Protein phosphorylation in mammalian spermatozoa.

Abstract

Spermatozoa undergo a series of changes before and during egg binding to acquire the ability to fuse with the oocyte. These priming events are regulated by the activation of compartmentalized intracellular signalling pathways, which control the phosphorylation status of sperm proteins. Increased protein tyrosine phosphorylation is associated with capacitation, hyperactivated motility, zona pellucida binding, acrosome reaction and sperm-oocyte binding and fusion. The main tyrosine phosphorylated proteins during the course of capacitation and fertilization are localized to the flagellum, although tyrosine phosphorylation of less abundant proteins may also be regulated in the sperm head. Spermatozoa bound to the zona pellucida and fusing with the oocyte plasma membrane are characterized by a tyrosine phosphorylated flagellum. Protein phosphorylation in the flagellum is linked to hyperactivated motility in spermatozoa, but may also regulate additional functions involved in sperm-oocyte fusion. Factors involved in the appearance of phosphorylation more likely arise from the milieu surrounding the spermatozoa, but their uptake and processing are likely to be regulated differentially at specific steps within the female genital tract and during penetration of the egg vestments. One of these factors is glucose, the metabolic products of which (ATP and NADPH) appear to participate in signalling pathways by supporting a precise onset of tyrosine phosphorylation in the sperm flagellum leading to successful fertilization.

Full text

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Reproduction (2003) 125, 17–26
Review
Protein phosphorylation in mammalian spermatozoa
Franc ¸oise Urner1and Denny Sakkas2
1Clinic of Sterility, Department of Obstetrics and Gynecology, University Hospital of Geneva,
1211 Geneva 14, Switzerland; and2Department of Obstetrics and Gynecology, Yale University
School of Medicine, New Haven, CT 06520-8063, USA
Spermatozoa undergo a series of changes before and during egg binding to acquire the
ability to fuse with the oocyte. These priming events are regulated by the activation of
compartmentalized intracellular signalling pathways, which control the phosphorylation
status of sperm proteins. Increased protein tyrosine phosphorylation is associated with
capacitation, hyperactivated motility, zona pellucida binding, acrosome reaction and
sperm–oocyte binding and fusion. The main tyrosine phosphorylated proteins during the
course of capacitation and fertilization are localized to the flagellum, although tyrosine
phosphorylation of less abundant proteins may also be regulated in the sperm head.
Spermatozoa bound to the zona pellucida and fusing with the oocyte plasma membrane
are characterized by a tyrosine phosphorylated flagellum. Protein phosphorylation in
the flagellum is linked to hyperactivated motility in spermatozoa, but may also regulate
additional functions involved in sperm–oocyte fusion. Factors involved in the appearance
of phosphorylation more likely arise from the milieu surrounding the spermatozoa, but
their uptake and processing are likely to be regulated differentially at specific steps within
the female genital tract and during penetration of the egg vestments. One of these factors
is glucose, the metabolic products of which (ATP and NADPH) appear to participate in
signalling pathways by supporting a precise onset of tyrosine phosphorylation in the sperm
flagellum leading to successful fertilization.
Protein phosphorylation is a post-translational modifi-
cation of proteins that allows the cell to control various
cellular processes. In eukaryotic cells, most phosphoryla-
tion occurs on serine or threonine residues and to a lesser
extent on tyrosine residues. The phosphorylation state of
phosphoproteins is controlled by the activity of protein
kinases and phosphatases.
Mature spermatozoa have their own particular idio-
syncrasies as highly specialized cells. They are highly
compartmentalized, transcriptionally inactive and un-
able to synthesize new proteins. Therefore, it could
be argued that the reliance of mature spermatozoa on
protein phosphorylation as a means of altering their
function is greater than in many other types of cell.
During fertilization, sperm function is regulated by the
activation of intracellular signalling systems that control
protein phosphorylation. Serine/threonine and tyrosine
phosphorylation occur in spermatozoa, but only a few
phosphorylated proteins have been identified. Although
cAMP-dependent protein kinase A plays a central role in
sperm function and has been studied in detail (Visconti
Email: francoise.urner@hcuge.ch
and Kopf, 1998), knowledge about tyrosine kinases and
other seronine/threonine kinases remains limited.
The processes regulated by protein phosphorylation
include capacitation, hyperactivated motility and the
acrosome reaction, all of which are required for
spermatozoa to reach and fuse with the oocyte. In
general, protein phosphorylation in mammalian sperma-
tozoa has been studied by examining phosphorylated
proteins in sperm populations during capacitation and
after exposure to inducers of the acrosome reaction
using western blot analysis. This approach has led
to the accumulation of information on the molecular
weight of the phosphorylated proteins and their
regulation during capacitation and acrosome reaction.
Phosphorylation in individual spermatozoa has also
been examined by immunocytochemistry, which has
revealed compartmentalization of the phosphoproteins
in individual spermatozoa and heterogeneity in sperm
populations. Knowledge of the phosphorylation pattern
in fertilizing spermatozoa and its regulation during
gamete interaction remains one of the most important
issues in this area of investigation. This review
summarizes the accumulated knowledge concerning
regulation of protein tyrosine phosphorylation in the
c ?2003 Society for Reproduction and Fertility
1470–1626/2003

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18 F. Urner and D. Sakkas
ZP
(a) Capacitating
spermatozoa
(c) Sperm–oocyte fusion
(b) Spermatozoa
bound to ZP
MP
PP
Fused spermatozoa
Bound spermatozoa
non-phosphorylated domain
phosphorylated domain
Oolemma
Fig. 1. Protein tyrosine phosphorylation localized to the mouse
sperm flagellum during capacitation and interaction with the
oocyte. (a) During capacitation, tyrosine phosphorylation appears
first in the principal piece (PP) and then in the midpiece (MP) in
about 20% of spermatozoa, whereas 15% of spermatozoa remain
phosphorylated in the principal piece only. (b) After binding to the
zona pellucida (ZP), tyrosine phosphorylation is stimulated in the
midpiece,resultinginnearly100%ofspermatozoaphosphorylated
in the whole flagellum. (c) After binding to the oolemma,
tyrosine phosphorylation is also stimulated in the midpiece. Soon
after fusion, the spermatozoa undergoing decondensation present
tyrosine phosphorylation in the whole flagellum or restricted to the
principal piece. Tyrosine phosphorylation limited to the principal
piece in fused spermatozoa may be explained by a process of
dephosphorylation of the flagellum after fusion, culminating in the
dephosphorylation of the entire flagellum.
different sperm compartments during capacitation and
interaction of spermatozoa with the oocyte.
Protein tyrosine phosphorylation in the flagellum
It is necessary to differentiate the localization of the
tyrosine phosphorylated proteins in spermatozoa to
understandthelinkbetweenthedifferentphosphorylated
proteins andthecorresponding
function. Except in boars (Petrunkina et al., 2001;
Tardif et al., 2001), the flagellum appears to be
the principal sperm compartment presenting tyrosine
phosphorylated proteins. Immunocytochemistry has
been used to localize tyrosine phosphorylated proteins
to the flagellum in human (Naz et al., 1991; Carrera
et al., 1996; Leclerc et al., 1997), monkey (Mahony
and Gwathmey, 1999), hamster (Si and Okuno, 1999),
rat (Lewis and Aitken, 2001), and mouse (Urner et al.,
2001a) spermatozoa.
regulatedsperm
Protein tyrosine phosphorylation during capacitation
and interaction with the oocyte
Protein tyrosine phosphorylation increases in sper-
matozoa during capacitation in a number of species
including mice, humans, cattle, pigs, hamsters and cats
(Leyton and Saling, 1989; Visconti et al., 1995a; Leclerc
et al., 1997; Galantino-Homer et al., 1997; Kalab et al.,
1998; Pukazhenthi et al., 1998; Si and Okuno, 1999).
Protein tyrosine phosphorylation appears to be a
necessary prerequisite for a spermatozoon to fertilize an
egg (Visconti et al., 1995a; Urner et al., 2001).
Immunofluorescence was used to localize tyrosine
phosphorylated proteins
capacitation, zona pellucida binding, oolemma binding
and gamete fusion (Urner et al., 2001). A summary
of the changes in tyrosine phosphorylation in the
mouse flagellum (Fig. 1) shows that the proportion
of spermatozoa presenting phosphorylated proteins in
the whole flagellum increases with capacitation, and
phosphorylation in the principal piece is a prerequisite
for phosphorylation in the midpiece (Fig. 1a). A similar
increase has been observed in human spermatozoa but
was restricted to the principal piece (Sakkas et al.,
in press). In both mice and men, approximately 50%
of spermatozoaremain
capacitation, indicating that there are subpopulations
of spermatozoa that exhibit a different susceptibility
to undergo tyrosine phosphorylation. The possibility
that the phosphorylation-positive spermatozoa have a
competitive advantage to fertilize an egg remains to
be determined. This finding also has implications for
certain types of infertility in males, as the proportion
of spermatozoa in an ejaculate that are unable to
undergo the correct sequence of phosphorylation to
achieve capacitation may render them unable to fertilize
an egg.
Although tyrosine phosphorylation during capacita-
tion is not required for spermatozoa to bind to the
zona pellucida in an in vitro system (Pukazhenthi et al.,
1998), most mouse spermatozoa binding to the zona
pellucida display a tyrosine phosphorylated principal
piece, indicating that this pattern of phosphorylation is
totheflagellumduring
non-phosphorylatedafter

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Protein phosphorylation in mammalian spermatozoa19
beneficial for binding. Binding to the zona pellucida
maintains the principal piece phosphorylation and
promotes tyrosine phosphorylation in the midpiece
(Fig. 1b). Similarly, most of the spermatozoa bound
to the oolemma display a tyrosine phosphorylated
principal piece, whereas tyrosine phosphorylation in
the midpiece is stimulated and maintained until fusion
with the oocyte. Nearly all fused spermatozoa display
a tyrosine phosphorylated flagellum that is subsequently
dephosphorylated (Fig. 1c). In the absence of glucose,
both sperm–oocyte fusion and tyrosine phosphorylation
in the midpiece are inhibited, indicating that tyrosine
phosphorylation in the whole flagellum is mandatory for
fusion (Urner et al., 2001).
Significance of protein tyrosine phosphorylation
in the flagellum
Tyrosine phosphorylated proteins in the flagellum
are linked to sperm hyperactivated motility (Mahony
and Gwathmey, 1999; Nassar et al., 1999; Si and
Okuno, 1999), which is a motility pattern required for
spermatozoa to penetrate the cumulus and the zona
pellucida of the oocyte. Exposure of spermatozoa to
drugs that increase intracellular cAMP enhances both
hyperactivated motility and the proportion of spermato-
zoa presenting tyrosine phosphorylation in the flagellum
in human (Nassar et al., 1999), monkey (Mahony
and Gwathmey, 1999) and rat (Lewis and Aitken, 2001)
spermatozoa. Glucose, which is required for hyper-
activated motility (Cooper, 1984; Williams and Ford,
2001) and zona pellucida penetration (Urner and
Sakkas,1996a),promotestyrosinephosphorylationinthe
flagellum in mouse spermatozoa (Urner et al., 2001).
Hyperactivated motility and the associated tyrosine
phosphorylation in the flagellum are initiated during in
vitro capacitation,andtheirmaintenanceinspermatozoa
bound to the zona pellucida is of great importance
(Stauss et al., 1995) and explains why the zona pellucida
promotes the tyrosine phosphorylation of proteins in the
flagellum. After penetration through the zona pellucida,
the fertilizing spermatozoon binds to the oolemma
and membrane fusion occurs subsequently between
the equatorial segment of the sperm head and the
microvilli of the oocyte. In addition, the sperm flagellum
appears to fuse at multiple sites with the oocyte plasma
membrane (Simerly et al., 1993). This process requires
calcium (Yanagimachi, 1978) and glucose (Urner and
Sakkas, 1996b; Redkar and Olds-Clarke, 1999), but the
molecular events occurring during binding and fusion
have yet to be elucidated. In contrast to the events
during sperm penetration through the zona pellucida,
hyperactivated motility is not required for spermatozoa
to fuse with the oolemma. It is possible that the tyrosine
phosphorylated proteins in the flagellum are linked
to another sperm function important for both zona
pellucida and oocyte penetration and that a common
pathway is activated in spermatozoa interacting with
the zona pellucida and the oolemma. The identification
of the tyrosine phosphorylated proteins in the mouse
midpiece would provide further insight into the specific
role of this pattern of phosphorylation and how it
influences membrane fusion occurring between the
sperm equatorial segment and the oolemma.
Tyrosine phosphorylated proteins in the flagellum
In human spermatozoa, the A-kinase anchoring
proteins (AKAPs) localized to the fibrous sheath AKAP82,
its precursor pro-AKAP82, and FSP95 are the most
prominent tyrosine phosphorylated proteins during
capacitation (Carrera et al., 1996; Mandal et al., 1999),
indicating that their affinity for the regulatory subunit
of protein kinase A or other molecules is regulated
by their phosphorylation status. An 86 kDa calcium-
binding and tyrosine phosphorylation-regulated protein
(CABYR) is localized to the principal piece of human
spermatozoa (Naaby-Hansen et al., 2002). Tyrosine
phosphorylated CABYR acquires the capacity to bind
calcium during capacitation and may therefore play
a role in calcium sequestration and release in the
principal piece, establishing a link between tyrosine
phosphorylation and calcium, which is itself involved in
regulatingsperm-hyperactivatedmotility(HoandSuarez,
2001). Although both AKAPs and CABYR are associated
with the fibrous sheath, soluble proteins may also be
associated with motility, as exemplified by the 55 kDa
tyrosinephosphorylatedsolubleproteinlinkedtomotility
in bovine spermatozoa (Vijayaraghavan et al., 1997a).
Protein tyrosine phosphorylation in the sperm head
Protein tyrosine phosphorylation during capacitation
Tyrosine
represents a minor pattern of phosphorylation in mouse
spermatozoa. Non-capacitated spermatozoa are all
non-phosphorylated, except for about 5–10% which
display protein tyrosine phosphorylation restricted to the
acrosomal region (Leyton and Saling, 1989; Urner et
al., 2001). In contrast to tyrosine phosphorylation in
the flagellum, the proportion of spermatozoa displaying
a tyrosine-phosphorylated acrosome does not increase
with capacitation (Urner et al., 2001).
The only tyrosine-phosphorylated protein detected by
western analysis in non-capacitated mouse spermatozoa
is hexokinase, indicating that this enzyme is probably
the tyrosine-phosphorylated protein localized to the
acrosome in uncapacitated spermatozoa by immuno-
fluorescence. In addition, both the phosphorylation
status of hexokinase and the proportion of tyrosine
phosphorylated acrosome are constant during capa-
citation. In mice, the germ-cell specific hexokinase
(Kalab et al., 1994) is the major membrane tyrosine
phosphorylation inthesperm head

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20 F. Urner and D. Sakkas
phosphorylated protein (116/95 kDa, reducing/non-
reducing condition). Although hexokinase was localized
to the head, midpiece and principal piece (Travis et al.,
1998),thelocalizationandtheroleofthephosphorylated
form of the enzyme have not been determined.
The significance of tyrosine phosphorylation in the
head during capacitation is unclear, but does not
appear to be influenced by capacitation. In rats, tyrosine
phosphorylation in the acrosomal region is characteristic
of immature spermatozoa from the caput epididymidis
(LewisandAitken,2001).Thispatternofphosphorylation
decreases from 100 to 10–20% with maturation in the rat
epididymis and the acrosome-positive spermatozoa that
persist in the cauda epididymidis have been interpreted
as representative of remaining immature spermatozoa.
Protein tyrosine phosphorylation upon zona pellucida
interaction
A tyrosine phosphorylation step is necessary for
the acrosome reaction induced by the zona pellucida,
as inhibition of tyrosine phosphorylation prevents the
acrosome reaction (Leyton et al., 1992; Pukazhenthi
et al., 1998). In addition, exposure to progesterone,
which induces the acrosome reaction, promotes an
increase in tyrosine phosphorylation in the head of
human spermatozoa (Tesarik et al., 1993). Solubilized
zona pellucida has been used to observe an increase
in tyrosine phosphorylation of a 95 kDa protein in
mouse spermatozoa (Leyton and Saling, 1989; Leyton
et al., 1992). This protein, which is not hexokinase,
localizes to the head surface in the acrosomal region and
becomes tyrosine phosphorylated upon binding to the
zona pellucida (Leyton et al., 1995). In humans, tyrosine
phosphorylation of a protein of approximately 95 kDa
has also been observed after exposure to solubilized and
recombinant ZP3 (Burks et al., 1995; Brewis et al., 1998).
In contrast to the above studies, a high percentage of
mouse spermatozoa bound to intact zona pellucida do
not present any sign of tyrosine phosphorylation in the
acrosomal region (Urner et al., 2001). This discrepancy
mightbeexplainedifthetyrosinephosphorylatedprotein
localized to the head is considered as a less abundant
protein in which changes in phosphorylation are not
detected easily by conventional immunofluorescence. In
boars, the tyrosine phosphorylated plasma membrane
proteins bound to the zona pellucida were found to
be minor phosphorylated proteins compared with the
tyrosine phosphorylated proteins of the whole sperm
cells, even though they may still play a key role in sperm–
zona pellucida interaction (Flesch et al., 1999, 2001).
Protein tyrosine kinases in the sperm head
The 95 kDa sperm membrane protein that becomes
tyrosine phosphorylated upon binding to the zona
pellucida in mice possesses the characteristics of a
receptor protein tyrosine kinase (Leyton et al., 1992).
This protein displays intrinsic tyrosine kinase activity and
contains phosphotyrosine that increases upon binding
to ZP3; because of this property it has been referred
to as zona receptor kinase (ZRK). A similar protein,
Hu9, has been described in humans (Burks et al., 1995)
but the Hu9 cDNA used in this study encoded for
the human proto-oncogene c-mer instead of the zona
receptor kinase, bringing into question the existence of a
human homologue of mouse ZRK (Bork, 1996).
Receptorproteintyrosine
transmembrane proteins presenting an extracellular
binding domain and an intracellular tyrosine kinase
domain. Upon extracellular ligand binding, kinase
isactivated and phosphorylates
onthe RPTK itself(autophosphorylation)
other proteins. After autophosphorylation of RPTK,
intracellular signalling proteins such as phosphoinositide
3-kinase and phospholipase C? may be recruited to
the phosphorylated domain of RPTK and activated.
The targeting of a phospholipase C? to the plasma
membrane and its tyrosine phosphorylation-dependent
activationin mousespermatozoa
1996), and phosphoinositide 3-kinase activity operating
downstream of tyrosine phosphorylation in human
spermatozoa (Fisher et al., 1998) indirectly indicate
that a receptor protein tyrosine kinase is operative in
spermatozoa.
Non-receptor protein tyrosine kinases may also be
present in the sperm head, as c-yes, which is a member of
the nonreceptor Src family of tyrosine kinases, has been
detected in the human sperm head (Leclerc and Goupil,
2002). The activity of the c-yes kinase depends on cAMP,
indicating that tyrosine phosphorylation of proteins in
the sperm head results from the cross-talk between the
cAMP pathway and tyrosine-kinases, as it does in the
flagellum.
kinases(RPTK)are
tyrosine residues
oron
(Tomes
et al.,
Regulation of protein tyrosine phosphorylation
Tyrosine
spermatozoa in vitro, provided they are diluted in
culture medium supporting capacitation (Urner et al.,
2001) and freed of seminal plasma (Tomes et al.,
1998).Invivo,theappearanceofcapacitation-associated
tyrosine phosphorylation is probably regulated by sperm
binding to oviductal epithelial cells before fertilization
(Petrunkina et al., 2001). The oocyte itself triggers
changes in tyrosine phosphorylation that are linked
to the acrosome reaction and other functions related
to gamete fusion. The sequence of events responsible
for capacitation, including removal of cholesterol from
the sperm plasma membrane, increase in bicarbonate
uptake, activation of adenylate cyclase, and the cross-
talk between the cAMP pathway and protein tyrosine
phosphorylation have been described in reviews by
Visconti and Kopf (1998) and Visconti et al. (2002). This
phosphorylation occursspontaneously in

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Protein phosphorylation in mammalian spermatozoa21
section describes recent contributions to our knowledge
on protein kinases and glucose metabolism in regulating
tyrosine phosphorylation.
cAMP-dependent protein kinase A
The cAMP-dependent protein kinase A (PKA) plays
a central role in sperm capacitation, motility and the
acrosome reaction (Bielfeld et al., 1994; Visconti et al.,
1995b; Harrison et al., 2000; Skalhegg et al., 2002).
PKA regulates protein tyrosine phosphorylation in sper-
matozoa by either direct or indirect effects on tyrosine
kinase or phosphatases (Visconti et al., 2002). PKA
is a tetrameric enzyme composed of two regulatory
and two catalytic subunits. Two isoforms of the regu-
latory subunit (RI and RII) are present in spermatozoa
(Vijayaraghavan et al., 1997b; Visconti et al., 1997). The
activity of PKA is dependent on the amount of cAMP,
which is in turn regulated by adenylate cyclase and
phosphodiesterase. The binding of cAMP to the regu-
latory subunit promotes the dissociation and activation
of the catalytic subunits that catalyse phosphoryla-
tion on serine/threonine residues.
PKA is anchored to specific sperm compartments
through binding of its regulatory subunit to the AKAPs. In
general,AKAPsareimplicatedintheregulationofprotein
phosphorylation by the tethering of protein kinases and
phosphatases in close proximity to their target proteins
within specific cell compartments. Different types of
AKAP have been found in spermatozoa (for review, see
Moss and Gerton, 2001). AKAP82 (Carrera et al., 1994;
Johnson et al., 1997) and FSP95 (Mandal et al., 1999)
are both localized to the fibrous sheath of the principal
piece and anchor the RII subunit of PKA. AKAP110,
localized to the principal piece and the acrosomal
region (Vijayaraghavan et al., 1999), presents unique
characteristics in spermatozoa. In addition to binding
to the regulatory subunit of PKA, the sperm AKAP110
interacts with other signalling proteins (such as ropporin)
that may regulate motility independently of PKA (Carr
et al., 2001). Two other AKAPS have been identified
in the midpiece: S-AKAP84, which is associated with
mitochondria (Lin et al., 1995) and AKAP220, which is
anchored to cytoskeletal structures and binds both the RII
and RI subunits (Reinton et al., 2000). The localization
of AKAPs in all sperm compartments indicates that they
participate indirectly (through scaffolding of PKA or
other signalling molecules) in the regulation of tyrosine
phosphorylation in both the flagellum and the sperm
head. In addition to participating in the regulation
of protein phosphorylation in spermatozoa, AKAPs
are themselves phosphorylated on tyrosine residues in
humans (AKAP82, FSP95; Carrera et al., 1996; Mandal
et al., 1999) and on serine/threonine residues in mice
(AKAP 82; Johnson et al., 1997) during capacitation,
indicating that their functions are modified by their
phosphorylation status.
Mitogen-activated protein kinases
The mitogen-activated protein kinases (MAPK), also
knownasextracellularsignal-regulatedkinases(ERK),are
serine/threonine kinases involved in signal transduction
of several extracellular messengers. Their activity is
regulatedbyaphosphorylationcascadeinitiallytriggered
by the GTP-binding protein Ras or protein kinase C.
MAPK-kinase-kinase (Raf), which is the first enzyme of
this cascade, phosphorylates MAPK-kinases (MEK) on
serine/threonine, which in turn phosphorylates MAPK
on threonine and tyrosine residues. MAPK requires
phosphorylation on both threonine and tyrosine to
become active.
The MAPK pathway has been identified in sperma-
tozoa and appears to play a role in capacitation
(de Lamirande and Gagnon, 2002) and the acrosome
reaction (Luconi et al., 1998; du Plessis et al., 2001).
Thezona pellucida- and
induced acrosome reactions are prevented when MAPK
activity is inhibited (du Plessis et al., 2001; de
Lamirande and Gagnon, 2002). Progesterone promotes
the phosphorylation–activation of the MAPK isoform
ERK2 in human spermatozoa and its redistribution from
the post-acrosomal region to the equatorial segment
(Luconi et al., 1998). The MAPK isoform ERK2 (Luconi
et al., 1998), the adaptor protein Shc (Morte et al., 1998)
and Ras (Naz et al., 1992) have all been localized to the
sperm head, indicating that this pathway is required for
controlling protein phosphorylation in the sperm head.
Although the proteins phosphorylated by MAPK in
spermatozoa remain to be identified, de Lamirande
and Gagnon (2002) reported a 75 and 80 kDa
protein presenting a phosphorylated serine/threonine–
proline motif recognized by the MPM-2 antibody, which
is consistent with a MAPK-phosphorylated epitope.
MPM2-reactive proteins in the 77–85 kDa range have
been detected in the rabbit sperm midpiece and
connective piece in different species (Pinto-Correia
et al., 1994; Long et al., 1997) and may be components
of the centrosome in the connecting piece (Simerly
et al., 1999). Therefore, centrosomal proteins may
be considered as candidate substrates of MAPK in
spermatozoa. In addition, MAPK may phosphorylate
proteins that influence protein tyrosine phosphorylation
indirectly, as inhibition of MAPK prevents protein
tyrosine phosphorylation associated with capacitation
(de Lamirande and Gagnon, 2002).
lysophosphatidylcholine-
Protein phosphatases
Tyrosine phosphorylation is stimulated indirectly by
activation of phosphorylation on serine/threonine (by
PKA or MAPK) and, therefore, it is conceivable that
dephosphorylation by phosphatases of these residues
inhibits tyrosinephosphorylation.
specific protein phosphatase (PP) activities have been
Serine/threonine-