Download Expression of the Catalytic and Regulatory Subunits of Protein

[CANCERRESEARCH54, 4879-4884, September15. 19941
Expression of the Catalytic and Regulatory Subunits of Protein Phosphatase
Type 2A May Be Differentially Modulated during Retinoic Acid-induced
Granulocytic Differentiation of HL-60 Cells1
Masakatsu Nishikawa,2 Serdar B. Omay, Hideki Toyoda, Isao Tawara, Hiroshi Shima, Minako Nagao,
Brian A. Hemmings,
Marc C. Mumby, and KatsUmi Deguchi
The 2nd Department of internal Medicine. Mie University School of Medicine, E4obashi, Tsu, Mie 514 Japan (M. N.. S. B. 0., H. T., i. T., K. DI; Carcinogenesis Division,
National Cancer Center Research Institute, Chuo-ku, Tokyo, 104 Japan (H. S., M. N.]; Friedrich Miescher-institut, CH-41X12, Base!, Switzerland (B. A. H.]; and Department of
Pharmaco!ogy, University of Texas. Southwestern Medica! Center, Da!!as, Texas 75235-9041 (M. C. M.]
ABSTRACT
To elucidate the regulation of protein phosphatases types 1 (PP1) and
2A (PP2A) during all-trans retinoic acid (ATRA)-induced granulocytic
differentiation of HL-60 cells, the phosphatase activity, proteins, and gene
expressions
ofPPl
and PP2A were examined.
Treatment
with 1 pM ATRA
caused an 85% decrease in the PP2A activity In extracts from HL-60 cells,
while the PP1 activity
was constant.
This reduction
in PP2A activity
appeared to parallel phenotypic and functional changes of HL-60 cells
Induced by A1'RA. Western blot analysis showed that the level of PP2A
catalytic subunit (PP2A-C) decreased
during the course of ATRA-induced
differentiation, whereas expressions of A and B (Mr 55,000) regulatory
subunits
subunit
of PP2A
were
relatively
isozymes (PP1a,
Unaltered.
Expressions
of PP1 catalytic
PP1'y, and PP18) were not significantly
affected
by ATRA treatment. Northern blot analysisrevealedthat mRNA levelsof
PP2A-C@and Aa regulatory subunits were decreased following treatment
with ATRA, while levels of PP2A-Ca and B (Mr 55,000) a regulatory
subunit transcripts were relatively constant. Selective down regulation of
PP2A-Cf3 preceded
the granulocytic
maturation
induced
by ATRA. Ex
pressions of PP2A-C isoforms and A and B regulatory subunits may be
differentially
modulated
during
ATRA-induced
granulocytic
differentia
don ofHL-60cells.
INTRODUCTION
ATRA3 induces granulocytic differentiation in cultured leukemic
HL-60 cells (1, 2) and is clinically effective as the differentiation
therapy in inducing high remission rates in patients with acute pro
myelocytic leukemia (3, 4). The biological effects of ATRA appear to
be mediated through a number of closely related nuclear retinoic acid
receptors that possess discrete DNA-binding and retinoic acid-binding
domains (5). Although the exact mechanism of the ATRA-induced
granulocytic differentiation remains to be elucidated, protein phos
phorylation/dephosphorylation
is also thought to be a regulatory de
vice eminently suited for the control of differentiation processes (6).
The levels of both protein kinase C activity and the expression of its
isoforms have been shown to increase during HL-60 cell differentia
tion induced by dimethyl sulfoxide and ATRA (7). However, little is
known concerning protein phosphatases responsible for reversing the
actions of protein kinase-catalyzed phosphorylation reactions in
HL-60 cell differentiation.
The protein serine/threonine phosphatase catalytic subunits of
mammalian cells comprise four forms which have been designated
type 1 (PP1), type 2A (PP2A), type 2B (calcineurin), and type 2C (8,
9). They differ in metal ion requirements and sensitivities to two
heat-stable protein inhibitors, inhibitor-i and inhibitor-2 (8, 9). Sev
eral isoforms of PP1 catalytic subunit, PP1a, PP1'y, and PP1@ have
been cloned from rat cDNAs (10). PP1s consist of multimeric struc
tures composed of a catalytic subunit complexed to several regulatory
subunits (8, 9). Formation of heteromeric complexes is thought to play
an important role in regulating the activity of PP1 catalytic subunit, as
yet not defined in HL-60 cells. The PP2A catalytic subunit (PP2A-C,
Mr 36,000)
Received 3/16/94; accepted 7/11/94.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
was
supported
in
part
by
grants
for
research
from
The
Ministry
of
Education, Science, and Culture, Japan.
2 To
3 The
whom
requests
abbreviations
for
used
reprints
are: ATRA,
MATERIALS
AND METHODS
superoxide,
as monitored
by the reduction
of NBT (Sigma)
and surface
antigen
analysis. The ability of NBT reduction was evaluated as described elsewhere
Surface
antigens
were assessed
by cytofluorometry
in a fluorescence
activated cell sorter scan (Becton Dickinson, Mountain View, CA), using
be addressed.
alh-trans-retinoic
forming a heterotrimer
Cells, Culture Conditions, and Evaluation of Differentiation. Proce
dures for the maintenance of HL-60 cells and determination of variable cell
counts have been described previously (19). Cells in the logarithmic growth
phase were used for experiments. The extent of differentiation by ATRA
(Sigma Chemical Co., St Louis, Mo) was assayed by the ability to produce
(20).
should
is mainly present as a holoenzyme
with A (Mr 65,000) regulatory and different B regulatory subunits; the
basic form is the PP2A-C/A complex, and the B subunit is associated
with the dimer through A subunit (9, 11, 12). The A and B regulatory
subunits were shown to modulate the phosphatase activity of the
PP2A-C in vitro. cDNA clones for the two respective isoforms of
PP2A-C and A subunit, PP2A-Ca and PP2A-C(3 and Aa and A(3,
respectively, have been isolated from several animal species (13—15).
Based on biochemical evidence, the B subunit of PP2A holoenzyme
is comprised of several distinct families of proteins of B (M1 55,000),
B' (Mr 54,000), B―
(Mr 74,000), and Mr 72,000 (9). Three cDNAs for
isoforms of the B (Mr 55,000) subunit (a, @3,
‘y)
have been cloned from
human, rabbit, rat, and yeast libraries (9, 16). A, B, and PP2A-C
subunits show a high degree of sequence conservation among species
(9). Although the effect of the regulatory subunits of PP2A on the
substrate specificity has clearly been demonstrated in vitro (8,9), the
roles of individual regulatory proteins in the regulation of metabolism,
growth, differentiation, and development remain largely unknown.
We reported that OKA and calyculin-A, both potent and specific
inhibitors of PP1 and PP2A, augment the granulocytic differentiation
of HL-60 cells induced by ATRA but not the monocytic differentia
tion induced by phorbol diester (17). In addition, PP2A-C is down
regulated during ATRA-induced
granulocytic differentiation of
HL-60 cells, whereas the PP1 catalytic subunit is unchanged (18). To
determine the effects of ATRA treatment on regulation of cellular
PP2A holoenzyme, we analyzed PP2A activity and expressions of the
A and B regulatory subunits in addition to that of PP2A-C, following
ATRA treatment.
acid;
PP1,
protein
phosphatase
type 1; PP2A, protein phosphatase type 2A; PP2A-C, the catalytic subunit of PP2A; MLC,
myosin light chain; OK.A,okadaic acid; NBT, nitrobluetetrazolium;cDNA, complementary
DNA, SDS, sodium dodecyl sulfate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
monochonal
antibodies,
including
CD! lb (0KM!;
Ortho
Diagnostic
System
Inc., Raritan, NJ), CDllc (LeuM5; Becton Dickinson), and CD54 (anti-ICAM;
Cosmo Bio Co., Tokyo, Japan).
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PROTEIN PHOSPHATASE 2A IN GRANULOCYI1C DIFFERENTIATION
Preparation of CytosohicFractions of HL-60 Cells. HL-60 cells (2 X l0@
cells each) were washed
saline
three times with ice-cold
and then disrupted
Tris-HC1-buffered
in 1 ml of homogenization
buffer
consisting
ATRA-induced Granulocytic Differentiation in HL-60 Cells.
of 250
mM sucrose and buffer A (20 mM Tris-HC1, pH 7.5, 2 mM dithiothreitol, 2 mM
EDTA, 2 mMethylene glycol bis-(13-aminoethyhether)-N-N'-tetraacetic acid,
and protease
inhibitors,
including
75 mg/liter
phenylmethyh-sulfonylfluoride,
10 mg/liter heupeptin, 10 mg/liter N-tosyl hysinechloromethyl ketone, and 5
mg/liter N-tosyhphenylalanine chhoromethylketone) by a glass-glass Potter
Ehvehjem homogenizer
1000
at 4°C(19). The sample was then centrifuged
X g for 10 mm. The resulting
fraction.
The supernatant
pellet
was centrifuged
was used
at 100,000
as the crude
at
nuclear
X g for 1 h to separate
the
cytosol and membrane fractions. The membrane and nuclear fractions were
rinsed with homogenization
nization
buffer
buffer and subsequently
containing
1% Nonidet
resuspended
P-40. Following
in homoge
a 1-h incubation
on
ice, these fractions were sonicated and further centrifuged at 100,000 X g for
1 h to obtain solubihized fractions. In preliminary experiments, we have found
that Nonidet P-40 is most suitable for solubilization
does not inhibit
the activity
of phosphatase
of phosphatase
at concentrations
because
it
as high as 1%.
Assay of Protein Phosphatases. Activities of PP1 and PP2A were assayed
in the absence
of divahent
cations
using 32P-phosphorylated
isolated
MLC of
chicken gizzard as a substrate, because it is known to be a good substrate for
both mammalian PP1 and PP2A (21). Phosphatase activity was determined by
the liberation of inorganic 32P from the substrate (32P-labehed Mr 20,000
at 30°C according
phatase
activity
preparation
to the method of Pato and Kerc (22). Inhibition
by OKA
was determined
by adding
OKA
MLC)
of phos
to the enzyme
10 mm prior to adding the substrate. The extent of dephosphory
lation was restricted
to <10%.
Under
these conditions,
the rates of phospho
ryhationwere linear with respect to time and enzyme dilution.
Immunobhot Analysis. Antisera against PP1 catalytic subunit, PP2A-C,
and A, B (Mr 55,000),
by immunizing
@
B' (Mr 54,000),
and Mr 72,000
rabbits with synthesized
(23, 24), A (25) regulatory,
fragments
B (Mr 55,000),
subunits were obtained
of PPla,
PPl@y, and PP1&
and B (M, 55,000)
(3 (12), B' (M,
54,000) (26), or Mr 72,000 (27) regulatory subunits. HL-60 cells were incu
bated with
1 ,iM ATRA,
trichloroacetic
acid-lO
and the reaction
mM dithiothreitoh-2
ethyl ether)-N,N'-tetraacetic
twice
with
acetone-lO
was terminated
with cold
mM ethyheneglycol
10%
bis-(f3-amino
acid solution. The resulting cell pellet was washed
mM dithiothreitol
and then dried.
The dried
acetone
powder was dissolved with Laemmhi sample buffer (28). The sample was
subjected
to 0.1% SDS-12.5%
electrophoretical
transfer
polyacrylamide
to a nitrocehlulose
gel electrophoresis
membrane
RESULTS
(pH 7.4)
followed
by
Treatment of HL-60 cells with 1 ,.LMATRA for 4 days led to
acquisition of a mature phenotype resembling that of a granulocyte
(17). The mature phenotype in HL-60 cells was confirmed by flow
cytometric assessments with various monoclonal antibodies and an
increased NBT reduction. HL-60 cell proliferation was inhibited after
treatment with 1 ,.LMATRA. As shown in Fig. 1, treatment of HL-60
cells with ATRA resulted in a time-dependent increase in NBT
reduction and increase in reactivities of the cells with CD1 lb. CDS4,
and CD1 ic, although the kinetics of each differentiation marker
acquisition were not necessarily identical. The reactivity of HL-60
cells with CD54 reached a plateau faster than that of CD1 lb or
CD11c.
Alterations of Activity and Expression of PP1 and PP2A during
ATRA-induced Differentiation. The activity of protein serine/thre
onine protein-phosphatase in HL-60 cells was assayed in the absence
of divalent cations with 32P-phosphorylated MLC as substrate (21).
The activities of cytosol, membrane, and nuclear fractions in HL-60
cells were 17.6 ±3.1, 4.90 ±0.65, and 5.84 ±0.58 nmol/mg/min
(mean ±SE; n = 3), respectively. In untreated HL-60 cells, approx
imately 60-70% of the total activity of MLC phosphatase was present
in the cytosol fraction, with the remaining activity located in the
membrane and nuclear fractions. Incubation of HL-60 cells with 1 @M
ATRA resulted in a 66% decrease in MLC phosphatase activity in the
cytosol fraction, as shown in Fig. 2, but the phosphatase activity of the
membrane or nuclear fraction was unaltered (data not shown), thereby
suggesting that the translocation of phosphatase from the cytosol
fraction to membrane or nuclear fractions did not occur. PP2A is
inhibited completely by S n@tOKA, while PP1 is hardly affected at
this concentration and complete inhibition requires 500 n@i(21). The
varying concentrations producing 50% inhibition for different phos
phatases mean that OKA can be used to distinguish between these
activities in cell-free systems. Therefore, we examined effects of
OKA (5 and 500 ni@i)on MLC phosphatase activities of HL-60 cells
during ATRA-induced differentiation to estimate the relative amounts
(29). The membrane
was reacted with the avidin-biotin peroxide complex method (Vectastain;
Vector
Laboratories,
Burhingame,
CA).
Prestained
SDS-polyacrylamide
gel
ehectrophoresis standards (Bio-Rad, Richmond, CA) were used as molecular
100
weight standards. Quantitative estimation of the level of PP1 and PP2A was
carried out densitometrically
with a Bio-Rad videodensitometer
by scanning
the immunoreactive
band after immediately photographing the band. The area
of an individual
peak
was
measured
tracings and expressed as absorbence
above
background
80
in densitometric
X mm (29).
RNA Isolation and Northern Blot Analysis. Total cellular RNA was
extracted by the guanidinium isothiocyanatecesium chloride method (30).
Total RNA (20 @g)
of HL-60 cells was electrophoresed through 0.8% agarose
with 18% formaldehyde
and transferred
to Nytran
membranes
(Schleicher
>1
>
&
Schuell, Dassel, Germany). After baking at 95°Cfor 2 h under vacuum, the
blots were hybridized at 42°Cin 50% formamide, 5X Denhardt's solution, SX
0
0.
SSPE (1 x SSPE is 0.15 MNaCh-lOmMNaH2PO4-0.lmr@i
EDTA), 0.5% SDS,
200
@g/mhof denatured
salmon
@40
20
sperm DNA, and the 32P-habeled cDNA probe.
The probes used were as follows: PsrI-EcoRI fragment of rat PP2A-Ca cDNA
and synthetic ohigomer (3'-noncoding
region) of rat PP2A-C@3 cDNA (31),
0.
EcoRl fragments of rat Aa and A@3regulatory subunit cDNAs (32), Hind!!
fragment of rat B (M1 55,000) a and AccI fragment of rat B (Mr 55,000) 13
regulatory
fragment
subunit cDNAs
(32), EcoRI
of rat PP1 y, and EcoRI fragment
fragment
of rat PPI1a,
0
EcoRV-PstI
of rat PP1@ (10). These probes were
labeled with [a-32P]dCFP by a multiprime labeling system kit (Amersham,
Buckinghamshire, England). Hybridized blots were finally washed with
0.1 x standard saline citrate (1X is 0.15 MNaCl-15 mMtrisodium citrate) and
0.5% SDS at room temperatureand autoradiographed.Quantificationof hy
bridization
was determined
by scanning
densitometry
(18).
24
48
72
96
Time ( hr)
Fig. 1. Induction of markers of cell differentiation in HL-60 cells after treatment with
ATRA. HL-60 cells were incubated with 1 @M
ATRA. Percentages of NBT-positive cells
(•)
and
surface
markers
using
CD11b
(0),CD11c
(L.@),
and
CD54(0)were
analyzed
by
flow cytometry, as described in “Materials
and Methods.―
Points, means of three separate
experiments expressed as percentages of positive cells.
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PROTEIN PHOSPHATASE 2A IN GRANULOCYI1C DIFFERENTIATION
@
@I
20
Expressions
of
mRNAs
of
PP2A
during
ATRA-induced
cell
differentiation
of HL-60 cells. To determine whether the ATRA
induced down regulation of PP2A-C in HL-60 cells was reflected at
the level of RNA transcripts, Northern blot analyses using 32P-labeled
cDNA probes for PP2A catalytic subunits (PP2A-Ca and PP2A-C@3)
and regulatory subunits (Aa, A(3, B (Mr 55,000) a and B (Mr 55,000)
I
15
f3subunits)
wereperformed
on totalcellularRNAsobtained
from
10
5 @A
@
A
A
A
A
__________________________________________
,
______________________________________________
,—
0
0
24
48
Time ( hr )
72
96
Fig. 2. Time course of protein phosphatase activity during ATRA-induced differenti
ation of HL-6O cells. HL-60 cells (2 x 108 cells) were treated with 1 @LM
ATRA, and
cytosolic fractions were prepared at the indicated times. Protein phosphatase activity was
measured in the absence (•)or presence of 5 nsi OKA (A) and 500 nsi OKA (I), as
described in “Materials
and Methods.―Point, mean from three separate experiments.
SD, <10%.
of PP1 and PP2A (Fig. 2). OKA (5 flM) inhibited about 68% of the
MLC phosphatase activity in the cytosol of untreated HL-60 cells, and
this OKA -sensitive phosphatase portion (5 nM) was decreased by
85% during ATRA-induced differentiation of HL-60 cells. Because
the MLC phosphatase activity of HL-60 cells in the presence of 5 nM
OKA was relatively constant for 4 days after the addition of ATRA,
@
the decreased MLC phosphatase activity of HL-60 cells after treat
ment with ATRA is probably mainly due to decreased PP2A activity,
even though the PP1 activity is relatively constant. Decrease in PP2A
activity appeared to coincide with an increase in NBT reduction (Figs.
1 and 2). We then examined the effects of ATRA on the regulation of
PP2A activity, protein expressions of PP2A-C, A, and B regulatory
subunits in whole cell extracts by Western blot analysis using poly
clonal antibodies specific for PP2A-C, A regulatory, and various B
regulatory subunits [B (Mr 55,000), B' (Mr 54,000), and 72,000J of
PP2A. Because precise titers of these antibodies against PP2A-C and
A and B (Mr 55,000) regulatory subunits await purification of these
subunits to homogeneity from HL-60 cells, it is impossible, at present,
to estimate the accurate molar ratio for each subunit, but one can
compare change in the amount of individual PP2A subunits during
ATRA-induced differentiation. As shown in Fig. 3, the major immu
noreactive species of PP2A-C, A, and B (Mr 55,000)
subunits were
bands of Mr 35,000, 65,000, and 55,000, respectively. Antibodies
against the B (Mr 55,000)13, B' (Mr 54,000), or Mr 72,000 regulatory
subunit detected no protein in HL-60 cells. The time-dependent and
dramatic decrease in immunoreactive PP2A-C was evident in HL-60
cells after the addition of ATRA, whereas expressions of both A and
B (M@55,000) a subunits were relatively constant (Fig. 3B). In
contrast, the expression of each isozyme of PP1 catalytic subunit
(PP1a, PP1y1, PP16) appeared to be unchanged in HL-60 cells after
ATRA treatment (Fig. 3A). Quantitative immunoblot analysis using
purified
recombinant
proteins
as
standard
revealed
that
PP2A-C,
HL-60 cells following treatment with ATRA (Fig. 4). HL-60 cells
expressed mRNAs of PP2A-Ca, PP2A-C@, and Aa and B (Mr
55,000) a subunits. The 2.0-kilobase mRNA was a major transcript of
HL-60 cells after hybridization to the PP2A-C@3 probe. The down
regulation of PP2A-C(3 mRNA was evident within 3 h after the
addition of 1 p.M ATRA. The level of PP2A-C@3mRNA was further
decreased to approximately one-third of the basal level of untreated
HL-60 cells within 6 h, when normalized to GAPDH mRNA expres
sion. Rehybridization of these blots with GAPDH cDNA probe con
firmed that relatively equal amounts of RNA were loaded in each lane.
The PP2A-Ca probe also detected a major band of 2.0 kilobase and
the expression of PP2A-Ca 2.0-kilobase transcript was relatively
unaltered by treatment with ATRA. Thus, our data suggest that two
isotypes of PP2A could be differently regulated during ATRA-in
duced differentiation. The Aa and B (M@55,000) a genes were also
expressed in HL-60 cells, and A a and B (M1 55,000) a transcripts
were approximately 3.1 and 2.6 kilobases, respectively. The levels of
Aa transcript gradually decreased by treatment of HL-60 cells with
ATRA, although expression of the Aa regulatory protein did not
fluctuate. The levels of B (Mr 55,000) a mRNA remained relatively
constant after ATRA treatment. Transcripts of the A(3 and B (M1
55,000) 3 subunits in HL-60 cells were not detectable. The mRNA
expressions of PP1a, PP1y, and PPl@ were unaltered during ATRA
induced differentiation of HL-60 cells (data not shown).
DISCUSSION
Our present results suggest that PP1 enzyme is not altered during
ATRA-induced granulocytic differentiation of HL-60 cells, while
PP2A activity is down regulated during ATRA-induced differentia
tion. Subunit composition and specific complex formation play im
portant roles in regulating the activity and specificity of PP2A-C (9).
Cellular PP2A is composed of a common PP2A-C/A complex that
interacts with different families of endogenous and exogenous regu
latory proteins including B (Mr 55,000), B' (Mr 54,000), B―
(Mr 74,000)
and Mr 72,000 proteins and tumor antigen (9). We found that the expres
sion of PP2A-C was markedly decreased during the course of granulo
cytic differentiation, whereas the levels of A and B (Mr 55,000) a
regulatory subunits were relatively constant. Other different B regulatory
subunits, including B (Mr 55,000) (3' B' (M1 54,000), and M1 72,000
regulatory proteins were not expressed in HL-60 cells before and after
treatment with ATRA. Although the molar ratio for the PP2A-C and A
and B (M@55,000) subunits in the cells is unknown, the selective down
regulation of PP2A-C may change the ratio ofdimeric and trimeric PP2A
holoenzymes and, therefore, may alter specific catalytic properties such
as substrate specificities or response to effectors.
HL-60 cells expressed RNA transcripts of PP2A-Ca, PP2A-C@3,and
Aa and B (Mr 55,000) a regulatory subunits. Treatment with ATRA led
to a dramatic reduction in mRNA expression of PP2A-C@3,whereas the
level of a isozyme mRNA was unchanged after exposure to ATRA. The
down regulation of PP2A-C@3 RNA transcript clearly preceded the
ATRA-induced granulocytic differentiation. The amino acid sequences
of PP2A-Ca and PP2A-C(3 proteins, deduced from cDNA sequences, are
PP1a, PP1'y, and PP1@ accounted for 5.0, 4.2, 3.9, and 1.4 @tg/mg 97% identical (8, 9). Although mRNAs encoding both isoforms have
protein (means of three determinations), respectively, of the total
been detected in mammalian cells and tissues (14, 33), the steady-state
cytosol fractions in untreated HL-60 cells.
PP2A-Ca mRNA is more abundant than PP2A-C13 mRNA in various cell
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PROTEIN PHOSPHATASE2A IN GRANULOCYTIC DIFFERENTIATION
A
ATRA(1#)
B
49.5
@
32.5
@- •
.
HL-60
— —‘PPla
@
ATRA(1j@)
80.0'—
49.5-'—0
@
.@
49.5
PP2A-C
fl5.O
; Z5—@@---------—---—-‘PP17
@
-*
@
@
@
@
@lO6.0
@.
80.0-'—'-―
—‘A
49.5..-
Fig. 3. Time course of changes in immunoreac
tive PP1 and PP2A during ATRA-induced differ
entiation of HL-60 cells. HL-6O cells (1 X 10@
49.5
cells) were treated with 1 p@ A'I'RA, and the re
action was stopped by the addition of ice-cold
32.5
—‘PPlo
80.0
49.5
trichloroacetic acid at the times indicated (abscis
sas). The acid-precipitated proteins were analyzed
by immunoblot analysis, using polyclonal antibod
ies specific for PP2A and PP1 isozymes, as de
scribed in “Materialsand Methods.―A, blots incu
0
24
C
batedwithPP1a,PP1y,andPP1&antisera.B,blots
incubated with PP2A-C and A and B (M, 55,000) a
regulatory subunit antisera. All antibodies were
used at a 1:100 dilution. Ordinate, molecular
weight (MW.) standard markers (Bio-Rad). These
data are from one representative experiment, but
similar results were obtained in two other experi
ments. C, graphic display of PP2A-C (•),A (U),
48
72(1w)
-—B
@.
0 24 48 72 96 ( hr)
6
5
E
and B (M, 55,000)a (A) regulatory subunits shown
4
in B, quantitated by densitometry.
C
3
2
-e
I
0
0
24
48
TIme(hr)
72
96
lines other than hematopoietic cells, and this differential expression is
thought to be due to different promoter activities (14). PP2A-Ca and
PP2A-C13 are encoded by diStinCtgenes whose expression appears to be
differentially regulated (14, 31). Therefore, it is conceivable that the
may be resistant to proteolysis and, therefore, has a long life span in
the cell. The ATRA-induced granulocytic differentiation of HL-60
cells appears to be mediated through the nuclear retinoic acid receptor
expression
superfamily of transcription factors and possesses discrete retinoic
of PP2A-Ca
and
PP2A-C13
is separately
modulated
in the
(34).Theretinoicacidreceptor
isa member
of thethyroidhormone
process of ATRA-induced granulocytic differentiation. We did not dem
onstrate directly the differential expression of two PP2A-C isoforms at
protein levels, because a specific antibody against each isoform was not
available. The expression ratio of PP2A-Ca to PP2A-C(3 proteins may
possibly be increased in the PP2A activity of ATRA-induced mature
granulocytes.
The mRNA level of Aa regulatory subunit was also decreased
following treatment with ATRA, while that of B (Mr 55,000) a
regulatory subunit was relatively constant. These data suggest that
mRNA expressions of PP2A-C and Aa subunits are coordinately
regulated in HL-60 cells following treatment with ATRA. In spite of
the early decrease in mRNA expression of Aa regulatory subunit, the
level of expression of the A subunit protein did not fluctuate through
out ATRA-induced differentiation. While the reason for this discrep
acid-binding (ligand-binding) and DNA-binding domains that regu
late transcription of certain target genes (5). ATRA can influence
PP2A gene expression either directly, by activating transcription of
the PP2A gene, or indirectly, by altering expression of genes encoding
transcription factors.
In addition to the modulation of protein phosphatases, the levels of
both activity of protein kinase C and expression of its isoforms (a, @,
and y) of protein kinase C was shown to increase during granulocytic
differentiation of HL-60 cells induced by ATRA and dimethyl sulf
oxide (7). 1,25-Dihydroxyvitamin D3-induced monocytic differentia
tion of HL-60 cells is also associated with an increase in both activity
of protein kinase C and expression of protein kinase C-a and -@, as
well as steady-state levels of mRNA of protein kinase C-a and -@(35,
ancy
be involved in the regulation of HL-60 cell differentiation by TPA,
remains
unknown,
we
speculate
that
the
A regulatory
subunit
36).The activationof cAMP-dependentproteinkinasealsoappearsto
4882
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PROTEIN PHOSPHATASE 2A IN GRANULOCYTIC DIFFERENTIATION
@
A
@
3 5 12 24(1w)
PP2A-C@
—
@
@
0
.@ - 2.0kb
5
12
24(1w)
Act— .
3.1kb
GAPDH—
01
3
5
1224(1w)
@
@
3
Bar— ,@
Fig. 4. Time course of mRNA expressions of
PP2A-Cf3, PP2A-Ca, Aa and B (M, 55,000) a
subunits in HL-60 cells stimulated with ATRA.
PP2A@C@—
r@
,
, -26kb
—2.0kb
HL-60cells(2 x iO@
cells)weretreatedwith1 ps,
ATRA, and total RNA (20 pg) was isolated from
@
the cells at the indicated times (abscissas). A,
Northern blots of total RNA were hybridized with
GAPDH -
32Plabeled cDNA probes for PP2A-Ca, PP2A-C13,
Aa, and B (M, 55,000) a subunits. The same RNA
blots were hybridized to GAPDH cDNA probe for
assessment of RNA quantities in each lane. B,
graphic representation of relative intensity of hy
GAPDH
bridization signals of PP2A-Ca (0), PP2A-C@
(•),
Aa(U),
andB (M,55,000)
a (A)obtained
B
from densitometric scanning of the autoradiogram
pictured in A. The ratio of these signals to the
GAPDH signal was determined for each time point
following ATRA addition and the resulting value is
120
expressed relative to control untreated HL-60 cells.
Similar results were obtained in two independent
experiments.
0
5-
100
C
0
C-)
C
0
4)
Cl)
0)
80
60
5-
40
w
0)
>
20
0)
0@
1@_
0
6
12
18
24
Time(hr)
ATRA or dimethyl formamide (37, 38). Although the monocytic and
granulocytic inducers share protein kinases as target molecules (6), it
is also true that activation and/or modulation of protein kinases may
be solely insufficient to explain the mechanisms for bringing about
terminal differentiation into granulocytic or monocytic phenotypes.
Thus, the modulation of protein phosphatases may play important
roles in regulating the net phosphorylation of critical substrate(s) that
subsequently mediate the differentiation of HL-60 cells into either
phenotype. PP2A is thought to regulate multiple functions in vivo,
including several metabolic pathways, protein synthesis, DNA repli
cation, and the cell cycle (8, 9). Differentiation of HL-60 cells by
chemical agents, such as ATRA, 1,25-dihydroxyvitamin
D3, and
phorbol diester is accompanied by withdrawal from the cell cycle
(1, 2). All of these data taken together suggest that down regulation of
PP2A may be associated with ATRA-induced differentiation
HL-60 cells, although the modulation of protein phosphatases
only
one
of several
parallel
events
which
mediate
the
in
is
various
effects of ATRA. Further studies of HL-60 cells utilizing antisense
oligodeoxynucleotides
directed against PP2A-C and variant cell
lines (39) arrested at discrete points of differentiation may provide
insights into the mechanisms by which PP2A influences myeloid
differentiation.
ACKNOWLEDGMENTS
We thank M. Ohara for critical comments.
4883
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PROTEIN PHOSPHATASE 2A IN GRANULOCYrIC
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Expression of the Catalytic and Regulatory Subunits of Protein
Phosphatase Type 2A May Be Differentially Modulated during
Retinoic Acid-induced Granulocytic Differentiation of HL-60
Cells
Masakatsu Nishikawa, Serdar B. Omay, Hideki Toyoda, et al.
Cancer Res 1994;54:4879-4884.
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