MOLECULAR REPRODUCTION AND DEVELOPMENT 28:150-157 (1991)

In Vitro Phosphorylation of Sea Urchin Sperm Adenylate Cyclase by Cyclic Adenosine Monophosphate-Dependent Protein Kinase LOUIS H. BOOKBINDER, GARY W. MOY, AND VICTOR D. VACQUIER Marine Biology Research Division, Scripps Znstitution of Oceanography, University of California, Sun Diego, La Jolla, California Addition of [ Y - ~ ~ P I A Tto P a ABSTRACT 2% Brii-78 40,OOOg supernatant of sea urchin sperm results in the CAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC).An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a K, for ATP of 5.2 pM and is maximally stimulated by 4-8 pM CAMP. The protein kinase inhibitors H8 (9 kM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine CAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the K, or , ,V, of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2+-activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by CAMP-dependent protein kinase.

Key Words: Fertilization, Invertebrates, Strongylocentrotus purpuratus

INTRODUCTION Phosphorylation of enzymatic and nonenzymatic proteins plays a key role in cellular regulation. Increases in the activity of CAMP-dependent protein kinase appear necessary for the activation and maintenance of flagellar motility in animal spermatozoa (Garbers and Kopf, 1980; Brokaw, 1987; Tash and Means, 1987; Lindemann and Kanous, 1989; Tash, 1989; Morisawa, 1987). A description of the proteins phosphorylated in sperm flagella and the role this modification plays in the motility process has yet to appear. Studies of flagel-

0 1991 WILEY-LISS, INC.

lar phosphoproteins are potentially important to the understanding of the motility process. We have shown (Bookbinder et al., 1990) that membranes of sea urchin spermatozoa contain a n Mr 190 kD protein, which is adenylate cyclase (AC). A monospecific polyclonal antiserum to this protein inhibits 94% of the AC activity in a n eluate from a calmodulin (CaM)-agarose column to which detergent-solubilized sea urchin sperm membrane proteins were applied. This antiserum also blocks 90% of the activity of horse sperm AC (Bookbinder e t al., 1990). Indirect immunofluorescence shows that the sea urchin sperm AC is highly concentrated on the anterior portion of the sperm flagellum (Bookbinder et al., 1990). The present study began with the discovery that addition of [y3’P]ATP to sea urchin sperm membrane vesicles resulted in the CAMP-dependent phosphorylation of the AC. In a detergent extract of sea urchin sperm membranes, the AC is one of the few proteins to become heavily phosphorylated by the endogenous kinase or purified bovine CAMP-dependent protein kinase (A-kinase). In this paper, we characterize the in vitro phosphorylation of the sea urchin sperm AC.

Abbreviations used: AC, adenylate cyclase; A-Kinase, CAMP-dependent protein kinase; Hepes, N-[Z-hydroxyethyl]-piperazine-N’-[2-ethanesulfonic acid]; EGTA, ethyleneglycol-bis-(P-aminoethyl ether) N,N’-tetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; Brij-78, polyoxyethylene-20-stearyl ether; CaM, calmodulin; CaM-El, sperm proteins eluted from CaM-Sepharose by EGTA; SDS-PAGE, polyacrylamide gel electrophoresis under reducing and denaturing conditions; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; EDTA, ethylenediaminetetraacetic acid; kD, relative molecular mass (kilodaltons); CNBr, cyanogen bromide. Received July 23, 1990; accepted September 17, 1990. Louis H. Bookbinder’s present address is Department of Pharmacology SJ-30,School of Medicine, University of Washington, Seattle, WA 98195. Address reprint requests to V.D. Vacquier, Marine Biology Research Division A-002, Scripps Institute of Oceanography, University of California, San Diego, La Jolla, CA 92093.

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MATERIALS AND METHODS Cells Sperm of Strongylocentrotus purpuratus was collected by intracoelomic injection of 0.5 M KCl. The 40,OOOg supernatant of detergent-extracted sperm (buffer composition: 10 mM Hepes, pH 7.5; 150 mM NaC1; 1 mM EGTA; 5 mM PMSF; 1 mM benzamidine HC1; 1% w/v Brij-78) and the CaM-agarose affinity chromatography eluant (CaM-El) (buffer composition: 10 mM Hepes, pH 7.5; 150 mM NaC1; 10 mM KC1; 2 mM MgC1,; 5 mM p-mercaptoethanol; 0.125%Brij-78; 2 mM EGTA) were prepared as described and glycerol was added to 50% prior to storage a t -70°C (Bookbinder et al., 1990). Except where noted, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Radioisotopes were purchased from ICN (Irvine, CAI. Protein Kinase Activity Endogenous protein kinase activity was assayed in the 40,OOOg supernatant or CaM-El by a tenfold dilution in 10 mM Hepes, pH 7.5; 150 mM NaC1; 10 mM MgC1,; and 0.5% Brij-78. Cyclic nucleotides or other reagents (Ca2+,CaM, CaM antagonists, protein kinase inhibitors) were added, and the reaction mixture was incubated for 30 min at room temperature. The reaction was initiated by addition of 50 pM ATP containing 10 pCi [y3'P]ATP and terminated by addition of trichloroacetic acid (TCA) to 10% final volume. The precipitated protein was pelleted by centrifugation a t 16,OOOg for 5 min and washed twice with 80% acetone by resuspension/sedimentation. The washed pellet was solubilized by boiling for 5 min in Laemmli sample buffer (Laemmli, 1970). Proteins were separated in 5% SDS-PAGE gels (Bookbinder et al., 1990; Laemmli, 1970), stained with silver (Morrisey, 19811, and dried between cellulose sheets. Phosphorylated proteins were visualized on Kodak X-AR film. The phosphorylated protein bands were excised from dried gels, and the radiation was determined in a liquid scintillation counter. Phosphorylation by Purified A-Kinase CaM-El was diluted 1:lO into CaM-El kinase buffer (10 mM Hepes, pH 7.5; 10 mM NaC1; 10 mM MgC1,; 0.2 mM Ca2+; 200 pM CAMP; and 1 mg/ml BSA). The reaction was initiated by adding bovine A-kinase (Sigma Catalog P2645; 0.56 pg kinase in 1.5 mM dithiothreitol), followed by addition of ATP containing 15 pCi [y3'P1ATP to a final concentration of 1 mM. The reaction was linear for 1hr at 23°C. Reactions were stopped by TCA precipitation and the samples processed as described above. The CaM-El was also phosphorylated by small volumes of the 40,OOOg supernatant by the same procedure described above for bovine A-kinase. Phosphorylation of CaM-binding proteins while attached to the CaM-agarose beads (Bookbinder et al.,

Fig. 1. The in vitro phosphorylation of the 40,OOOgsupernatant of 2% Brij-78 solubilized sea urchin sperm by an endogenous CAMPdependent protein kinase. Lanes A X : 5% gel. Lanes D, E: 20%gel. Lane A, silver stain 5 pg total protein. Lane B, autoradiogram of phosphorylated proteins in lane A. Lane C, autoradiogram of immunoprecipitation of proteins in lane B by the antiserum to adenylate cyclase (Bookbinder et al., 1990). Lanes D and E, autoradiograms of CNBr-generated 32P-peptidesfrom the immunoprecipitate (D) and total phosphoproteins (E) in the Mr 190 kD region of lane B. Molecular mass standards (lanes A-C) marked a t left in kD.

1990) was performed by diluting 50 pl of beads that had been previously washed in CaM bead wash buffer (10 mM Hepes, pH 7.5; 150 mM NaC1; 10 mM KC1; 2 mM MgCl,; 5 mM P-mercaptoethanol; 0.2 mM CaCI,) into 450 pl CaM-El kinase buffer. The' reaction was initiated as above with either 0.56 pg of bovine A-kinase or 50 p1 of 40,OOOg supernatant, and allowed to proceed for up to 1h r a t room temperature (23°C). The

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ATP [uM] Fig. 3. Phosphorylation of adenylate cyclase by the endogenous kinase as a function of ATP concentration. Radioactivity in the adenylate cyclase was determined by excising the Mr 190 kD band from SDS-PAGE gels and liquid scintillation counting. The K, for ATP is approximately 5.2 FM.

Fig. 2. Phosphoamino acid analysis of the immunoprecipitated adenylate cyclase shows the label to be incorporated into seryl residues.

beads were sedimented by gentle centrifugation and washed three times with 1ml of CaM bead wash buffer. SDS-PAGE sample buffer was added to the washed beads, the beads were boiled for 5 min, and sedimented in a microfuge, and the proteins in the supernatant were separated in 5% gels (Bookbinder et al., 1990).

Peptide Mapping Phosphorylated protein samples were separated in a 5% SDS-PAGE gel, dried (nonstained), and exposed to film. The phosphorylated Mr 190 kD bands were excised from gel lanes and rehydrated overnight in 125 mM Tris, pH 6.8. Peptide mapping was performed as described (Cleveland et al., 1977). Briefly, rehydrated gel slices were added to 0.6 ml of 125 mM Tris, pH 6.8, in 1.5 ml microfuge tubes. Six hundred microliters of 0.6 N HC1 and 0.12 ml of CNBr (2.25 g CNBr/ml acetonitrile) were added to each gel slice, and the reaction

was allowed to continue for 2 h r at room temperature with shaking. Gel slices containing the digested Mr 190 kD protein were washed twice for 10 min with 5 ml of 125 mM Tris, pH 6.8, per gel slice, followed by boiling for 5 min in SDS-PAGE sample buffer (Laemmli, 1970). The slices were inserted into the wells of a 5% stacking gel, which was overlayed with 0.1% melted agarose in pH 6.8 sample buffer. The peptides were separated at 55 mA through a 20% resolving gel (Laemmli, 1970). Following electrophoresis, the gels were stained with silver and dried, and the phosphorylated peptides were visualized by autoradiography.

Phosphoamino Acid Analysis Phosphoamino acid analysis of electroeluted (Vacquier and Moy, 1986) Mr 190 kD protein from unstained, excised gel slices was performed as described (Cooper et al., 1983). Immunoprecipitation Fixed Staphylococcus aureus cells (Pansorbin; Calbiochem, La Jolla, CAI were added to a final concentration of 0.3% vlv to 32P-labeled phosphorylated 40,OOOg supernatant. The suspension was rotated for 30 min a t

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Fig. 4. Phosphorylation of the adenylate cyclase by the endogenous kinase as a function of cyclic nucleotide concentration. Radioactivity incorporated into the adenylate cyclase was determined as for Figure 3. Maximum stimulation of the kinase occurred between 4 and 8 pM CAMP.cGMP showed no effect.

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Fig. 5. The time course of phosphorylation by the endogenous kinase. Maximal phosphorylation (23°C) occurs at 10 min. The radiolabe1 cannot be chased off the adenylate cyclase by addition of 2 mM unlabeled ATP.

protein were diluted 1:50 into AC reaction buffer (Vacquier et al., 1988) containing 2 mM CaC1, and assayed for AC activity. Total protein concentration of samples was determined using BSA as a standard (Smith et al., 4°C to preabsorb the sperm membrane lysate. The S. 1985). aureus was then sedimented by centrifugation, and the supernatant was removed to another tube that conRESULTS tained a n equal volume of antiadenylate cyclase polyThe Endogenous A-Kinase clonal antiserum (Bookbinder et al., 1990) diluted in Addition of CAMP to the Brij-78 40,OOOg supernatant TBSA (10 mM Tris-base, pH 7.5; 150 mM NaC1; 10 mM of sea urchin sperm activates a n endogenous A-kinase, sodium azide). The tube containing the 40,OOOg super- which phosphorylates primarily eight to ten proteins natant and antiserum was incubated at 4°C with rota- ranging in Mr from about 32 to 190 kD (Fig. 1, lanes A, tion for 45 min, after which the S. aureus cells were B). The Mr 190 kD band is heavily phosphorylated in added to 0.3%vlv and the tube was rotated for a n ad- this preparation. The addition of cGMP or CaM and ditional 45 min. The s. aureus antibody-antigen com- Ca2 reveals no additional phosphorylated proteins plexes were sedimented, and the pellet was washed under these conditions. This endogenous A-kinase does twice by resuspensionhedimentation in 1) IP lysis not appear to be tightly associated with the Mr 190 kD buffer, 2) IP wash buffer A, 3) IP wash buffer B as given protein because A-kinase activity does not elute with by Owens et al. (1980). The final washed pellet was the Mr 190 kD protein from CaM-agarose in the prepresuspended in 100 ~1 SDS-PAGE sample buffer and aration of the CaM-El. boiled for 5 min. The fixed S. aureus cells were sediImmunoprecipitation of the phosphorylated 40,OOOg mented and the proteins in the supernatant separated supernatant with the antiserum specific for the sea urby SDS-PAGE. chin sperm AC (Bookbinder et al., 1990) shows that the Mr 190 kD phosphoprotein is the AC (Fig. 1, lane C). AC Assay We have previously shown that this antiserum to the AC activity was assayed as in Vacquier et al. (1988). AC reacts with only one protein of Mr 190 kD on twoThe CaM-El or CaM-agarose beads with bound sperm dimensional Western immunoblots (Bookbinder et al., +

L.H. BOOKBINDER ET AL.

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Fig. 6. Inhibition of phosphorylation of the adenylate cyclase by the endogenous kinase by H8. Fifty percent inhibition occurs at 9 pM.

1990). The immunoprecipitated AC (lane C) and the total Mr 190 kD band (lane B) yield identical CNBr peptide maps (lanes D and E), showing that the only phosphoprotein a t Mr 190 kD is the AC. Phosphoamino acid analysis of the AC excised from gels as in Figure 1 shows the label to be incorporated into serine (Fig. 2). Characterization of the endogenous A-kinase was performed with respect to its ability to phosphorylate the sperm AC in the 40,OOOg supernatant. The endogenous A-kinase has a K, for ATP of 5.2 pM (Fig. 3) and shows maximal stimulation by CAMPat 4-8 pM (Fig. 4).Under the conditions of the assay, maximal phosphorylation of the AC occurs by 10 min (Fig. 5). Addition of 2 mM unlabeled ATP a t 10 min followed by sampling for a n additional 10 min shows no decrease in radiolabelling of the AC, indicating that the label does not turn over in vitro (not shown).

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PKI [units/ml] Fig. 7. Inhibition of phosphorylation of the adenylate cyclase by the endogenous kinase by the A-kinase inhibitor PKI. Fifty percent inhibition occurs at 30 U/ml.

30 U/ml (Fig. 7). The phosphorylation of AC in this in vitro preparation occurs in the presence of excess EGTA. However, as low as 0.1 pM buffered free Ca2+ totally blocks the radiolabeling of the AC (not shown). The radiophosphate labeling of the AC is not affected by the CaM antagonists calmidazolium (200 pM), trifluorperazine (200 pM), W7, W12, and W13 (100 pM).

Phosphorylation of the Sperm AC by the Catalytic Subunit of A-Kinase We previously showed that approximately 10% of the protein in the CaM-El is the Mr 190 kD AC (Bookbinder e t al., 1990). The AC in the CaM-El is depleted of the endogenous A-kinase. The CaM-El consists of a t least 20 sperm membrane proteins (Fig. 8, lane A; Bookbinder et al., 1990). The AC and two other proteins of the CaM-El can be phosphorylated by the catalytic subunit of bovine A-kinase (Fig. 8, Lane B). Effects of Inhibitors on the Comparison of CNBr generated one-dimensional pepEndogenous A-Kinase tide maps of the AC phosphorylated by either the enThe protein kinase inhibitor H-8 (Hidaka et al., dogenous A-kinase or bovine A-kinase are identical, 1984; Seikagaku America, Inc., St. Petersburg, FL) in- demonstrating that the endogenous kinase is in fact a hibits the phosphorylation of the AC in the 40,OOOg genuine A-kinase (Fig. 8, lanes E, F). The AC plus one other sperm protein can be phossupernatant, with a Ki of approximately 9 pM (Fig. 6). The more specific A-kinase inhibitor PKI (Walsh e t al., phorylated by bovine A-kinase while bound to CaM1971) inhibits the phosphorylation of AC, with a Ki of agarose beads (Fig. 8, lanes C, D). This preparation of

PHOSPHORYLATION OF SPERM ADENYLATE CYCLASE

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ATP [uM] Fig. 9. The effect of in vitro phosphoryation by the bovine A-kinase on the activity of adenylate cyclase bound to CaM-agarose. 0,Minus A-kinase; A,plus A-kinase. No differences in cyclase activity resulted from phosphorylation.

easily change buffers and modifying enzymes by washing the beads, was utilized to attempt to determine if phosphorylation affects AC activity. In vitro phosphorylation of the AC while bound to the CaM-agarose beads does not change the apparent K, or V,,, of the AC (Fig. 9). Fig. 8. Phosphorylation of the adenylate cyclase in the CaM-El by the catalytic subunit of bovine heart CAMP-dependent protein kinase. Lanes A-D: 5% gel. Lanes E, F: 20% gel. Lane A, silver stain gel 2 kg total CaM-El protein. Lane B, autoradiogram of lane A. Lanes C and D, autoradiograms of CaM-binding proteins phosphorylated while they are bound to the CaM-agarose; C, plus bovine A-kinase; D, minus bovine A-kinase. Lanes E and F, proteins of the CaM-El were phosphorylated by bovine A-kinase (El or the A-kinase in the 40,OOOg supernatant of sea urchin sperm (F).The Mr 190 kD adenylate cyclase bands were excised from wet gels and subjected to CNBr cleavage. The phosphopeptides were separated by SDS-PAGE 20% gels. The autoradiograms show that the same peptides are labeled by the two kinase preparations. A trace of incompletely cleaved protein occurs in the Mr 190 kD range.

DISCUSSION In this study we show that a sea urchin sperm AC of Mr 190 kD becomes phosphorylated in vitro by action of a n endogenous CAMP-dependent protein kinase. Of the large number of proteins in the 40,OOOg 2% Brij-78 supernatant, only eight to ten become heavily phosphorylated, the AC being one of the two most prominent phosphoproteins (Fig. 1, lanes A, B). The endogenous A-kinase is not tightly associated with the AC, since the AC binds to CaM-agarose, whereas the Akinase does not coelute from the column with the AC. As is typical for A-kinase, phosphate is incorporated into seryl residues. The K, of the A-kinase for ATP is AC is stable for at least 6 months when stored in 50% 5.2 pM, and maximal stimulation occurs between 4 and glycerol at - 70°C. This solid-phase preparation of AC 8 pM CAMP.These values are low enough to be within has high AC activity (3 pMol CAMP formed/min/pl their physiological concentration range. Both A-kinase beads). The ability to phosphorylate the AC, and to inhibitors used in this study, H8 and PKI, inhibit phos-

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phorylation of AC a t concentrations similar to those of other systems (Hidaka et al., 1984; Walsh et al., 1971). Bovine A-kinase phosphorylates the sea urchin sperm AC in the CaM-El, which is devoid of endogenous kinase activity. The CNBr peptide maps of the AC phosphorylated by both endogenous and bovine A-kinase are identical. The AC is the most heavily phosphorylated band in the CaM-El. The one-dimensional CNBrgenerated peptide maps show a minimum of five sites of phosphorylation (Fig. 1, lanes D, E). No apparent of the AC activity occur as changes in the K, or V,, a result of phosphorylation with the bovine A-kinase. Attempts to dephosphorylate the AC with calf alkaline phosphatase were unsuccessful. An interesting finding was that submicromolar concentrations of Ca2 completely blocked the phosphorylation of the AC by the endogenous A-kinase in the 40,OOOg supernatant. This is suggestive of the presence of a Ca2+-dependentphosphatase in this preparation that competes with the Akinase for the phosphorylation sites. The action of such a calcineurin-like phosphatase in sperm has been postulated to play a role in the mechanism of flagellar motility (Tash, 1989). We attempted to label the AC in vivo by incubation of undiluted S. purpuratus sperm in 32P-orthophosphate (Porter and Vacquier, 1986; Bentley et al., 1987). These experiments were unsuccessful, in that no incorporation of label was seen in the Mr 200 kD range either before or after the addition of S. purpuratus egg jelly. The sperm of this species of sea urchin is extremely impermeable to orthophosphate compared with another sea urchin species, Arbaciapunctulata. In vivo labeling of sperm of this latter species with orthophosphate labels a n unknown protein, which is slightly smaller than Mr 200 kD (Bentley et al., 1987). Thus, in our study on S.purpuratus sperm, we cannot show that the phosphorylation of the sperm AC is relevant to the role of this important enzyme in cell function. However, the K, value for ATP of 5.2 pM and the failure to label the AC in 0.1 pM Ca2+ are suggestive of physiological relevance. The phosphorylation of frog red cell AC (Yoshimasa et al., 1987) has been demonstrated. In this study the phosphorylation of AC occurs by action of protein kinase C in response to extracellular ligands. Associated with this phosphorylation is more than a doubling of AC activity. It has been postulated that this phosphorylation may provide a synergistic mechanism by which AC and the phosphatidylinositol transmembrane signalling systems interact (Yoshimasa et al., 1987). The question of the relevance of an in vitro study to possible in vivo function is important. Sea urchin sperm AC undergoes a n activation when the cells are treated with soluble egg jelly (Garbers and Kopf, 1980). This can result in large increases in CAMPconcentrations and a s much as a 50-fold increase in A-kinase activity. In S. purpuratus sperm the major substrate for in vivo phosphorylation by the activated A-kinase is +

sperm histone H 1 (Porter and Vacquier, 1986). Monoclonal antibodies to a 210 kD (Mr 210-240 kD) sea urchin sperm membrane protein also activate the sperm AC and the phosphorylation of H1 (Vacquier et al., 1988). To our knowledge, this is the first demonstration of the phosphorylation of a n AC by A-kinase. Although we could not demonstrate that the phosphorylation of the AC occurs in vivo, and because in vitro phosphorylation has no effect on the kinetic properties of this AC, the possible significance of these findings remains uncertain. However, we believe that they are worth reporting because of the importance of AC in cellular regulation. If phosphorylation of this AC occurs in vivo, it could affect the association of the AC with other regulatory elements of the cell membrane in the signal transduction pathway (Sibley e t al., 1986). Although G proteins are known to exist in sperm (Kopf et al., 1986; Bentley et al., 1986), there is as yet no evidence for their interaction with sperm AC (Garbers, 1989).

ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant HD12986 to V.D.V. REFERENCES Bentley JK, Garbers DL, Domino SE, Noland TD, Van Dop C (1986): Spermatozoa contain a guanine nucleotide-binding protein ADPribosylated by pertussis toxin. Biochem Biophys Res Commun 138: 728-734. Bentley JK, Khatra AS, Garbers DL (1987):Receptor-mediated phosphorylation of spermatozoan proteins. J Biol Chem 262:1570815713. Bookbinder LH, Moy GW, Vacquier VD (1990): Identification of sea urchin sperm adenylate cyclase. J Cell Biol 111:1859-1866. Brokaw CJ, (1987):Regulation of sperm flagellar motility by calcium and CAMP-dependent phosphorylation. J Cell Biochem 35175-184. Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK (1977): Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252:1102-1106. Cooper JA, Sefton BM, Hunter T (1983):Detection and quantification of phosphotyrosine in proteins. Methods Enzymol 99:387-402. Garbers DL (1989): The regulation of spermatozoon function by the egg. In Schatten H, Schatten G (eds): “The Molecular Biology of Fertilization.” San Diego: Academic Press, pp 3-19. Garbers DL, Kopf GS (1980): The regulation of spermatozoa by calcium and cyclic nucleotides. Adv Cyclic Nucleotide Res 13:251-306. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y (1984): Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23: 5036-5041. Kopf GS, Woolkalis MJ, Gerton GL (1986): Evidence for a guanine nucleotide-binding regulatory protein in invertebrate and mammalian sperm. Identification by islet-activating-protein catalyzed ADP-ribosylation and immunochemical methods. J Biol Chem 261: 7327-733 1. Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Lindemann CB, Kanous KS (1989): Regulation of mammalian sperm motility. Arch Androl 23:l-22. Morisawa M (1987): The process of initiation of sperm motility a t spawning and ejaculation. In H Mohri (ed): “New Horizons in Sperm Cell Research.” Tokyo: Japan Science Societies Press, pp 137-157. Morrisey J (1981): Silver stain for proteins in polyacrylamide gels: A

PHOSPHORYLATION OF SPERM ADENYLATE CYCLASE modified procedure with enhanced uniform sensitivity. Anal Biochem 117:307-310. Owen MJ, Kissonerghis AM, Lodish H F (1980):Biosynthesis of HLAA and HLA-B antigens in vivo. J Biol Chem 255:9678-9684. Porter DC, Vacquier VD (1986):Phosphorylation of sperm histone H1 is induced by the egg jelly layer in the sea urchin Strongylocentrotus purpuratus. Dev Biol 116:203-212. Sibley DR, Strasser RH, Benovic JL, Daniel K, Lefkowitz RJ (1986): Phosphorylatioddephosphorylation of the p-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution. Proc Natl Acad Sci USA 83:9408-9412. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gortner FH, Provenazno MD, Fujimoto EK, Goeke NM, Klenk DC (1985):Measurement of protein using bicinchonic acid. Anal Biochem 150:76-85. Tash JS (1989):Protein phosphorylation: The second messenger signal transducer of flagellar motility. Cell Motil Cytoskel 14:332339.

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Tash JS, Means AR (1987):Ca2+ regulation of sperm axonemal motility. Methods Enzymol 139:808-823. Vacquier VD, Moy GW (1986):Stoichiometry of phosphate loss from sea urchin sperm guanylate cyclase during fertilization. Biochem Biophys Res Commun 137:1148-1152. Vacquier VD, Moy GW, Trimmer JS, Ebina Y, Porter DC (1988): Monoclonal antibodies to a membrane glycoprotein induce the phosphorylation of histone H1 in sea urchin spermatozoa. J Cell Biol 107:2021-2027. Walsh DA, Ashby CD, Gonzalez C, Calkins D, Fischer EH, Krebs EG (1971):Purification and characterization of a protein inhibitor of adenosine 3',5'-monophosphate-dependentprotein kinases. J Biol Chem 246:1977-1985. Yoshimasa T, Sibley DR, Bouvier M, Lefkowitz RJ, Caron MG (1987): Cross-talk between cellular signallingpathways suggested by phorbolester-induced adenylate cyclase phosphorylation. Nature 327: 67-70.

In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase.

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten prot...
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