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1572
BIOLOGY OF REPRODUCTION 69, 1572–1579 (2003)
Published online before print 25 June 2003.
DOI 10.1095/biolreprod.103.018739
Binding and Inactivation of the Germ Cell-Specific Protein Phosphatase PP1g2
by sds22 During Epididymal Sperm Maturation
1
Sanjay Mishra, Payaningal R. Somanath, Zaohua Huang, and Srinivasan Vijayaraghavan
2
Biological Sciences Department, Kent State University, Kent, Ohio 44242
ABSTRACT
Testis- and sperm-specific protein phosphatase, PP1g2, is a
key enzyme regulating sperm function. Its activity decreases
during sperm maturation in the epididymis. Inhibition of PP1g2
leads to motility initiation and stimulation. Our laboratory is
focused on identifying mechanisms responsible for the decline
in PP1g2 activity during sperm motility initiation in the epidid-
ymis. Previously, using immuno-affinity chromatography, we
showed that a mammalian homologue of yeast sds22 is bound
to PP1g2 in motile caudal spermatozoa (Huang Z, et al. Biol
Reprod 2002; 67:1936–1942). The objectives of this study were
to determine: 1) stoichiometry of PP1g2-sds22 binding and 2)
whether PP1g2 in immotile caput epididymal spermatozoa is
bound to sds22. The enzyme from caudal and caput sperm ex-
tracts was purified by column chromatography. Immunoreactive
PP1g2 and sds22 from both caudal and caput spermatozoa were
found in the flow-through fraction of a DEAE-cellulose column.
However, PP1g2 from caudal spermatozoa was inactive, where-
as in caput spermatozoa it was active. The DEAE-cellulose flow-
through fractions were next passed through a SP-sepharose col-
umn. Caudal sperm sds22 and PP1g2 coeluted in the gradient
fraction. In contrast, caput sperm sds22 and PP1g2 were sepa-
rated in the flow-through and gradient fractions, respectively.
Further purification through a Superose 6 column showed that
PP1g2-sds22 complex from caudal sperm was 88 kDa in size.
Caput sperm sds22 and PP1g2 eluted at 60 kDa and 39 kDa,
respectively. SDS-PAGE of these purified fractions revealed that
in caudal sperm, the 88-kDa species is composed of sds22 (43
kDa) and PP1g2 (39 kDa), suggesting a 1:1 complex between
these two proteins. PP1g2 bound to sds22 in this complex was
inactive. Caput sperm sds22 eluting as a 60-kDa species was
found to be associated with a 17-kDa protein (p17). This sug-
gests that dissociation of sds22 from p17 or some other post-
translational modification of sds22 is required for its binding
and inactivation of PP1g2. Studies are currently underway to
determine the mechanisms responsible for development of
sds22 binding to PP1g2 during epididymal sperm maturation.
gamete biology, phosphatases, signal transduction, sperm matu-
ration, testis
INTRODUCTION
We discovered that testis-specific protein phosphatase,
PP1
g
2, is the predominant protein phosphatase in sperma-
tozoa [1, 2]. High PP1
g
2 activity results in low motility,
1
Supported by NIH grant RO1 HD38520.
2
Correspondence: Srinivasan Vijayaraghavan, Biological Sciences Depart-
ment, Kent State University, Kent, Ohio 44242-0001. FAX: 330 672 3713;
e-mail: svijayar@kent.edu
Received: 29 April 2003.
First decision: 21 May 2003.
Accepted: 17 June 2003.
Q 2003 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
and low PP1
g
2 activity is associated with vigorous motility
[2]. This decline in PP1
g
2 activity during sperm maturation
is not due to a decline in the amount of the enzyme but to
a decrease in its catalytic activity. We found that the protein
phosphatase inhibitors, okadaic acid and calyculin A, ini-
tiate and stimulate motility of epididymal spermatozoa. The
enzyme PP1
g
2 is involved in the regulation of spermatozoa
from all mammalian species studied so far, including hu-
man [1–3].
At least four different somatic cell serine/threonine phos-
phatase types, PP1, PP2A, PP2B, and PP2C, are known [4–
6]. Four catalytic subunit isoforms of PP1, encoded by three
genes, are PP1
a
, PP1
g
1, PP1
g
2, and PP1
d
[6–8]. PP1
g
1
and PP1
g
2 are alternatively spliced isoforms generated
from a single gene [6–8]. Although PP1
g
1 is ubiquitous,
PP1
g
2 is present only in germ cells and spermatozoa [8,
9]. These two PP1 variants are identical in all respects ex-
cept that PP1
g
2 has a unique 21 amino acid carboxy ter-
minus extension. It is noteworthy that the carboxy terminus
extension of PP1
g
2, which is lacking in somatic cell PP1
isoforms, is conserved in all mammalian spermatozoa we
have studied so far, suggesting sperm-specific roles for this
amino acid sequence domain. Disruption of the PP1
g
gene
in mice causes sterility in males because of arrest of sper-
matogenesis at the spermatid stage [9]. This observation
shows that other isoforms of PP1 can compensate for the
lack of PP1
g
1 in somatic cells and for the lack of PP1
g
2
in germ cells until the final stages of spermatogenesis. Tes-
tis-specific PP1
g
2 is indispensable in the final stages of
spermatogenesis.
Our studies are focused on understanding mechanisms
regulating PP1
g
2 and determination of the basis for the
decline in PP1
g
2 activity during epididymal sperm matu-
ration. Protein phosphatases, in general, are regulated by
their binding and targeting proteins [10, 11]. Following
identification of PP1
g
2, we speculated that one or more
somatic cell protein regulators of PP1 might be present in
spermatozoa. Our initial efforts focused on proteins I1 and
I2, which are ubiquitous PP1 inhibitors found in all somatic
cells [11]. Surprisingly, sperm do not contain any detectable
I1 or I2 suggesting that mechanisms regulating PP1
g
2
could be unique (unpublished data, [2]).
We first used immunoaffinity chromatography to isolate
PP1
g
2 and its associated proteins [12]. Antibodies to PP1
g
2
immunoprecipitate sds22, a mammalian homologue of the
yeast PP1-binding protein [12]. Sds22 is a prototypic mem-
ber of a family of proteins containing leucine-rich repeats
[13]. Human sds22 contains 11 repeats of a leucine-rich 22
amino acid segment. The leucine-rich repeat is a structural
motif used in such diverse molecular recognition processes
as signal transduction, cell adhesion, cell development and
RNA processing [13]. In caudal epididymal spermatozoa,
sds22-bound PP1
g
2 is catalytically inactive [12].
In the immunoprecipitation study, PP1
g
2 antibodies
1573
REGULATION OF PP1
g
2 BY sds22
were found to bind to only a portion of the total PP1
g
2in
sperm extracts [12]. Coimmunoprecipitation suggested, but
did not prove, that sds22 was bound to PP1
g
2. Stoichi-
ometry of PP1
g
2 binding to sds22 was not known. It was
also necessary to determine why PP1
g
2 activity is lower in
caudal, compared with caput spermatozoa. Therefore, the
present study was undertaken to purify PP1
g
2 and sds22,
by column chromatography, from both caput and caudal
spermatozoa and to understand how sds22 may regulate
PP1
g
2 activity during sperm maturation and motility initi-
ation. These studies show that in caudal spermatozoa, a
proportion of PP1
g
2 present as a heterodimer with sds22,
is inactive. In caput epididymal spermatozoa, sds22 is not
bound to PP1
g
2. Caput sperm sds22 may be bound to a
17-kDa protein, p17. A significant proportion of caput
sperm PP1
g
2 is therefore in its catalytically active state.
These studies suggest that dissociation of sds22 from p17
or some other modification of sds22 is followed by its bind-
ing and inactivation of PP1
g
2. Because inhibition of PP1
g
2
activity induces motility, binding and inactivation of PP1
g
2
by sds22 during sperm maturation in the epididymis is pre-
sumably one of the biochemical events responsible for mo-
tility initiation.
MATERIALS AND METHODS
Preparation of Sperm Extracts
Testes with intact tunica from mature bulls were obtained from a local
slaughterhouse. Spermatozoa were isolated from caudal and caput epidid-
ymis and washed as previously described [3] in buffer A (10 mM Tris-
HCl [pH 7.2] containing 120 mM NaCl, 10 mM KCl, 5 mM MgSO
4
).
Sperm pellets were suspended in a homogenization buffer (buffer B) (10
mM Tris [pH 7.2] containing 1 mM EDTA, 1 mM EGTA, 10 mM ben-
zamidine-HCl, 1 mM PMSF, 0.01 mM N-p-tosyl-
L
-phenylalanine chloro-
methyl ketone [TPCK], and 5 mM
b
-mercaptoethanol). The sperm sus-
pension was sonicated with three 5-sec bursts of a Biosonic II sonicator
(Bronwell Scientific, Rochester, NY) at maximum setting. The sperm son-
icate was then centrifuged at 16000
3
g for 10 min. The 16 000
3
g
supernatants were supplemented with 10% glycerol and stored at
2
20
8
C
until further use for column procedures.
Column Chromatography
All column procedures were conducted at 4
8
C. Total protein in caudal
and caput sperm extracts and fractions obtained from column chromatog-
raphy was measured with Coomassie brilliant blue dye reagent (Bio-Rad
laboratories, Hercules, CA) as described previously [14]. Caudal sperm
extract (50 ml prepared from 5
3
10
10
spermatozoa in buffer B) was
passed through a DEAE-cellulose (0.5cm
3
13 cm) column pre-equili-
brated with buffer C (buffer B with 0.05 M KCl and additional protease
inhibitors-pepstatin A [1
m
g/ml], aprotinin [2
m
g/m], and leupeptin [0.5
m
g/ml]). The column was washed with 20 ml of buffer C followed by
elution with a linear gradient of 0.05–0.65 M KCl in buffer C. Flow-
through and gradient fractions (0.185–0.35 M KCl) containing PP1
g
2 ac-
tivity and PP1
g
2 and/or sds22 immunoreactivity were pooled separately.
The pooled fractions were concentrated to 12.5 ml using Centricon-10
filter (Millipore Corp., Bedford, MA). A SP-sepharose column (5 ml pre-
packed, Pharmacia, Piscataway, NJ) was pre-equilibrated with buffer C.
The DEAE-cellulose flow-through fraction was applied to this SP-sephar-
ose column. The column was then washed with 10 ml buffer C followed
by elution with a linear gradient of 0.05–0.65 M KCl in buffer C. Fractions
containing sds22 and PP1
g
2 were pooled, concentrated to 1 ml, and ap-
plied in five batches to Superose 6 (24 ml, prepacked high-resolution
FPLC, Pharmacia, Piscataway, NJ) column pre-equilibrated with buffer C.
The elution was performed with buffer C. Immunoreactive fractions of all
five batches were pooled, concentrated to 1 ml, and applied to Mono S (1
ml, prepacked high-resolution FPLC, Pharmacia, Piscataway, NJ) column.
The column was washed with 5 ml buffer C followed by elution with a
linear gradient of 0.05–0.65 M KCl in buffer C. Immunoreactive fractions
(0.140–0.215 M KCl) containing sds22-PP1
g
2 complex were pooled, and
concentrated to 0.5 ml. For purification of sds22 and PP1
g
2 from caput
epididymal spermatozoa, we followed the same protocol as described
above for caudal sperm, except that 50 ml of extract derived from 1
3
10
10
spermatozoa was used. The final step for purifying caput sds22-p17
complex through a heparin sepharose (1 ml, prepacked Pharmacia, Pis-
cataway, NJ) column was similar to the protocol used for Mono S column
chromatography. Purified and concentrated caudal sds22-PP1
g
2 complex
and caput sds22 and free PP1
g
2 fractions were stored at
2
20
8
C until
further use.
Protein Phosphatase Assay
Preparation of radiolabeled phosphorylase a, and its use as a substrate
for measurement of PP1 activity is based on a protocol described previ-
ously [3]. The substrate, aliquots from sperm extracts, and fractions ob-
tained after column chromatography were incubated (in a total volume of
40
m
l) at 30
8
Cwith1mMMn
2
1
and with or without inhibitors for 10
min. At the end of this incubation, the reaction was terminated with 180
m
l of 20% trichloro acetic acid, after which the tubes were centrifuged for
5 min at 12000
3
g at 4
8
C. The supernatants were analyzed for
32
PO
4
released from phosphorylase a. One unit of enzyme activity was defined
as the amount of enzyme that catalyzed the release of 1 nmol of
32
PO
4
/
min under conditions of the enzyme assay. This assay is specific for the
protein phosphatases PP1 and PP2A [3].
SDS-PAGE and Western Blot Analysis
Sperm extracts (30–50
m
g) and aliquots from fractions obtained from
column chromatography (2–10
m
g) were separated by SDS-PAGE through
12% acrylamide slab gels based on the protocol of Laemmli [15]. One
milliliter of soluble extracts (prepared from 10
9
cells) contain approxi-
mately 1 mg/ml and 1.9 mg/ml protein for caudal and caput sperm, re-
spectively. After electrophoresis, proteins were electrophoretically trans-
ferred to Immobilon-P, PVDF membrane (Millipore Corp.). Nonspecific
protein binding sites on the membrane were blocked with 5% nonfat dry
milk in Tris buffered saline (TBS; 25 mM Tris-HCl, pH 7.4, 150 mM
NaCl). The blots were washed twice for 15 min each with TBS containing
0.1% Tween 20 (TTBS) and then incubated with anti-PP1
g
2 (1:5000) or
anti-sds22 (1:2000). Both antibodies were commercially prepared (Zymed
Laboratories, San Francisco, CA) by using a synthetic carboxyl terminus
extension of PP1
g
2 (22 amino acids of the carboxyl terminus) and amino
acid residues 329–342 of sds22 as antigens. Antibodies were affinity pu-
rified with the synthetic peptides conjugated to a sulfo-link column (Pierce,
Rockford, IL). After washing, the blots were incubated with the appropri-
ate secondary antibody conjugated to horseradish peroxidase at 1:2000
dilution for 1 h at room temperature. Blots were washed with TTBS twice
15 min each and four times 5 min each. Blots were then developed with
an enhanced chemiluminescence kit (Amersham, Piscataway, NJ). Protein
composition of purified samples was also assessed by Coomassie blue or
silver staining following SDS-PAGE. Coomassie staining was done with
0.1% Coomassie brilliant blue R250 in 40% methanol and 10% acetic
acid. Silver staining was performed using a commercial kit (Bio-Rad Lab-
oratories) as per instructions provided by the manufacturer.
RESULTS
Purification of PP1
g
2 and sds22 From Caudal
Epididymal Spermatozoa
We used DEAE-cellulose, SP-sepharose, high-resolution
Superose 6, and Mono S columns for purification of PP1
g
2
and sds22 from caudal spermatozoa. Column fractions were
analyzed for PP1
g
2 and sds22 immunoreactivity and for
PP1
g
2 catalytic activity. Following at least three pilot pu-
rification runs, 50 ml of sperm extracts prepared from 5
3
10
10
bovine caudal epididymal spermatozoa were used to
isolate enough PP1
g
2 and sds22 for biochemical studies.
Specific activity of PP1
g
2 in the pooled extracts was 1.71
U/mg protein. A summary of the purification steps is shown
in Table 1.
The sperm extract was first passed through a DEAE-
cellulose column. A portion of the enzyme was found in
the flow-through fraction, and the rest was absorbed in the
column. In addition to PP1
g
2, the DEAE-cellulose flow-
through fraction also contained immunoreactive sds22. The
enzyme in the DEAE-cellulose flow-through fraction was
inactive whereas that released from the column by a salt
1574
MISHRA ET AL.
TABLE 1. Summary of sds22-PP1
g
2 purification from caudal epididymal sperm extracts.*
Purification step Protein (mg) Total activity (U)
Specific activity
(U/mg protein)
Caudal sperm extract 152 260 1.71
DEAE-cellulose flow-through 65 Inactive —
DEAE-cellulose gradient (0.185–0.350 M KC1)
(PP1
g
2 without sds22) 71 155 2.18
SP sepharose gradient (0.125–0.275 M KC1)
of DEAE-cellulose flow-through 21 Inactive —
Superose 6 of SP-sepharose gradient 1.05 Inactive —
Mono S gradient
(0.140–0.215 M KC1) of SP-sepharose gradient 0.525 Inactive —
* Table summarizes purification of three batches of 50 ml each of caudal sperm extract as outlined under
Materials
and Methods
. The total number of cells corresponding to 153 mg protein is 1.5 3 10
11
. Activity of PP1
g
2 in caudal
sperm extracts and fractions obtained after column procedures was measured in the presence of 1 mM Mn
2
1
.
FIG. 1. Western blot analysis showing caudal sperm PP1g2-sds22 com-
plex during different stages of purification through column chromatogra-
phy. The same blot was probed with PP1g2 and sds22 antibodies. Lane
1 is caudal extract (30 mg); lane 2 is DEAE-cellulose flow-through fraction;
lane 3 is SP-sepharose flow-through of DEAE-cellulose flow-through frac-
tion; and lane 4 is SP-sepharose gradient of DEAE-cellulose flow-through
fraction. In lanes 2–4, 25 ml each of the concentrated column fractions
as described in
Materials and Methods
were loaded.
FIG. 2. Molecular weight determination of the caudal sperm PP1g2-
sds22 complex. A) Western blot of the fractions (25 ml each) showing
PP1g2-sds22 complex eluted in the fractions 22–29 in Superose 6. B)
Elution profile of PP1g2-sds22 complex from caudal sperm extracts in
Superose 6. PP1g2-sds22 complex eluted in Superose 6 at 88 kDa. Cal-
ibration of the column for its void volume was performed by running blue
dextran. Standardization of the column was done by resolving a mixture
of aldolase (5 mg/ml), albumin (5 mg/ml), ovalbumin (10 mg/ml), and
chymotrypsinogen (10 mg/ml).
gradient (0.185–0.35 M KCl) had a specific activity of 2.18
U/mg protein (Table 1). Details of purification of PP1
g
2
released from the DEAE-cellulose column are not outlined
in this report. The focus in this report is on PP1
g
2 asso-
ciated with sds22. The DEAE-cellulose flow-through frac-
tion containing PP1
g
2 and sds22 was concentrated and
passed through a SP-sepharose column. Here PP1
g
2 and
sds22 were bound in the column. All the bound PP1
g
2 and
sds22 were released by a salt gradient between 0.125–0.275
M KCl. The presence of sds22 and PP1
g
2 in the DEAE-
cellulose and SP-sepharose column fractions is shown in
Figure 1 (lanes 2 and 4).
The SP-sepharose salt gradient fractions containing both
PP1
g
2 and sds22 were concentrated and passed through a
Superose 6 column. In the Superose 6 column, sds22 and
PP1
g
2 coeluted as a 88-kDa species in fractions 26, 27,
and 28 (Fig. 2). Coelution of sds22 and PP1
g
2 through
these three columns, DEAE-cellulose, SP-sepharose, and
Superose 6, suggested that they could be complexed. To
confirm their association, we further purified sds22 and
PP1
g
2 containing fractions through a Mono S column.
Sds22 and PP1
g
2 were absorbed on the Mono S column
and were coeluted in a 0.140–0.215 M KCl gradient. These
fractions containing PP1
g
2 and sds22 were pooled and con-
centrated and analyzed by SDS-PAGE and Western blot.
Coomassie blue and silver staining following SDS-PAGE
(Fig. 3A) showed that the complex eluting from Mono S
column contains two major protein bands at 43 kDa and
39 kDa. These two protein bands were confirmed to be
1575
REGULATION OF PP1
g
2 BY sds22
FIG. 3. Caudal sperm PP1g2 is bound to protein sds22. A) Coomassie
blue (lane 1) and silver stained gels (lane 2) showing PP1g2 and sds22
after SDS-PAGE of purified PP1g2-sds22 complex obtained after Mono S
column chromatography (2.5 mg). Silver staining was performed on the
same gel following Coomassie blue. Lane 3 shows protein molecular
weight markers. B) Western blots showing immunoreactive PP1g2 (left
panel) and sds22 (right panel) in caudal extracts (lane 1: 30 mg protein)
and purified PP1g2-sds22 complex (lane 2: 2.5 mg protein).
FIG. 4. Western blot analysis showing caput sperm PP1g2 and sds22
during different stages of purification through column chromatography.
Blot was probed with both PP1g2 and sds22 antibodies. Lane 1 is caput
extract (50 mg); lane 2 is DEAE-cellulose flow-through fraction; lane 3 is
SP-sepharose flow-through of DEAE-cellulose flow-through fraction; and
lane 4 is SP-sepharose gradient of DEAE-cellulose flow-through fraction.
In lanes 2–4, 25 ml each of the concentrated column fractions as de-
scribed in
Materials and Methods
were loaded.
sds22 and PP1
g
2, respectively, by Western blot analysis
(Fig. 3B, lane 2 in the left and right panels). All column
fractions containing sds22 and PP1
g
2 had virtually no pro-
tein phosphatase activity (Table 1).
Purification of PP1
g
2 and sds22 From Caput
Epididymal Spermatozoa
Next, we subjected caput sperm extracts to essentially
the same purification scheme used for caudal sperm ex-
tracts. Caput sperm extracts have higher PP1
g
2-specific ac-
tivity: 7.26 U/mg protein, compared with 1.71 U/mg protein
in caudal sperm. A summary of the purification of sds22
and PP1
g
2 from caput sperm extracts is shown in Table 2.
Similar to caudal sperm extracts, PP1
g
2 in caput sperm
extracts also separated into flow-through and gradient frac-
tions on DEAE-cellulose. In addition to PP1
g
2, the DEAE
-cellulose flow-through fraction also contained immunore-
active sds22. However, unlike caudal sperm (Table 1),
PP1
g
2 in the DEAE-cellulose flow-through fraction of ca-
put sperm extracts was catalytically active: 14.7U/mg pro-
tein (Table 2). The enzyme released by a salt gradient
(0.17–0.305 M KCl) from the DEAE-cellulose column had
a specific activity of 9.6U/mg protein. Further purification
of DEAE-bound pool of PP1
g
2 is not outlined here. The
DEAE-cellulose flow-through fraction, containing sds22
and PP1
g
2, was next passed through a SP-sepharose col-
umn. In this column, sds22 was found in the flow through
fraction, but PP1
g
2 was absorbed by the matrix and was
eluted by a salt gradient (0.41–0.5 M KCl). Specific activity
of enzyme eluting from SP-sepharose was 19.8 U/mg pro-
tein (Table 2). Western blot (Fig. 4) shows the fractionation
of sds22 and PP1
g
2 through DEAE-cellulose and SP-se-
pharose columns. It can be seen that sds22 and PP1
g
2 pres-
ent together in the DEAE-cellulose flow-through fraction
(Fig. 4, lane 2) are separated in the SP-sepharose column
(Fig. 4, lanes 3 and 4).
The SP-sepharose column fractions containing sds22 and
PP1
g
2 were concentrated and passed through a Superose 6
column. Caput sperm sds22 eluted as a 60-kDa protein,
fractions 31, 32, and 33 (Fig. 5A). This 60-kDa protein was
next passed through a heparin sepharose column, in which
immunoreactive sds22 was found in the flow-through frac-
tion. SDS-PAGE followed by Coomassie blue and silver
staining shows that heparin Sepharose-purified 60-kDa spe-
cies is composed of two major protein bands at 43 kDa and
1576
MISHRA ET AL.
TABLE 2. Summary of sds22 and PP1
g
2 purification from caput epididymal sperm extracts.*
Purification step Protein (mg) Total activity (U)
Specific activity
(U/mg protein) Recovery (%)
Purification
fold
Caput sperm extract 54.6 396 7.26 100 1
DEAE-cellulose flow-through
(sds221PP1
g
2) 21.5 31.5 14.65 72 2
DEAE-cellulose gradient
(0.17–0.305 M KC1) (PP1
g
2
without sds22) 26.6 255 9.6 64 1.3
SP sepharose flow-through of
DEAE cellulose flow-through
(free sds22) 11.8 Inactive — — —
Superose 6 (sds22) 1.1 Inactive — — —
Heparin sepharose flow-through
(sd22) 0.49 Inactive — — —
SP sepharose gradient (0.410–
0.5 M KC1) of DEAE cellu-
lose flow-through
(free PP1
g
2) 5.2 103 19.81 26 2.7
Superose 6 (free PP1
g
2) 0.15 85 567 22 78
* Table summarizes purification of three batches of 50 ml each of caput sperm extract as outlined under
Materials and Methods
. The total number of
cells corresponding to 54.6 mg protein is 3 3 10
10
. Activity of PP1
g
2 in caput sperm extracts and fractions obtained after column procedures was
measured in the presence of 1 mM Mn
2
1
. Experimental details are given in
Materials and Methods
.
17 kDa (p17) (Fig. 5, B, C, and D). The SP-sepharose frac-
tions containing PP1
g
2 eluted through the Superose 6 col-
umn at about 39 kDa: a molecular size in close correspon-
dence to the calculated size based on its amino acid se-
quence (39 kDa) (Fig. 5A). Identities of purified PP1
g
2 and
sds22 were also confirmed by Western blot analysis (Fig.
5D).
Caput Sperm sds22-p17 Complex Does Not Bind
to PP1
g
2
Caudal but not caput sperm sds22 is bound to PP1
g
2. A
logical question is whether the lack of sds22-PP1
g
2 binding
is due to some modification in PP1
g
2 in caput spermatozoa.
If this were the case, purified caput sperm sds22 should
bind to recombinant PP1
g
2 expressed in bacteria. We in-
vestigated whether the purified caput sperm sds22-p17
complex can inhibit recombinant PP1
g
2 in vitro. Varying
concentrations of heparin sepharose-purified sds22-p17
complex (0 to 1
m
g) were preincubated with PP1
g
2 (Table
2 and Fig. 5D) followed by measurement of protein phos-
phatase activity. Catalytic activity of PP1
g
2 activity was
not significantly inhibited at any concentration of sds22
(Fig. 6). Both calyculin A (5 nM) and protein I2 (10 ng),
known inhibitors of PP1, do inhibit catalytic activity of the
recombinant enzyme (Fig. 6), showing that the recombinant
enzyme is capable of binding to these inhibitors. Identical
results to those shown in Figure 6 were also obtained when
recombinant PP1
a
or PP1
g
2 purified from caput sperm ex-
tracts (Superose 6 eluate; Fig. 5A) instead of recombinant
PP1
g
2 was used (data not shown).
DISCUSSION
In somatic cells, PP1 isoforms are regulated by several
ubiquitous and cell-specific regulatory and targeting pro-
teins [11]. Germ cell-specific protein phosphatase PP1
g
2is
the predominant serine/threonine phosphatase in sperma-
tozoa [1–3]. The activity of this enzyme is inversely cor-
related with motility: low activity in vigorously motile cau-
dal spermatozoa and high activity in immotile caput sper-
matozoa [2]. Inhibition of the enzyme with okadaic acid
and calyculin A causes motility initiation and stimulation
[1–3]. Our focus was to understand how PP1
g
2 is regulated
and how the activity of the enzyme declines during epidid-
ymal sperm maturation.
Antibodies to PP1
g
2 coprecipitate sds22 from sperm
extracts suggesting that sds22 is bound to PP1
g
2 [12]. One
of the objectives of this study was to confirm this obser-
vation by column purification techniques and determine
stoichiometry of the PP1
g
2-sds22 complex. Caudal sperm
sds22 copurifies with PP1
g
2 through four different col-
umns: DEAE-cellulose, SP-sepharose, Superose 6, and
Mono S. The fact that sds22 and PP1
g
2 copurify in col-
umn chromatography and immunoprecipitation [12]
strongly supports the conclusion that sds22 is complexed
to PP1
g
2. The apparent molecular weight of the sds22 and
PP1
g
2 complex, determined by Superose 6 column chro-
matography, is 88 kDa. This is in close correspondence
with a molecular weight of 82 kDa, for a 1:1 PP1
g
2-sds22
complex, calculated on the basis of their amino acid se-
quences.
Our data do not rule out the possibility that some of
caudal sperm sds22 may exist as a homodimer (86 kDa).
It is, however, unlikely that this sds22 will coelute with the
sds22-PP1
g
2 complex through the four different column
matrices. Furthermore, free sds22 (i.e., sds22 not bound to
PP1
g
2) exists as a 60-kDa species in caput spermatozoa
(Fig. 5). It is noteworthy that immunoreactive sds22 is not
detected in any other column fraction except those also con-
taining PP1
g
2 (Table 1 and Fig. 1), suggesting that all of
the sds22 present in caudal sperm extracts is bound to
PP1
g
2 (i.e., sds22 in sperm extracts is a PP1
g
2 regulatory
protein).
In accordance with our earlier observations made with
crude caudal sperm extracts [12], we also determined that
column-purified sds22-PP1
g
2 complex does not also bind
to microcystin (data not shown). Microcystin can bind to
PP1 and PP2A catalytic subunits alone or when the cata-
lytic subunits are associated with a variety of their regula-
tory proteins [16]. In particular, the sds22 complex with the
somatic cell PP1 isoform, PP1
a
, can bind to microcystin
[17]. The inability of the sperm sds22-PP1
g
2 complex to
bind to microcystin suggests that sds22 masks the micro-
cystin-binding site on PP1
g
2. Purified PP1
g
2-sds22 com-
plex and the complex present in the different column frac-
tions are virtually devoid of catalytic activity (Table 1). The
1577
REGULATION OF PP1
g
2 BY sds22
FIG. 5. Molecular weight determination of the caput sperm PP1g2 and
sds22. A) Elution profile of sds22-p17 complex and PP1g2 from caput
sperm extracts in Superose 6. PP1g2 and sds22-p17 complex eluted in
Superose 6 at 39 kDa and 60 kDa, respectively. Calibration of the column
for its void volume was performed by running blue dextran. Standardi-
zation of the column was performed as described in Figure 2. B and C)
Coomassie blue and silver stained gels showing sds22 and p17 after SDS-
PAGE of purified sds22-p17 complex (5 mg) obtained after heparin se-
pharose column chromatography. C) Western blots showing immunore-
active PP1g2 and sds22 in caput extract (50 mg protein), purified PP1g2
fraction and sds22-p17 complex (5 mg).
FIG. 6. Effect of caput sperm sds22-p17 complex on catalytic activity of
recombinant PP1g2. The indicated amounts of sds22-p17 complex puri-
fied from caput sperm extracts, 5 nM calyculin A or 10 ng of I2 were
added to recombinant PP1g2 (150 mU) and incubated at 308C for 10
minutes. After this incubation, the phosphatase assay was initiated with
phosphorylase
a
as described in
Materials and Methods.
Results are ex-
pressed as mean 6 SEM (n 5 3).
sds22-PP1
g
2 complex isolated by immunoaffinity chro-
matography from crude sperm extracts was also inactive
[12]. Thus sds22 is a PP1
g
2 inhibitor in spermatozoa. It is
noteworthy that spermatozoa do not contain the ubiquitous
somatic cell PP1 inhibitors I1 [2] and I2 (unpublished data).
Data presented in this report provide strong evidence that
in motile caudal spermatozoa PP1
g
2, present as a 1:1 com-
plex with sds22, is catalytically inactive.
The next objective of this study was to examine wheth-
er sds22 in caput epididymal spermatozoa is also bound
to PP1
g
2. Data in Table 2 and Figure 4 clearly show that
caput sperm sds22 is not bound to PP1
g
2. Sds22 in caput
sperm extracts is present in the flow-through fraction of
SP-sepharose column, which does not contain any detect-
able PP1
g
2 (Fig. 4). The enzyme PP1
g
2, free of any de-
tectable sds22, is released by a salt gradient from SP-se-
pharose (Fig. 4). Unlike caudal sperm extracts, PP1
g
2
from caput spermatozoa in the DEAE cellulose flow-
though fraction and SP-sepharose gradient fractions is cat-
alytically active. The specific activity of caput sperm
PP1
g
2, not bound to sds22, following Superose 6 column
purification, is 567 U/mg protein. The enzyme at this stage
of purification (78-fold purification, compared with crude
extracts, Table 2) is not expected to be homogenous. Our
goal here was not purification of the enzyme to homoge-
neity but to assess whether the enzyme was bound to
sds22 or some other regulatory protein. The apparent mo-
lecular weight of the active subunit of the enzyme eluting
through the Superose 6 column is 39 kDa in correspon-
dence with the calculated molecular weight (39 kDa)
based on its amino acid sequence. In caput sperm, active
PP1
g
2 is not bound to sds22 or any other regulatory pro-
tein. One of the reasons for the high protein phosphatase
activity in caput extracts is due to this 39-kDa free cata-
lytic subunit form of PP1
g
2.
Our studies also show that a portion of PP1
g
2 in both
caput and caudal sperm extracts is bound to DEAE-cellu-
lose. Specific activities of PP1
g
2 released from DEAE-cel-
lulose are 9.6 and 2.18 U/mg protein in caput and caudal
sperm, respectively (Tables 1 and 2). We have not described
further purification of this DEAE-cellulose bound form of
sperm PP1
g
2 in this study. These results have been pre-
sented elsewhere [18, 19].
One of the important questions emerging from this study
is why sds22 from caput spermatozoa is not bound to
PP1
g
2. Caput sperm sds22 elutes as a 60-kDa species
through the Superose 6 column (Fig. 5A). This 60-kDa spe-
cies consists of sds22 (43 kDa) and another 17-kDa protein
(p17) (Fig. 5, B and C). This suggests that sds22 may exist
as a complex with p17 in caput spermatozoa. It is therefore
possible that p17, bound to sds22, prevents sds22 from
binding to PP1
g
2. Caput sperm sds22 complexed to p17 is
also unable to inhibit recombinant PP1
g
2 in vitro (Fig. 6),
1578
MISHRA ET AL.
presumably because it cannot bind to it. Partially purified
caput sperm PP1
g
2 behaves like recombinant PP1
g
2 with
respect to inhibition by calyculin A and inhibitor I2 and
binding to microcystin. Thus, the reason for inability of
sds22 to bind to PP1
g
2 probably is not due to a modifi-
cation of the PP1
g
2 subunit but most likely is due to the
fact p17 bound to sds22 prevents formation of the sds22-
PP1
g
2 complex. Dissociation of the sds22-p17 complex
and sds22-PP1
g
2 complex formation may be caused by
phosphorylation or some other chemical modification of
sds22 or p17. Alternatively p17 may be proteolyzed during
epididymal sperm maturation (i.e., p17 may be absent in
caudal spermatozoa). Molecular characterization of p17,
development of antibodies against p17, and determination
of the status of p17 in caudal spermatozoa will help us to
explore mechanisms underlying sds22 dissociation from
p17 followed by binding to PP1
g
2 during epididymal
sperm development.
Our data conclusively demonstrate that purified caput
sds22 is free of PP1
g
2. However, coelution of p17 and
sds22 does not necessarily imply that they are complexed
to each other (Fig. 5). It could be argued that p17 may not
be bound to sds22 but is a protein contaminant coeluting
with sds22 in the Superose 6 column. Other mechanisms
may be responsible for the development of sds22 binding
to PP1
g
2. It may be possible that sds22 phosphorylation
may be necessary for its binding to PP1
g
2 (i.e., sds22 may
be phosphorylated in caudal but not caput spermatozoa).
Insights into these questions might have been provided if
we were able to immunoprecipitate sds22 from sperm ex-
tracts. However, antibody preparations against two regions
in the carboxy terminus region of sds22 were unsuitable for
immunoprecipitation [12].
Whatever the reason for the lack of binding of sds22 to
PP1
g
2 may be, our data clearly show that all of sds22 in
caudal sperm extracts is bound to PP1
g
2, whereas in caput
sperm extracts, sds22 is free of any detectable PP1
g
2. A
consequence of this difference is that a proportion of caput
sperm PP1
g
2 is in its free (39 kDa) catalytically active
form, with a specific activity of 567 U/mg protein after
partial purification through Superose 6 column (Table 2).
Thus, one of the reasons for the higher PP1
g
2 activity in
caput than in caudal spermatozoa is that PP1
g
2isnot
bound to sds22.
Although sds22 is present in yeast and is ubiquitous in
animal cells [20], several features of sperm sds22 are
unique. In somatic cells, sds22 is localized to the nucleus
and only a small proportion of cellular PP1 is bound to
sds22 [17]. In spermatozoa, sds22 and sds22-bound PP1
g
2
are cytoplasmic [12]. Unlike complexes of sds22 with so-
matic cell isoforms of PP1 [16], sperm PP1
g
2-sds22 com-
plex does not bind to microcystin [12]. This suggests that
PP1
g
2 binding to sds22 may involve a unique amino acid
domain in PP1
g
2. Finally, in yeast, PP1 binding to sds22
activates the enzyme [20], whereas in spermatozoa, sds22-
bound PP1
g
2 is inactive. These differences suggest that
unique isoform- or sperm-specific mechanisms may operate
with respect to sds22 regulation of PP1
g
2. Furthermore, the
significance of sds22-bound PP1 in somatic cells is unclear
because it is not known whether free sds22 is found in
somatic cells. It is therefore also unclear whether sds22
binding to PP1 is regulated. Our studies are the first to show
that a significant change in the composition of sds22 occurs
during sperm development.
Studies described here clearly show that a portion of
PP1
g
2 in motile caudal epididymal spermatozoa is inac-
tive and present as a heterodimer with sds22. In immotile
caput spermatozoa, sds22 is not bound to PP1
g
2 but is
either free or bound to a 17-kDa protein. Therefore, a por-
tion of caput sperm PP1
g
2 is in its catalytically active and
free form. It is therefore very likely that the reason for the
higher PP1
g
2 activity in caput, compared with caudal
spermatozoa [1–3] is due to the inability of sds22 to bind
and inactivate PP1
g
2. That is, the ability of sds22 to bind
to PP1
g
2 develops during sperm maturation. We hypoth-
esize that the change in binding partners of sds22 from
p17 to PP1
g
2 and the development of the binding capacity
of sds22 for PP1
g
2 are key biochemical events responsible
for the decline in protein phosphatase activity during ep-
ididymal sperm maturation and motility initiation. Studies
are underway to determine molecular mechanisms and in-
tracellular signals regulating sds22 binding to PP1
g
2 dur-
ing sperm development.
ACKNOWLEDGEMENTS
We thank Dr. Balwant Khatra, University of California Long Beach,
for his generous supply of phosphorylase b and phosphorylase kinase. We
also thank Shannan Jack, Brian Sapola, John Ferrara, and Kimberly Mey-
ers for their assistance in the laboratory and useful discussions.
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