Phosphoprotein Phosphatase Inhibits Flagellar Movement of Triton Models of Sea Urchin Spermatozoa

CELL STRUCTURE AND FUNCTION 10, 327-337 (1985) C by Japan Society for Cell Biology Phosphoprotein Phosphatase Inhibits Flagellar Movement of Triton Models of Sea Urchin Spermatozoa Daisuke Takahashi1, Hiromu Murofushi, Koichi Ishiguro2, Jun Ikeda and Hikoichi Sakai Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ABSTRACT. Phosphoprotein phosphatase prepared from bovine cardiac muscle was used to study the roles of axonemal phosphoproteins in the flagellar motility of sea urchin spermatozoa. When isolated axonemes were incubated with cyclic AMP-dependent protein kinase, ƒÁ[32P]ATP and cyclic AMP, more than 15 polypeptides were phosphorylated. Most were dephosphorylated by treatment with phosphoprotein phosphatase. When Triton models of sea urchin spermatozoa were treated with phosphoprotein phosphatase followed by an addition of ATP, the flagellar motility of the models was drastically reduced in comparison with that of the untreated models. The motility of the phosphatase-treated Triton models was partially restored by an addition of cyclic AMP and cyclic AMP-dependent protein kinase. These data give strong support to the idea that the motility of eukaryo- tic flagella is controlled by a protein phosphorylation-dephosphorylation system. For several years much attention has been paid to the control of flagellar and ciliary movement in eukaryotes by the cyclic nucleotide system. Morton et al. (24) reported that flagellar movement of hamster cauda epididymal spermatozoa could be induced by diluting the semen with a medium containing calcium and that the initiation of beat was accompanied by an increase in the concentration of cyclic AMP within the cells. Garbers et al. (8) and Hoskins (14) reported that cyclic AMP or inhibitors of cyclic nucleotide phosphodiesterase added exogenously to bovine epididymal spermatozoa not only stimulated energy metabolism but activated flagellar motility as well. In addition, the spermatozoa of a wide variety of animals have been shown to contain enzymes related to the cyclic nucleotides such as adenylate cyclase (2, 7, 12, 24), guanylate cyclase (5, 12), cyclic nucleotide phosphodiesterase (2, 12, 24) and phosphoprotein phosphatase (28). An especially large amount of cyclic AMP- dependent protein kinase is included in sperm cells (6, 15, 19, 20, 25). To investigate the role of the cyclic nucleotide system in the regulation of flagellar motility, we used the Triton models of sea urchin spermatozoa developed by Gibbons 1 Present address: Department of Radiation Biophysics , Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 2 Present address: Mitsubishi-Kasei Institute of Life Science , 11 Minamioya, Machida, Tokyo 327 328 D. Takahashi, et al. and Gibbons (9). This system is of particular advantage because macromolecules related to cyclic nucleotide system, such as protein kinase and protein phosphatase, are easily introduced to the motile apparatus. We found that the cyclic AMP-dependent protein kinase and a protein factor prepared from a detergent-extract of sea urchin or starfish spermatozoa reactivated the motility of the Triton models of sea urchin spermatozoa (16, 26). A similar protein factor was found by Tash et al. (29). It was phosphorylated by cyclic AMP-dependent protein kinase and induced the movement of Triton models of dog spermatozoa. The need for protein phosphorylation to initiate the flagellar beat also has been shown in Triton models of bull (21), fish (23) and tunicate (27) spermatozoa. If phosphorylation is required to initiate or activate flagellar movement, dephosphorylation of phosphoproteins in the flagellum should affect motility. No one has yet done this type of experiment which is necessary for confirmation of the significance of protein phosphorylation in flagellar motility. We used the phosphoprotein phosphatase of bovine cardiac muscle (4) for our experiments because it is easily be purified and gives a higher yield than the enzyme present in spermatozoa (28). We found that protein dephosphorylation of the axonemal phosphoproteins drastically reduced the motility of freshly prepared Triton models of sea urchin spermatozoa and that the original motility was partially restored by an addition of cyclic AMP and protein kinase. This result supports the belief in the significance of protein phosphorylation in the control of flagellar motility in spermatozoa and sheds light on unknown functions of the phosphoprotein phosphatase present in spermatozoa. MATERIALS AND METHODS Preparation of Triton models of sea urchin spermatozoa. Spermatozoa from the sea urchins, Pseudocentrotus depressus and Hemicentrotus pulcherrimus were collected by injecting 0.5 M KC1 into the coelom and were used within several hours. About 30 eul of "dry" sperm was suspended in 0 .5 ml of calcium-free sea water. A 40 ƒÊl portion of the suspension was added to 0.5 ml of 0.04 % (v/v) Triton X-100 in 50 mM Tris-HCl (pH 8.1)- 0.1 M KCl-2 mM MgCl2-0.1 mM EDTA (TKME) then gently swirled at room temperature for 30 s. The demembranated spermatozoa were precipitated by centrifugation at 1,000 g for 5 min after which they were suspended carefully in 0.5 ml of TKME to make a suspension of Triton models free from Triton X-100 and spermatozoal materials that have been made soluble by the detergent. The models were reactivated by mixing 40 ƒÊl of the suspension with 2 ml of 1 mM ATP in TKME. The percent motility of the Triton models, defined as (the number of motile Triton models/total Triton models) •~ 100, was checked with a microscope equipped with a dark-field condenser and a 100 W Hg arc lamp. Fresh Triton models that had a percent motility greater than 60 % (usually 60-90 %) were used routinely in our experiments. Preparation of axonemes and purification of dynein. Axonemes were prepared from spermatozoa of the sea urchin Pseudocentrotus depressus. Spermatozoa that had been washed in calcium-free sea water were homogenized with 0.5 % (v/v) Triton X-100 in TKME to remove the membranes and separate the tails from the heads. The demembranated heads and tails were sedimented by centrifugation at 10,000 g for 10 min. After discarding the supernatant, the upper, white pellet consisting mainly of tails was suspended in TKME leaving the lower, hard pellet that contained mostly heads. The tail suspension was centri- Control of Flagellar Motility 329 fuged and its upper pellet was washed several times in TKME, which gave an axoneme fraction free from the contamination by heads. Dynein was prepared from Hemicentrotus spermatozoa by the method of Gibbons and Fronk (11) with some modification. Dynein was extracted from the axonemes by use of a solution containing 0.5 M KCl, 50 mM Tris-HCl (pH 8.1), 2 mM MgCl2 and 0.1 mM EDTA. It was purified by ultracentrifugation through a 5-20 % (w/v) sucrose density gradient in 0.5 M KCl-50 mM Tris-HCl (pH 8.1)-2 mM MgCl2-0.1 mM EDTA. The specific activity of the purified dynein was about 1.1 ƒÊmol/mg/min, evidence that activation had already occurred during purification. No latent type of dynein has yet been obtained from the sperm of this sea urchin. Purification of phosphoprotein phosphatase from bovine cardiac muscle. Phosphoprotein phosphatase was purified from bovine cardiac muscle according to the method of Chou et al. (4). The purified enzyme was dialyzed against TKME then stored at -80•Ž. Its activity was measured with casein as the substrate. A 0.2 ml mixture containing 7 mg/ml casein (Merck) and phosphoprotein phosphatase in TKME was incubated at 30•Ž for 10 min, after which 0.2 ml of 15 % (w/v) trichloroacetic acid was added to terminate the reaction. The precipitates formed were removed by centrifugation, and the amount of inorganic phosphate in the supernatant was measured by the method of Chen et al. (3). The specific activity of the purified enzyme was 1.9 ƒÊmol of Pi released/min/mg protein under our assay conditions. Purification of cyclic AMP-dependent protein kinase from sea urchin spermatozoa. Cyclic AMP-dependent protein kinase was purified from spermatozoa of the sea urchin Anthocidaris crassispina according to the method of Ishiguro et al. (16). The specific activity of the purified enzyme was 13 nmol of Pi transferred/min/mg protein. Electrophoresis and autoradiography. Electrophoresis was performed after Laemmli (18) using 5-15 % (w/v) acrylamide density gradient gels. Proteins were stained with Coomassie Blue R-250. After being destained in 7 % (v/v) acetic acid solution, the gels were dried then autoradiographed using Kodak X-Omat S films. Other procedures. The Mg-ATPase activity of the dynein was assayed by the procedure described elsewhere (13). Proteinase activity was assayed in a reaction mixture containing 7 mg/ml of casein and 0.1 mg/ml of phosphoprotein phosphatase in TKME. This mixture was incubated at 30•Ž for 1 h, after which trichloroacetic acid was added to precipitate the proteins in the mixture. The UV absorbance of the supernatant then was measured. Protein concentration was determined by the method of Lowry et al. (22) with bovine serum albumin as a standard. RESULTS Phosphorylation and dephosphorylation of axonemal proteins by protein kinase and phosphoprotein phosphatase. Chou et al. (4) reported that the phosphoprotein phosphatase prepared from bovine cardiac muscle dephosphorylates the regulatory subunit of cyclic AMP-dependent protein kinase, histone, casein and phosphorylase. They did not, however, examine the substrate specificity of the enzyme toward axonemal proteins. Therefore, before applying this enzyme to the Triton models of spermatozoa, we examined whether the enzyme is capable of dephosphorylating axonemal proteins. We prepared 32P-labeled axonemes using ƒÁ-[32P]ATP and the cyclic AMP- dependent protein kinase purified from sea urchin spermatozoa. When the axonemes 330 D. Takahashi, et al. Fig. 1. In vitro phosphorylation of axonemal proteins by protein kinase and phosphoprotein phosphatase.

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