www.elsevier.com/locate/ydbio

Developmental Biology 2

Tandem mass spectrometry identifies proteins phosphorylated by

cyclic AMP-dependent protein kinase when sea urchin sperm

undergo the acrosome reaction

Yi-Hsien Sua,*, Sheng-Hong Chenb,c, Huilin Zhoub,d, Victor D. Vacquiera

aCenter for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,

University of California San Diego, La Jolla, CA 92093-0202, USAbLudwig Institute for Cancer Research, University of California San Diego, La Jolla, CA 92093-0359, USA

cDivision of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0359, USAdDepartment of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093-0359, USA

Received for publication 27 April 2005, revised 17 May 2005, accepted 6 June 2005

Available online 20 July 2005

Abstract

The exocytotic acrosome reaction (AR), which is required for fertilization, occurs when sea urchin sperm contact the egg jelly (EJ) layer.

Among other physiological changes, increases in adenylyl cyclase activity, cAMP and cAMP-dependent protein kinase (PKA) activity occur

coincident with the AR. By using inhibitors of PKA, a permeable analog of cAMP and the phosphodiesterase inhibitor IBMX, we show that

PKA activity is required for AR induction by EJ. A minimum of six sperm proteins are phosphorylated by PKA upon exposure to EJ, as

detected by a PKA substrate-specific antibody. The phosphorylation of these proteins and the percentage of acrosome reacted sperm can be

regulated by PKA modulators. The fucose sulfate polymer (FSP), a major component of EJ, is the molecule that triggers sperm PKA

activation. Extracellular Ca2+ is required for PKA activation. Six sperm proteins phosphorylated by PKA were identified by tandem mass

spectrometry (MS/MS) utilizing the emerging sea urchin genome. Based on their identities and localizations in sperm head and flagellum, the

putative functions of these proteins in sperm physiology and AR induction are discussed.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Fertilization; Acrosome reaction; Tandem mass spectrometry; Cyclic nucleotides; Invertebrate reproduction

Introduction

For successful fertilization, sea urchin sperm must

activate flagellar motility, swim to the egg, undergo the

acrosome reaction (AR), bind to the egg vitelline layer,

penetrate the vitelline layer and fuse with the egg plasma

membrane (Vacquier, 1998). The AR of sea urchin sperm

involves the exocytosis of the acrosomal vesicle and the

polymerization of actin to form the acrosomal process. The

EJ-induced AR depends on a net influx of Ca2+ and Na+ and

a net efflux of K+ and H+ (Darszon et al., 2001, 2005; Neill

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2005.06.007

* Corresponding author. Fax: +1 858 534 7313.

E-mail address: yhsu@ucsd.edu (Y.-H. Su).

and Vacquier, 2004). Among other changes, EJ increases

sperm adenylyl cyclase activity 50-fold (Watkins et al.,

1978), elevates cAMP levels over 100-fold and increases

PKA activity 4- to 8-fold (Garbers et al., 1980; Garbers and

Kopf, 1980).

In mammalian sperm, PKA induces the phosphorylation

of several proteins required for capacitation, a series of

changes that enable sperm to undergo the AR (Harrison,

2004; O’Flaherty et al., 2004; Visconti et al., 1995). PKA is

also involved in the human sperm AR, induced by the egg

zona pellucida (Bielfeld et al., 1994), progesterone (Harri-

son et al., 2000), follicular fluid and oviductal fluid (De

Jonge et al., 1993) and the calcium ionophore A23187

(Lefievre et al., 2002). Gene deletion of the PKA catalytic

subunit, Ca2, results in infertile male mice (Nolan et al.,

85 (2005) 116 – 125

Y.-H. Su et al. / Developmental Biology 285 (2005) 116–125 117

2004). Ca2 is required for the enhancement of sperm

motility by bicarbonate. However, whether sperm of the

Ca2 knock out mice can undergo the AR remains unknown.

In both sea urchins and mammals, although PKA

pathways seem to be generally important in sperm

physiology, preparing the sperm for fertilization, few

proteins have been identified as substrates for PKA

phosphorylation (Bracho et al., 1998; Harrison, 2004;

Porter et al., 1988; Tash and Bracho, 1999). Here we

show that PKA-mediated phosphorylation of sea urchin

sperm proteins is required for the EJ-induced AR and we

use tandem mass spectrometry to identify six of these

phosphoproteins.

Materials and methods

Materials

Alexa Fluor 488 phalloidin, Alexa Fluor 546 goat

anti-rabbit IgG, fura-2 AM and Pluronic F-127 were from

Molecular Probes. H-89 and myristoylated protein kinase

A inhibitor peptide, 14–22 amide, were from Calbio-

chem. H-85 was from Seikagaku Corporation. cAMP-AM

and protease inhibitor cocktail were from Sigma. Phos-

pho-(Ser/Thr) PKA substrate antibody was from Cell

Signaling Technology (Catalog number 9621). Speract

was from Peninsula Laboratories. Anti-bindin antibody

was prepared as described (Moy and Vacquier, 1979). All

other reagents and chemicals were from Sigma. H-89 and

H-85 were made as 10 mM stock solutions in DMSO

and diluted into sperm suspensions so that the final

DMSO concentration was 0.3%. The 0-AM control also

contained 0.3% DMSO.

Gametes

Sea urchin (Strongylocentrotus purpuratus) gametes

were spawned by injection of 0.5 M KCl into adults.

Undiluted semen was collected and kept on ice for no longer

than 24 h. Egg jelly (EJ), fucose sulfate polymer (FSP) and

EJ sialoglycans (SG) were prepared and quantified as

described (Hirohashi and Vacquier, 2002).

Acrosome reaction assay

Undiluted semen was diluted 1:100 in ASW (486 mM

NaCl, 10 mM CaCl2, 10 mM KCl, 27 mM MgCl2, 29 mM

MgSO4, 2.5 mM NaHCO3 and 10 mM HEPES, adjusted

to pH 8.0 with 1 N NaOH). Sperm suspensions were

mixed with various reagents before exposure to EJ (15-C)for 2 min. Fifty-microliter sperm suspensions were fixed

for 30 min by adding 750 Al of 3% paraformaldehyde in

ASW. A recently published method to score AR (Bier-

mann et al., 2004) was slightly modified as follows. Fixed

sperm were washed twice by resuspension in 500 Al of PBS

followed by a 2-min centrifugation at 5000 � g. The sperm

pellet was then stained with 0.4 units Alexa Fluor 488

phalloidin in 100 Al 1 mg/ml BSA in PBS for 2 h in the

dark. The cells were washed three times with 1 ml PBS and

resuspended in 70% glycerol in PBS. Acrosome reacted

sperm were then scored by fluorescence microscopy.

Immunoblots

For whole sperm protein preparation, sperm suspensions

were precipitated with acetone (80% final concentration),

sedimented by centrifugation for 5 min at 21,000 � g and

the pellet dissolved in 10% SDS. Isolation of sperm heads

from flagella was done as previously described (Vacquier

and Hirohashi, 2004). Sperm proteins were separated on

4–15% SDS/PAGE precast gels (BioRad) and transferred

to PVDF. The PVDF membranes were probed with the

phospho-(Ser/Thr) PKA substrate antibody, detected with

an HRP-conjugated goat anti-rabbit secondary antibody

(following the manufacturer’s instructions) and developed

with SuperSignal West Dura Extended Duration Substrate

(Pierce).

Measurement of intracellular Ca2+

Sperm were loaded with fura-2 AM and intracellular

Ca2+ was measured as previously described (Darszon et al.,

2004; Su and Vacquier, 2002). Briefly, undiluted semen was

diluted 5-fold in ASWand incubated with 12 AM fura-2 AM

overnight on ice in the dark. Free fura-2 AM was removed

by centrifugation at 1000 � g for 10 min at 4-C. The cell

pellets were washed twice with ASW. Ca2+ measurements

were at 15-C under constant stirring in a FluoroMax-2

fluorometer with excitation at 340 and 380 nm and emission

at 500 nm.

Identification of proteins by MS/MS

For immunoprecipitation, EJ-treated sperm were

extracted for 30 min in RIPA buffer (1% NP40, 1%

sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M

sodium phosphate pH 7.2, 2 mM EDTA, 50 mM NaF,

0.2 mM sodium vanadate, 100 nM okadaic acid, 10 Ag/ml aprotinin, 1 mM benzamidine and 1:100 dilution of

protease inhibitor cocktail). The RIPA-solubilized proteins

were obtained after centrifugation at 26,000 � g for 90

min. Two hundred microliters supernatant (2 mg/ml) was

precleared by incubation with 2 Al of normal rabbit IgG

(100 Ag/ml) and 20 Al of 50% Protein-A Sepharose

CL4B beads (Amersham Biosciences) for 2 h. The

precleared supernatants were incubated overnight with

the phospho-(Ser/Thr) PKA substrate antibody at a 1:100

dilution of the commercial stock. Twenty microliters of

50% Protein-A Sepharose CL4B beads was added to the

antibody-supernatant mixture and incubated for 2 h.

Precipitated immunocomplexes, bound to the Protein-A

Y.-H. Su et al. / Developmental Biology 285 (2005) 116–125118

beads, were washed five times in RIPA buffer and the

bound proteins eluted by boiling in Laemmli sample

buffer. After the proteins were separated by SDS–PAGE, the

bands were visualized by silver staining (Shevchenko et al.,

1996). Specific protein bands were excised, in-gel digested

with trypsin and the peptides were extracted and analyzed by

microcapillary liquid chromatography and tandem mass

spectrometry (Zhou et al., 2004). An in-house MS system

consisting of an HPLC and LCQ-ion trap mass spectrometer

(Thermo Finnigan) was used. The SEQUEST software

package (Han et al., 2001) was used to analyze tandem mass

spectra using a sea urchin ORF database (Neill, 2005) with no

restriction on the protease used. Only tryptic peptides

identified after database searches were further manually

inspected for correct identification of the expected peptide

fragments in the tandemmass spectra. The ORF database was

constructed by downloading the assembly of the sea urchin

genome from the Human Genome Sequencing Center at

Baylor College of Medicine (http://www.hgsc.bcm.tmc.edu/

projects/seaurchin/).

Fig. 1. PKA modulators regulate the EJ-induced AR. (A) Alexa Fluor 488

phalloidin was used to visualize the AR, four reacted sperm being shown

with rod-like acrosomal processes. Two non-acrosome reacted sperm are

indicated by arrowheads. (B) The PKA inhibitor H-89 blocks the EJ-

induced AR. Sperm were incubated with various concentrations of H-89

(filled circles) or H-85 (open circles) for 2 min before the addition of EJ

(final concentration 708 ng fucose/ml). Two minutes after EJ addition,

sperm were fixed and stained with Alexa Fluor 488 phalloidin for AR

scoring. At 30 AM H-89, AR dropped from 96% to 13%. In 30 AM H-85,

AR dropped from 96% to 77%. (C) The myristoylated protein kinase A

inhibitor 14–22 amide blocks the EJ-induced AR. Sperm were incubated

with various concentrations of the myristoylated peptide for 1 h at 15-C

before treatment for 2 min with EJ. (D) IBMX and cAMP-AM induce the

AR. Sperm were incubated with the permeabilizing reagent Pluronic F-127

(final concentration 0.5%), 100 AM IBMX, with or without 120 AM cAMP-

AM for 4 h at 15-C. At least 200 sperm cells were counted for each sample.

The error bars indicate standard errors from at least three independent

experiments.

Results

Effects of PKA modulators on the acrosome reaction

Acrosome reacted and non-acrosome reacted sperm can

be distinguished by fluorescent phalloidin staining (Bier-

mann et al., 2004). Completely acrosome reacted sperm

have a ¨1 Am long, rod-like acrosomal process labeled with

phalloidin, whereas non-acrosome reacted sperm show a

circular dot of weak phalloidin staining (Fig. 1A). Preincu-

bation of sperm for 2 min with the PKA inhibitor H-89

inhibits the EJ-induced AR in a dose-dependent manner

(Fig. 1B). At 30 AM of H-89, only 13% AR occurs

compared to 96% in the no inhibitor control (spontaneous

AR are usually about 4%). H-85 closely resembles H-89 in

its chemical structure, but is not specific to PKA and has

similar inhibitory effects on other kinases (Chijiwa et al.,

1990). Therefore, H-85 serves as a negative control for H-

89. H-85 at 30 AM results in 77% AR compared to 96% in

the control (Fig. 1B). Myristoylated PKA peptide inhibitor,

14–22 amide, was also used to test for involvement of PKA

in the AR. Incubation of sperm with this inhibitor for 1 h

before adding EJ also blocks the EJ-induced AR in a dose–

dependent manner. At 80 AM, it lowers the EJ-induced AR

to 8% (Fig. 1C). Conversely, 100 AM IBMX (1-methyl-3-

isobutylxanthine), a phosphodiesterase inhibitor which

increases cyclic nucleotide concentrations, induces the AR

to about 40% of the cells. Co-incubation in IBMX and the

membrane permeable form of cAMP (120 AM cAMP-AM)

further increases the AR to about 81% (Fig. 1D). These

results show that PKA inhibitors block the EJ-induced AR,

whereas PKA activators induce the AR. From these data we

conclude that PKA activity is required for the sea urchin

sperm AR.

Sperm protein phosphorylation induced by egg jelly

Phosphorylation of sperm proteins by PKA was

detected with the phospho-(S/T) PKA substrate antibody.

This antibody detects proteins containing a phospho-Ser/

Thr residue in RXXT or RRXS motif (where X is any

amino acid). In sperm suspensions without EJ, there is

only one major phosphoprotein at 100 kDa recognized

by the antibody under these conditions of protein load

and antibody concentration. Although there is experi-

mental variation in the phosphorylation pattern of minor

protein bands, upon treatment with EJ, several distinct

bands, including those of relative molecular masses 320,

230, 130, 75, 70, 43 and 22 kDa, are almost always

detected with this antibody. Phosphorylation of these

proteins is induced in 5 s after EJ addition (15-C). Thesame blot was stripped and reprobed with an anti-bindin

rabbit antibody to show equal protein loads in each lane

(Fig. 2).

Fig. 2. Time course of EJ-induced protein phosphorylation by PKA. Sperm

were incubated with EJ and precipitated with 80% acetone after the time

shown on top (from 5 s to 8 min) and resuspended in 10% SDS. Eight

micrograms of protein was loaded in each lane on a 4–15% gradient gel

and Western blotting was performed with the phospho-(Ser/Thr) PKA

substrate antibody at a 1:5000 dilution (upper panel). The same blot was

stripped and reprobed with the anti-bindin antibody as a loading control at a

1:50,000 dilution (lower panel). Molecular mass standards are indicated on

the left in kilodaltons. Under these conditions of detection, only one protein

at ¨100 kDa is phosphorylated before treatment with EJ.

Y.-H. Su et al. / Developmental Biology 285 (2005) 116–125 119

The effects of PKA inhibitors and activators were then

tested on sperm protein phosphorylation. H-89 at 30 AMtotally blocks the EJ-induced phosphorylation of sperm

proteins (Fig. 3A). Similar effects are observed with the

myristoylated peptide inhibitor at 80 AM, although the

phosphorylation of the 100-kDa protein also decreased

(Fig. 3B). IBMX alone partially induces sperm protein

Fig. 3. PKA modulators regulate EJ-induced sperm protein phosphorylation. (A)

with sperm for 2 min before EJ treatment for 2 min. Eight micrograms of protei

performed with phospho-(Ser/Thr) PKA substrate antibody. (B) The myristoy

concentrations (AM) was incubated with sperm for 1 h before EJ addition for 2 m

gels. (C) Sperm were incubated with 0.5% Pluronic F-127, 100 AM IBMX with o

the same phosphorylation pattern as seen in EJ.

phosphorylation (Fig. 3C), which explaining why IBMX

induces AR to only 40% (Fig. 1D). Co-incubation of

IBMX and cAMP-AM induces sperm protein phosphor-

ylation with a pattern similar to EJ-induced phosphor-

ylation (Fig. 3C). These results support the hypothesis

that protein phosphorylation by PKA is required for the

sea urchin sperm AR.

FSP and extracellular Ca2+ trigger protein phosphorylation

by PKA

Sea urchin EJ is composed of polysaccharides, glycopro-

teins and peptides. We tested what component of EJ triggers

the phosphorylation of sperm proteins by PKA. Trypsinized

or boiled EJ still induce sperm protein phosphorylation (Fig.

4A), indicating that the component of EJ inducing phosphor-

ylation is probably not protein. FSP is a major polysaccharide

of EJ that induces the AR (SeGall and Lennarz, 1979;

Vacquier and Moy, 1997). Purified FSP also induces sperm

protein phosphorylation and the phosphorylation pattern is

the same as that induced by EJ (Fig. 4A). Also, sialic acid-rich

glycans (sialoglycans, SG) from EJ, and the sperm respiration

activating peptide, speract (1 AM for 2 min), do not induce

sperm protein phosphorylation. In the presence of GTP,

speract (100 AM for 10 min) will induce phosphorylation in

Lytechinus pictus sperm when isolated sperm membranes are

labeled with 32P (Bentley et al., 1987). At higher concen-

trations (100 AM) and longer incubation time (10 min), we

found that several phosphoproteins were detected by our

PKA substrate antibody. However, the phosphorylation

pattern induced by speract is different from the EJ- and

FSP-induced patterns (data not shown). Therefore, as the AR

inducer in EJ, FSP is also the inducer of sperm protein

phosphorylation as detected by this antibody.

Various concentrations of H-89 (AM indicated above lanes) were incubated

n was loaded per lane on a 4–15% gradient gel and Western blotting was

lated protein kinase A inhibitor peptide 14–22 amide at the indicated

in before extracting the cells in 80% acetone and preparing the samples for

r without 120 AM cAMP-AM for 4 h. Treatment with both reagents yielded

Fig. 5. H-89 blocks Ca2+ entry into sperm. Fura-2 AM-loaded sperm were

used for intracellular Ca2+ measurements. Various concentrations of the

PKA inhibitor H-89 were incubated with sperm for 2 min before the

addition of EJ at the arrow. At 30 AMH-89 there was no detectable increase

in intracellular Ca2+ when EJ was added. Data are from a representative

experiment repeated three times. The Ca2+ signal is presented as the

340-nm/380-nm ratio.

Fig. 6. Cellular locations of the phosphoproteins. Sperm heads and flagella

were separated by homogenization and differential centrifugation. Eight

micrograms of head (H) or flagellar (F) proteins was subjected to SDS–

PAGE and Western blotting with the phospho-(Ser/Thr) PKA substrate

antibody.

Fig. 4. FSP and Ca2+ are required for sperm protein phosphorylation by

PKA. (A) FSP is the component in the EJ that induces sperm protein

phosphorylation by PKA. Different components isolated from EJ were used

to test their ability to induce the phosphorylation of sperm proteins. Sperm

were incubated 2 min with EJ at 708 ng fucose/ml, EJ trypsinized for 4 h

(EJ/Tryp), EJ boiled for 5 min (EJ/Boiled), fucose sulfate polymer (FSP) at

10 Ag/ml, sialoglycans (SG) at 10 Ag heparin/ml or the 10 amino acid EJ

peptide speract at 1 AM. The pattern of phosphoproteins in SG and speract

was the same as the non-EJ-treated control samples. (B) Ca2+ is required for

EJ-induced sperm protein phosphorylation by PKA. Sperm were suspended

in ASW with (lane 2) or without EJ (lane 1) addition. In lane 3, EJ was

added into a sperm suspension in CaFSW. In lane 4, sperm were incubated

with 10 AM ionomycin for 5 min in ASW. Eight micrograms of protein was

loaded per lane and subjected to SDS–PAGE and Western blotting using

the phospho-(Ser/Thr) PKA substrate antibody.

Y.-H. Su et al. / Developmental Biology 285 (2005) 116–125120

Because extracellular Ca2+ is a necessity for AR induction,

we also tested the effect of Ca2+ influx on sperm protein

phosphorylation by PKA. Sperm protein phosphorylation by

EJ does not occur when sperm are suspended in Ca2+-free

seawater (Fig. 4B). The Ca2+ ionophore, ionomycin, which is

a potent AR inducer, also induces sperm protein phosphor-

ylation when sperm are in ASW, although compared to EJ,

the phosphorylation is weaker (Fig. 4B). Two pharmacolog-

ically distinct Ca2+ channels are required for AR induction.

The first channel, opening 1 s after EJ treatment, is blocked

by nifedipine. The second channel, opening 5 s after EJ

treatment, is blocked by Ni2+. Normally, the activity of the

first, transiently opened channel opens the second, long

duration (sustained) channel. It is the opening of the second

channel that is mandatory for induction of the AR (Darszon et

al., 2001; 2005; Gonzalez-Martinez et al., 2001). In our

experiments, both nifedipine and Ni2+ inhibit sperm protein

phosphorylation by PKA (data not shown). These results

show that Ca2+ influx is required for sperm protein

phosphorylation by PKA just as it is for AR induction.

Although a pH increase of 0.25 units is also required for AR

induction, addition of 2.5–20 mM NH4Cl, which will

increase intracellular pH above permissive levels, does not

induce sperm protein phosphorylation (data not shown).

The effect of the PKA inhibitor H-89 on the EJ-

induced Ca2+ influx into sperm was also tested. Fura-2

AM-loaded sperm were used for intracellular Ca2+

measurements. Preincubation of sperm with H-89 for 2

min before EJ addition blocks the Ca2+ influx in a dose-

dependent manner (Fig. 5). The sustained influx after EJ

addition is clearly seen in the 0-AM H-89 control. As the

H-89 concentration increases the opening and closing of

the first transient channel is seen, as for example at 7.5

AM H-89. At this concentration of H-89, the sustained

second channel appears to be inhibited. At 15 AM a very

low level of activity of the first channel is still seen, but

the second channel appears to be totally inhibited. At 30

AM, H-89 completely blocks both Ca2+ channels. Thus,

H-89 appears to inhibit the second channel more than the

first.

Localizations of the phosphoproteins in sperm

To study the location of the sperm phosphoproteins, EJ-

treated sperm were homogenized and sperm head and

flagellum separated by differential centrifugation. Immuno-

Fig. 7. Immunoprecipitation of sperm phosphoproteins. Proteins were

extracted after sperm were incubated 2 min with (C, D) or without (A, B)

EJ. Immunoprecipitation was performed using normal rabbit IgG as a

negative control (A, C) or the PKA substrate antibody (B, D). The number

of proteins subjected to MS/MS analysis were indicated at right. Molecular

weight standards were indicated at left in kilodaltons.

Y.-H. Su et al. / Developmental Biology 285 (2005) 116–125 121

blots revealed that the 230- and 130-kDa proteins are

localized on both sperm head and flagellum. The 70-kDa

protein is specific to the head, whereas the 100-kDa protein

is mostly in the flagellum (Fig. 6).

Identification of sperm phosphoproteins

The PKA substrate-specific antibody was used to

immunoprecipitate phosphoproteins extracted from EJ-

treated sperm. The precipitated proteins were subjected to

SDS–PAGE and the gels stained with silver nitrate (Fig. 7).

In the control IP lanes, in which normal rabbit IgG were

used, only the heavy chain of IgG is seen on the gel at

approximately 50 kDa (lanes A and D). Several distinct

protein bands, including those at 230, 130, 100, 75, 70 and

Table 1

Peptide sequences identified by MS/MS and the results of BLASTp searches aga

Band no. MW (kDa) Identified peptide

1 230 No peptide identif

2 130 R.VVEANSDAAG

R.VTGFIHLDGTR

K.WNTFVENVK

K.DTSEREDDKE

R.IILVIGGPGSGK

R.EVEQGAEFEK

R.VDDNEETI

K.FLEDDSWK

R.GTGGVDTAST

3 100 K.EISDVDPEFR

R.GIFEEQTSLDN

K.QQVALDVLSY

K.VAELVFSEFFQ

4 75 No peptide identif

5 70 K.EGVVSVEDGI

6 43 K.LPSGLTVASLE

R.VAPEEHPVLLT

43 kDa, are precipitated by the PKA substrate antibody after

sperm were treated with EJ (lane D). In contrast, only the

constitutively phosphorylated 100-kDa protein was precipi-

tated and other phosphoproteins were almost undetectable in

non-EJ-treated sperm (lane B). The proteins were excised

from the gel, digested with trypsin and the peptides

analyzed by microcapillary liquid chromatography and

tandem mass spectrometry. A sea urchin ORF database

(Neill, 2005) was than used for searching peptide sequences

that match to the mass spectra data.

Table 1 summarizes the peptide sequences identified and

the results of BLASTp searches against the non-redundant

protein database. Except for bands 1 and 4, which did not

yield a peptide sequence, all the other phosphoproteins were

identified. There are eight peptides from band 2 that match to

either adenylate kinase-1 or -5 (AK). One peptide from band

2 matches to sperm creatine kinase. Four peptides from band

3 match to either phosphodiesterase 5 or 11A. The peptide

from band 5 matches to an EGF receptor pathway substrate,

EPS8. Two peptides were identified from band 6. One

matches to mitochondrial ubiquinol–cytochrome c reductase

complex core protein 2 and the other matches to actin.

Discussion

The commercial antibody used in this study reacts with

phosphorylated T or S residues in the sequence RXXT or

RRXS (where X is any amino acid). An R must be at the

�3 position for the antibody to bind the phospho-S or -T

residue. The antibody does not recognize other potential

PKA phosphorylation sites such as KRXT, KRXS, KKXT

or KKXS. The antibody therefore reports on only a subset

of the total possible PKA phosphorylation sites. In

addition, the antibody can also identify certain sites

inst the non-redundant protein database

BLASTp result

ied

FVLDSFPK Adenylate kinase 5

Adenylate kinase 1

Adenylate kinase 5

EDVVAR Adenylate kinase 5

Adenylate kinase 5

Adenylate kinase 5

Adenylate kinase 1

Adenylate kinase 5

DGTFDISNLDR Creatine kinase

PDE11A

VVHK PDE11A

HATAQPDEVSK PDE5A

QGDLER PDE5A

ied

R EPS8

NNSPVSR Ubiquinol–cytochrome

c reductase complex core protein 2

EAPLNPK Actin

Y.-H. Su et al. / Developmental Biology 285 (2005) 116–125122

phosphorylated by protein kinase C (PKC). However, the

PKC inhibitor chelerythrine (8 AM) has no effect on the

EJ-induced AR of sea urchin sperm. Moreover, a phospho-

(Ser) PKC substrate antibody (Cell Signaling Technology,

Catalog number 2261) detects several phosphoproteins in

both EJ-treated and non-treated sperm and the PKC

phosphorylation pattern is indistinguishable between the

two (data not shown). These results indicate that PKA, and

not PKC, is involved in the protein phosphorylation and

AR induction in sea urchin sperm.

Although much work has been published on cAMP

metabolism in sea urchin sperm (Garbers, 1989; Garbers

and Kopf, 1980), the question of whether PKA activation is

required for AR induction by EJ remained unknown. In this

study, we show that the PKA inhibitors H-89 and the

myristoylated peptide inhibitor, 14–22 amide, inhibit the

EJ-induced AR. In addition, the phosphodiesterase inhibitor

IBMX and cAMP-AM can induce the sperm AR and the

stimulatory effects of each are additive when combined.

Sperm protein phosphorylation by PKA is induced within 5

s after exposure to EJ. The PKA substrates include proteins

of relative molecular masses of 320, 230, 130, 75, 70, 43

and 22 kDa. The 100-kDa protein is constitutively

phosphorylated without EJ treatment and even before the

sperm are diluted from fully concentrated semen into

seawater (data not shown). EJ activates PKA activity to

maximal levels within 15 s (Garbers et al., 1980) and the

AR occurs within 5 s (Darszon et al., 2001). Therefore, our

observation that sperm protein phosphorylation occurs by

5 s after EJ treatment is within the physiologically relevant

time scale of AR induction. Sperm protein phosphorylation

and the AR are also modulated by PKA inhibitors and

activators, indicating that these phosphoproteins probably

have roles in mediating the sea urchin sperm AR.

FSP is the major polysaccharide in EJ that is an absolute

requirement for AR induction (SeGall and Lennarz, 1979;

Vacquier and Moy, 1997). Purified FSP induces sperm

protein phosphorylation by PKA to the same extend as

crude EJ, reinforcing the involvement of PKA activation in

AR induction. Sialoglycans (SG) from EJ, and speract

(1 AM for 2 min), do not induce sperm protein phosphor-

ylation. Speract elevates cAMP concentrations in sperm

with an IC50 of 0.02 AM (Hansbrough and Garbers, 1981).

The amount of speract we used in the experiments, 1 AM =

1.5 � 106 molecules/sperm, should be enough to saturate

the speract receptors (¨6 � 104 receptors/cell; Nishigaki

and Darszon, 2000). Treatment of sperm with 100 AMsperact for 10 min showed the phosphorylation of mem-

brane proteins of 210, 100, 75, 70, 52, 48, 35, 30, 23 and 15

kDa (Bentley et al., 1987). Some of these proteins might be

those we have found in sperm treated with EJ. Extracellular

Ca2+ is a necessity for the AR and for sperm protein

phosphorylation by PKA. Although ionomycin can trigger

the sperm AR, it only partially induces sperm protein

phosphorylation, suggesting that sperm receptor occupancy

by FSP may be part of the transmembrane signaling cascade

leading to the activation of PKA. By using the PKA

inhibitor H-89, we show that inhibition of PKA blocks Ca2+

influx through the second of the two Ca2+ channels

activated in AR induction (Darszon et al., 2001, 2005).

Given that Ca2+ is required for PKA activation, and

inhibition of PKA blocks Ca2+ influx, the first transient

Ca2+ influx could activate PKA and one role of PKA

activation in sea urchin sperm might be to activate the

second, long duration, ‘‘sustained’’ Ca2+ channel.

The constitutively phosphorylated 100-kDa protein is

localized on sperm flagellum and is phosphodiesterase

(PDE) 5A, or 11A (Table 1). PDE5A is a cGMP-specific

PDE (Soderling and Beavo, 2000), whereas PDE11A can

catalyze both cAMP and cGMP (Fawcett et al., 2000).

cAMP and cGMP regulate sea urchin sperm physiology in

many ways, including sperm activation, motility, chemo-

taxis and the acrosome reaction (Darszon et al., 2001; 2005;

Garbers, 1989; Morisawa, 1994; Neill and Vacquier, 2004).

Sea urchin sperm are also one of the richest sources of

guanylyl cyclase, the enzyme is localized in the flagellum

and its activity is controlled by phosphorylation (Garbers,

1989; Ward et al., 1985). PDE activity of sea urchin sperm

is known to be restricted to the flagellum (Sano, 1976).

Inhibitor studies show that PDE1 and 4 are present in

human sperm (Fisch et al., 1998). Moreover, RT-PCR

studies revealed that washed human sperm contain an

extended pattern of PDE mRNA transcripts, including those

for PDE1, 2, 3, 4, 5 and 8 (Richter et al., 1999). The

regulation of PDE5 by PKA phosphorylation has been

reported. For example, phosphorylation of partially purified

recombinant bovine PDE5 causes a 50–70% increase in its

activity (Corbin et al., 2000). PDE5 from smooth muscle

cells can also be phosphorylated and activated by PKA in

vitro (Rybalkin et al., 2002). The phosphorylation of sea

urchin sperm flagellar PDE may be one mechanism to

regulate cyclic nucleotide concentrations.

The 130-kDa protein is distributed on both sperm head

and tail, and by MS/MS analysis, eight peptides identify it

as adenylate kinase (AK). This is probably the same 130-

kDa phosphoprotein previously identified in sea urchin

sperm (Bracho et al., 1998; Tash and Bracho, 1999).

Adenylate kinase is a ubiquitous enzyme, which catalyses

the reaction AMP + ATP 6 2ADP. The reaction constant is

approximately 1, meaning that the reaction can go in either

direction depending on the concentrations of the three

nucleotides (Schoff et al., 1989). AK is thus perfectly suited

to regulate ATP concentrations in sperm. AK activity has

been detected in the flagella of sea urchin (Brokaw and

Gibbons, 1973) and bovine sperm (Schoff et al., 1989),

working in the direction of ATP synthesis. To our know-

ledge, the phosphorylation of AK by PKA has not been

reported. The phosphorylation of AK induced by EJ might

regulate the activity of AK and the homeostasis of

adenosine nucleotides. Increasing ATP production might

be required for sperm to swim through the viscous jelly

layer surrounding the egg. One peptide from this protein

Y.-H. Su et al. / Developmental Biology 285 (2005) 116–125 123

identified by MS/MS matched to creatine kinase. In sea

urchin sperm, a 145-kDa creatine kinase is a structural

protein bound to both the flagellar plasma membrane and

the axoneme. The phosphocreatine shuttle hypothesis posits

that ADP, produced by dynein, is rephosphorylated to ATP

by this unusual phosphocreatine kinase. This is thought to

be the mechanism by which ATP is transported down the

sperm flagellum, 40 Am from the sperm mitochondrion

(Wothe et al., 1990). To our knowledge, the phosphor-

ylation of creatine kinase has not been reported, although

sea urchin sperm creatine kinase (GenBank accession no.

NM_214522) contains three PKA sites and all of which are

KRGT. It is possible that both AK and the 145-kDa creatine

kinase are immunoprecipitated by the PKA substrate anti-

body. However, since eight peptides from this protein match

to AK, and only one peptide is from creatine kinase, it is

also possible that creatine kinase in a contaminant in the

immunoprecipitation complex despite the washing of the

Protein-A beads after immunoprecipitation. This unusual

creatine kinase, together with AK, probably play major roles

in ATP regulation in swimming sea urchin sperm.

One peptide identified by MS/MS from the 70-kDa

sperm head phosphoprotein is homologous to Eps8,

originally identified as a substrate for the epidermal growth

factor receptor kinase (Fazioli et al., 1993). Subsequent

work showed that Eps8 regulates actin elongation and cell

motility by capping the barbed ends of actin filaments

(Croce et al., 2004; Disanza et al., 2004). The phosphor-

ylation of Eps8 by PKA has not been studied. The fact that

the 70-kDa protein is localized in the sperm head suggests it

may function in regulating actin polymerization to form the

acrosomal process.

Two peptides were identified by MS/MS from the 43-

kDa band 6 (Table 1). One is similar to ubiquinol–

cytochrome c reductase complex core protein 2 and the

other matches to actin. It is possible that both proteins are

present in band 6. In the mitochondrial respiratory chain, the

ubiquinol–cytochrome c reductase complex (cytochrome

bc1 complex) transfers electrons from ubiquinol to cyto-

chrome c. This complex consists of 10 different subunits

and the core protein 2 (48 kDa in human, GenBank

accession no. BC000484) is one of them. In yeast, the

function of the core protein 2 is thought to be in assembly of

the cytochrome bc1 complex (Zara et al., 2004). However, in

Neurospora crassa and the plant cytochrome bc1 complex,

the core proteins are mitochondrial processing peptidases

(MPP) which process nuclear encoded mitochondrial

proteins (Braun and Schmitz, 1995). Phosphorylation of

the core protein 2 might induce its ability to assemble the

cytochrome bc1 complex, hence activating the mitochon-

drial electron transfer chain to modulate ATP production.

cAMP-induced actin phosphorylation has been reported in

S49 lymphoma cells (Steinberg, 1980) and IPC-81 cells

(Hovland et al., 1999). In both cases, actin is co-transla-

tionally phosphorylated by PKA. In our experiments, actin

could have co-immunoprecipitated with the actin capping

protein EPS8 and thus be merely a contaminant. Alter-

natively, actin might be phosphorylated by PKA in sea

urchin sperm since all sea urchin actins have one PKA site

(RKYS) that could be recognized by the antibody used in

this study.

Sea urchins and other echinoderms are invertebrate

deuterostomes, the branch of animal evolution leading to

the vertebrates. Morphologically, sea urchin sperm are much

less complex than mammalian sperm. In addition, the sea

urchin genome is one-quarter the size of the human genome.

The sea urchin genome has been sequenced and an

assembly is in progress. There is an excellent chance that

discoveries regarding the molecular regulation of sea urchin

sperm cell physiology will be applicable to mammalian

sperm. In depth studies of each of the phosphoproteins

identified here will aid in the elucidation of the molecular

mechanism of fertilization.

Acknowledgments

We thank Dr. Bartholomew M. Sefton at the Salk

Institute, La Jolla, California, and Dr. Jean Y. J. Wang at

the University of California San Diego, La Jolla, California,

for valuable discussions. This research was supported by

National Institutes of Health Grant HD12986 to YHS and

VDV. S. Chen and H. Zhou are supported by NHGRI K22

HG002604 faculty transition award and the Ludwig Institute

for Cancer Research.

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