae PP2A isoforms The region deleted in DN-Pph22p is framed.
Fig 2 Purification of Pph22p and DN-Pph22p from overexpressing
P pastoris cells Aliquots of Pph22p and DN-Pph22p overexpressed in
P pastoris and purified by using three steps of purification (protein
precipitation with ammonium sulfate, ion-exchange chromatography
on DEAE-Sephacel and affinity chromatography on Ni 2+
-nitrilotri-acetatic acid agarose were taken and analysed by polyacrylamide
(10%) gel electrophoresis and staining with Coomassie Brilliant
Blue St, molecular mass standard (kDa); lane 1, Pph22p; lane 2,
DN-Pph22p.
Trang 6residues involved in catalysis Some metal ions, e.g Co2+
and Ni2+, might interact with the N-terminal His-tag, but
this is unlikely to explain the effect on phosphatase activity,
because even (nonrecombinant) PP2A purified from rabbit
skeletal muscle is inhibited by 20–30% by these ions at a
concentration of 0.75 mM(data not shown)
Mammalian PP2Ac is inhibited by several naturally
occurring compounds in a way that allows this enzyme to be
distinguished from PP1 [33] In contrast, more recently
discovered protein phosphatases such as PP4 and PP6,
which are present in mammalian cells in much smaller
quantities, are inhibited similarly to PP2A Figure 3
presents inhibition of purified Pph22p by okadaic acid,
nodularin, cantharidin and endothall The IC50 values
calculated at 3 nMconcentration of Pph22p were 0.2, 0.5,
130 and 210 nM, respectively, and were very similar to those obtained for DN-Pph22p (data not shown) For PP2Ac applied at a similar concentration (4 nM) IC50values were very similar, 0.5, 0.6, 140 and 300 nM for okadaic acid, nodularin, cantharidin and endothall, respectively Thus, both Pph22p and PP2Ac are similarly affected by a panel of inhibitors reflecting the high degree of conservation of the catalytic core between these two enzymes and ruling out the involvement of the N-terminal extension in binding to these inhibitors As can be expected from the presence of a stretch
of acidic amino acids, the pH optimum of Pph22p (pH 7.5)
is slightly different from the pH optimum of PP2Ac (pH 7) Unique properties of Pph22p
In vivo PP2Ac associates with the scaffolding PR65/A subunit to form dimers, which can further associate into trimers by association with a variable B subunit Homo-logues of the PR65/A subunit and of various B subunits are present in yeast (reviewed in [23]) A classical biochemical approach to distinguish between PP2Ac and the dimer makes use of protamine It has been described that this compound inhibits the activity of the isolated catalytic subunit, but stimulates activity of the dimer [34] Unexpect-edly, however, protamine (both in the absence and in the presence of ammonium sulfate) stimulated the phosphatase activity of purified Pph22p (Fig 4A) At 66 lgÆmL)1, protamine (and 16 mM ammonium sulfate) an 11-fold activation of Pph22p was observed Protamine, in the presence of the same concentration of ammonium sulfate, had little effect on human PP2Ac and on DN-Pph22p (Fig 4A) In contrast, 33 lgÆmL)1, protamine together with
16 mM ammonium sulfate stimulated the activity of the PP2Ac-PR65/A dimer about 12-fold and that of the DN-Pph22p-PR65/A dimer around fourfold (Fig 4B) Hence, with respect to protamine stimulation of phospha-tase activity, the yeast catalytic subunit Pph22p behaved similarly to the PP2Ac-PR65/A dimer, but mutant DN-Pph22p behaved like PP2Ac As can be seen in Fig 4B, addition of PR65/A subunit to Pph22p increased the protamine activation a further 1.8-fold
Fig 3 Inhibition of Pph22p activity by okadaic acid, nodularin, can-tharidin and endothall Purified Pph22p applied at 3 n M was incubated with the indicated concentration of inhibitor at 30 C for 10 min before the reaction was initiated with 32 P-labelled phosphorylase a as substrate.
Table 1 The effects of metal ions on the activity of type 2A protein
phosphatases Activities of purified human PP2Ac (PP2Ac, specific
activity 1.7 lmolÆmin)1Æmg protein)1) and purified S cerevisiae
Pph22p (Pph22p, specific activity 1.3 lmolÆmin)1Æmg protein)1) were
measured against 10 l M32P-labelled phosphorylase a 100% activity
refers to activity measured in the absence of exogenous metal ions
added.
Metal
Relative activity (%) ions (m M ) PP2Aca Pph22p
Mn2+
Co2+
Fe 2+
Fe 3+
Ni2+
Mg2+
Ca 2+
Zn 2+
Cu2+
Trang 7The observation that in the absence of protamine PP2Ac,
Pph22p and DN-Pph22p are inhibited by increasing the
PR65/A subunit concentration (Fig 4C), indicates the
effi-cient formation of PP2Ac-PR65/A, DN-Pph22p-PR65/A
and Pph22p-PR65/A dimers We have shown previously
[29] that protamine exerts its effect on PP2Ac activity via an
interaction with the PR65/A subunit, which interacts much
more strongly with protamine-agarose than PP2Ac itself
Here we have confirmed these data, but we also showed
that, in agreement with the protamine stimulation of
Pph22p, this enzyme interacts strongly with
protamine-agarose (Fig 4D) The strong interaction between Pph22p
catalytic subunit and protamine-agarose is reflected by the
resistance to elution with 1MNaCl Denaturing conditions
(boiling of the gel in SDS sample buffer) are required to
dissociate Pph22p from the protamine-agarose column
This interaction is mediated by the acidic N-terminal
extension, since the DN-Pph22p protein cannot interact strongly with protamine-agarose and like PP2Ac is eluted with 0.5MNaCl from the gel (Fig 4D)
In order to determine the effect of other polycations on phosphorylase phosphatase activity of yeast and mamma-lian PP2Ac poly-L-lysine was added to the purified enzymes As presented in Fig 5 a peak of poly-L -lysine-stimulated phosphorylase phosphatase activity was observed at 20 lg poly-L-lysine per mL for both enzymes, but the extend of activation of Pph22p was much more pronounced (4.5-fold activation of Pph22p activity vs 2.5-fold activation of PP2Ac) PR65/A subunit (3 nM) increased activation of PP2Ac by poly-L-lysine by approximately 70% and it also increased stimulation of Pph22p by poly-L-lysine another 50% From Fig 5 it is clear that the activation (4.5-fold) of Pph22p by
20 lgÆmL)1 poly- -lysine is very similar to activation
Fig 4 Pph22p responds to protamine in a similar way as the mammalian PP2Ac-PR65/A dimer (A) Phosphatase activity of 0.5 n M Pph22p (squares), 0.5 n M DN-Pph22p (triangles) and 0.5 n M PP2Ac (circles) was assayed using 32 P-labelled phosphorylase a as a substrate in the presence
of the indicated concentrations of protamine and in the absence (open symbols) or presence (closed symbols) of 16 m M ammonium sulfate (SA) (B) Phosphatase activity of 0.5 n M Pph22p (squares), 0.5 n M DN-Pph22p (triangles) and 0.5 n M PP2Ac (circles) was assayed using 32 P-labelled phosphorylase a as a substrate in the presence of the indicated concentrations of purified PR65a/A subunit and in the absence (open symbols) or presence (closed symbols) of 33 lgÆmL)1protamine and 16 m M ammonium sulfate (C) Phosphatase activity of the indicated concentrations of Pph22p (squares), DN-Pph22p (triangles) and PP2Ac (circles) was assayed using32P-labelled phosphorylase a as a substrate in the absence (open symbols) or presence (closed symbols) of 3 n M purified PR65a/A subunit (D) Binding of PP2Ac, DN-Pph22p and Pph22p to protamine-agarose Immunodetection of Pph22p/DN-Pph22p/PP2Ac was carried out on a Western blot after separation of protein fractions on 10% SDS-poly-acrylamide gel Lanes 1 and 2, material loaded to the column; lane 3, flow-through; lanes 4, 5 and 6, material eluted with 50 m M , 500 m M and 1 M
NaCl, respectively; lane 7, material eluted with 1 · SDS/PAGE buffer.
Trang 8(4.5-fold) of the human PP2Ac-PR65/A dimer by
20 lgÆmL)1poly-L-lysine and much stronger than that of the free human catalytic subunit PP2Ac Interestingly the deletion mutant DN-Pph22p behaved more like PP2Ac DN-Pph22p is only around 70% activated by polyL-lysine and even the DN-Pph22p/PR65a dimer is less stimulated than Pph22p alone (Fig 5) Again these data point to a domain present in the yeast Pph22p N-terminus respon-sible for mimicking the polycation-stimulation effects exerted by the PR65/A subunit in mammalian PP2A Taken together we can conclude that activation of Pph22p by polycations is mediated by its N-terminal region The role of this region in vivo is unknown, but it might stabilize the structure of Pph22p, influence substrate specif-icity or exert a regulatory function
Effects of phospholipids on activity of Pph22p Some studies showed that phospholipids can stimulate or inhibit of PP2A and PP1 phosphatases [26] (reviewed in [23,24]) In this study we also checked the influence of several phospholipids (assembled in liposomes) on activity
of Pph22p and DN-Pph22p Lipids were tested at a
Fig 5 Effect of polylysine on the phosphatase activity of Pph22p,
DN-Pph22p, PP2Ac and the corresponding dimers Phosphatase activity
of 0.5 n M Pph22p (squares), 0.5 n M DN-Pph22p (triangles) and 0.5 n M
PP2Ac (circles) was assayed using 32 P-labelled phosphorylase a as a
substrate in the presence of the indicated concentrations of
poly-L -lysine and in the absence (open symbols) or presence (closed
symbols) of 3 n M purified PR65a/A subunit.
Fig 6 Influence phospholipids on Pph22p and DN-Pph22p activity Phosphatase assays were carried out as described in Materials and Methods (A) Influence of egg yolk phosphatidic acid (PA) and synthetic dioleoylphosphatidic acid (DOPA) on Pph22p and DN-Pph22p phosphatase activity (B) Influence of phosphatidylserine (PS) and (C) phosphatidylethanolamine (PE) on Pph22p and DN-Pph22p phosphatase activity (D) Effects
of dioleoylphosphatidylcholine (DOL) and phosphatidylcholine (L) on Pph22p and DN-Pph22p phosphatase activity Phosphatases at a con-centration of 2.3 n M were used to measure the effects of phospholipids on its activity Lipids were solubilized in chloroform and after evaporation
of chloroform were resuspended in 50 m M Tris, pH 7 4, 0.1 m M EDTA, 0.1 m M 2-mercaptoethanol buffer and sonicated Liposomes were added
to phosphatase and reactions were incubated on ice for 30 min and phosphatase activity in samples were determined (see Materials and methods).
Trang 9calculated concentration of 4–400 lM and after 10 min
incubation on ice, a time where effects were maximal
Phosphatidic acid from egg yolk (PA) stimulated Pph22p
activity, but it inhibited DN-Pph22p Inhibition reached
70% for DN-Pph22p at 320 lM concentration with IC50
around 80 lMof lipid (Fig 6A) The same lipid stimulated
Pph22p around twofold at 8 lMor higher concentrations
(Fig 6A)
Dioleoylphosphatidic acid (C18:1, [cis]-9) inhibited both
Pph22p and DN-Pph22p in a similar manner (IC50)40 lM
concentration of phospholipid for DN-Pph22p and 30 lM
of phospholipid for Pph22p) (Fig 6A) Phosphatidylserine
and phosphatidylethanolamine both stimulated Pph22p
phosphatase around 2.5-fold to threefold, but only slightly
affected DN-Pph22p (Figs 6B,C)
Dioleoylphosphatidyl-choline (C18:1, [cis]-9) and phosphatidylDioleoylphosphatidyl-choline were not
selective, and stimulated both Pph22p and DN-Pph22p in a
similar way (Fig 6D) These results indicate a specific
influence of the N-terminal extension of Pph22p on its
activity and on interactions with specific phospholipids and possibly membranes
Redox state of Pph22p Some authors reported an influence of reducing and oxidizing agents on PP2A and PP1 activity [27,28] Oxid-izing reagents like o-iodosobenzoate, dipiridyl disulfates or glutathione disulfide can inactivate of PP2A and PP1 isolated from rabbit skeletal muscle [27] Inactivation is different between PP2A and PP1 and depends on the oxidizing agent used [27] We tested the influence of the oxidizing agent GSSG on activity of Pph22p, DN-Pph22p and recombinant human PP2Ac catalytic subunit as control Figure 7A illustrates the influence of various concentrations of GSSG on PP2Ac, Pph22p and DN-Pph22p phosphatases All these enzymes are inactivated
in a similar way by GSSG, but interestingly only Pph22p activity is also inhibited by GSH GSH at 6 mM concen-tration has no effect on recombinant human PP2Ac and in low concentrations even slightly stimulates PP2Ac activity (Fig 7A) (data not shown)
We then checked the influence of reducing agents on the activity of phosphatases Dithiothreitol and 2-mercapto-ethanol can reactivate PP1 and PP2A phosphatases from rabbit skeletal muscle inactivated by GSSG [27] We tested whether both Pph22p and DN-Pph22p were reactivated by dithiothreitol and 2-mercaptoethanol As a control recom-binant PP2Ac was used Pph22p and DN-Pph22p were not reactivated, even at 50 mM concentration dithiothreitol or 2-mercaptoethanol, while PP2Ac was reactivated by both reducing agents to around 15% of its original activity (Fig 7B) This is in agreement with data reported previ-ously [27], where authors have shown slight reactivation of PP2A from rabbit skeletal muscle and very high levels of reactivation of rabbit PP1 Pph22p cannot be reactivated under our conditions by reducing agents, moreover 2-mercaptoethanol and in lesser extend dithiothreitol, even decrease the activity of Pph22p, in contrast to PP2Ac, which can be activated about twofold (data not shown) Serine-threonine phosphatases, e.g rabbit and human PP1c and PP2Ac, probably have disulfide bonds connecting their cysteine residues PP2Ac and Pph22p have 10 and 9 cysteine residues, respectively The different properties of both enzymes can probably be explained by two residues, Cys50 and Cys251, which are unique for PP2Ac, and Cys143, which is unique for Pph22p Because cysteine residues can influence secondary structure, the structure of both phosphatases might be slightly different More likely,
in contrast to Pph22p, PP2Ac might be regulated by reversible oxidation of cysteine residues; it is noteworthy that PP2A from rabbit tissue was isolated in complex with nucleoredoxin, an enzyme with high homology to thiore-doxins (S Zolnierowicz, N Andjelkovic, C Van Hoof,
J Goris & B A Hemmings, unpublished data) (reviewed in [24]) However, homologs of nucleoredoxin are not found in the yeast genome
C O N C L U S I O N
Although Pph22p has been studied thoroughly using genetic methods, no data are available regarding its enzymatic properties To fill this gap we overexpressed
Fig 7 Determination of influence of redox agents on phosphatase
activity (A) Effects of GSSG and GSH on activity of yeast and human
recombinant phosphatases Phosphatases (PP2Ac, Pph22p and
DN-Pph22p) at a concentration of 2.5 n M were added to the indicated
concentrations of GSSG and GSH Reactions were incubated at 30 C
for 10 min and phosphatase activity was determined (B) Reactivation
test of PP2Ac, DN-Pph22p and Pph22p phosphatases inactivated by
incubation with 20 m M GSSG After inactivation phosphatases were
dialysed extensively and assayed for activity after 10 min incubation
with dithiothreitol or 2-mercaptoethanol in the indicated
concentra-tions A 2.5-n M concentration of PP2Ac, Pph22p and DN-Pph22p was
used On the graph, the solid squares precisely overlap the open
squares.
Trang 10Pph22p in P pastoris, purified this phosphatase to
apparent homogeneity, determined its enzymatic
proper-ties and compared them to those of mammalian PP2Ac
This analysis shows that although both enzymes share a
number of characteristics (specific activity, sensitivity to
inhibitors, inhibition by high concentrations of metal
ions), they show a remarkably different response to
protamine, polylysine and reducing agents Using purified
Pph22p lacking the N-terminus, we can attribute most of
these differences to the unique N-terminal extension
present in Pph22p In contrast to PP2Ac and DN-Pph22p,
Pph22p strongly interacts with protamine resulting in
stimulation of enzymatic activity The stimulation of
catalytic activity of Pph22p by protamine and polylysine
reflects the stimulation of the mammalian PP2Ac-PR65/A
dimeric form of the phosphatase The N-terminus of
Pph22p also influences interactions of Pph22p with
specific phospholipids or membranes Our data therefore
indicate a possible regulatory function for the acidic
N-terminus of Pph22p and demonstrate that yeast Pph22p
has unique enzymatic characteristics compared to other
PP2A phosphatases studied so far
A C K N O W L E D G E M E N T S
This work was supported by grants from NATO (LST.CLG 974983)
and the Ministry of the Flemish Community (BIL99/26), by a
postdoctoral fellowship from the Research Fund of the Katholieke
Universiteit Leuven to S W., and by a grant BW# B000-5-0217-1 to
S Z The authors thank Prof Michal Wozniak for helping with the
preparation of liposomes.
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