Proteasome activity and its relationship with protein phosphorylation Proteasome activity and its relationship with protein phosphorylation during capacitation and acrosome reaction in human spermatozoa Patricio Morales, Emilce S. Díaz and Milene Kong Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile 1 We have shown that the proteasome is present in mammalian sperm and plays a role during fertilisation. In this work we studied the relationship between protein phosphorylation and proteasomal activity in human sperm. Aliquots of motile sperm were incubated for 0, 5 and 18 h at 37 C, 5% CO 2, with different concentration of the kinase inhibitors genistein, H89 or tamoxifen. Control aliquots were treated with the inhibitor solvent. The chymotrypsin-like activity of the proteasome was assayed using a fluorogenic substrate. Aliquots of spermatozoa capacitated during 18 h were incubated for 30 min with kinase inhibitors and then with 7 µm progesterone (P). The percentage of viable acrosome-reacted sperm was evaluated using FITC-labeled Pisum sativum agglutinin. The results indicate that spermatozoa treated with different concentrations of genistein and tamoxifen did not modify the chymotrypsin-like activity of the proteasome during capacitation. On the other hand, proteasome activity was significantly decreased by incubation with H89. Sperm treatment with genistein, H89 and tamoxifen significantly inhibited the P-induced acrosome reaction. Western blot analysis indicated that the proteasome inhibitor, epoxomicin, reduced serine protein phosphorylation. These results suggest that the enzymatic activity of the proteasome is modulated by protein kinase A, and that both enzymes are involved in the P-induced acrosome reaction. Introduction The phosphorylation/dephosphorylation of proteins is a universal postransductional biochemical mechanism that involves the reversible introduction of a phosphate group into an organic molecule. It is controlled by protein kinases and phosphatases, and takes place predominantly on serine, threonine and tyrosine residues (Hunter, 2000). The phosphorylation of proteins has demonstrated to play an important role in a variety of physiological processes including growth, differentiation, and cellular function. There are numerous studies which show that before and during mammalian fertilisation, the sperm undergo a series of changes that are regulated by the activation of intracellular signalling pathways, which control the phosphorylation status of many proteins (Baldi et al., 2002; Urner Corresponding author E-mail: pmorales@uantof.cl 1
2 P. Morales et al. and Sakkas, 2003; Visconti et al., 2002). Thus, several processes regulated by protein phosphorylation during sperm development and acquisition of fertilising capacity have been described, including sperm maturation, capacitation, hyperactivated motility, acrosome reaction, and gamete membrane fusion (Lewis and Aitken, 2001; Naz and Rajesh, 2004). Most of the changes that have been studied during sperm protein phosphorylation take place in tyrosine residues. Thus, it has described that human sperm experience a progression in the phosphorylation status of its proteins during capacitation and zona pellucida binding (Sakkas et al., 2003). The main proteins that are phosphorylated in tyrosine residues during capacitation correspond to the AKAPs and the CABYR, both families associated to the hyperactivated motility of the sperm (Vijayaraghavan et al., 1997). In contrast to the numerous studies related to sperm protein phosphorylation in tyrosine residues, little is known about the phosphorylation in serine/threonine residues. There are only a few studies that suggest an important role for the phosphorylation in ser/thr residues during the events of capacitation, hyperactivation and acrosome reaction (Jha and Shivaji, 2002; Naz and Rajesh, 2004). In addition, it has been shown that inhibition of the camp-dependent protein kinase (PKA) inhibit both capacitation and the acrosome reaction (reviewed in Baldi et al., 2002). This kinase modifies other proteins by phosphorylation in ser/thr residues. In addition, PKA participates in the cross-talk between different intracellular signalling cascades like tyrosine kinases, MAPKs, and ser/thr phosphatases. Sperm proteases play important roles during fertilisation. Recently, it was shown that spermatozoa from several mammalian species, including human, possess a multi-enzymatic protease complex or proteasome, which has trypsin-like, chymotrypsin-like and peptidylglutamyl peptide-hydrolyzing activities (Tipler et al., 1997; Pizarro et al., 2004). The proteasome degrades most nuclear and cytosolic proteins, after they have been covalently labelled to ubiquitin molecules, and the ubiquitin-proteasome pathway is responsible for most of the cell proteolysis (Ciechanover, 1998). Proteasome is a multi-subunit protease composed of two 19S regulatory complexes, capping both sides of a barrel-shaped 20 S proteasomal core. The 19S particle is composed of at least 17 proteasomal regulatory complex subunits, including ATP-dependent and ATP-independent subunits. The 20S core is composed of 14 subunits, 7 of α-type and 7 of ß-type. The α-subunits possess regulatory functions and the ß-subunits catalytic functions (Coux et al., 1996). In marine invertebrate sperm, the proteasome is involved in multiple steps of the fertilisation process, from the acrosome reaction induced by the egg jelly to penetration of the vitelline coat and fusion with the plasma membrane of the oocyte (Sawada et al., 2002). In human sperm, we demonstrated that the proteasome is involved in the zona pellucida- and progesterone (P)-induced acrosome reaction. The aim of this work was to examine the relationship between proteasomal activity and protein phosphorylation in human sperm during capacitation and the P-induced acrosome reaction. Protein kinase and proteasome activity During capacitation, the chymotrypsin-like activity of the proteasome was inhibited by treating human spermatozoa with the PKA inhibitor H89 (Fig. 1). This is in agreement with the work of Marambaud et al. (1996), which showed that PKA effectors (like forskolin) but not protein kinase C agonists increased the activity and phosphorylation state of the proteasome in HK293 cells. This observation suggests that the enzymatic activity of the proteasome depends on PKA activation and protein phosphorylation in ser/thr residues. In addition, treatment with H89 significantly reduced the P-induced acrosome reaction (7 µm P = 54 ± 3.9% vs. 50 µm H89 + 2
Proteasome activity and its relationship with protein phosphorylation 3 7 µm P = 30.5±3%). On the other hand, none of the kinase inhibitors affected proteasome activity during induction of the acrosome reaction with P. This suggests that phosphorylation of the proteasome is not required during the occurrence of the acrosome reaction stimulated by P. Control 10µ M 50µ M 100µ M Proteasome activity (% control) 100 75 50 25 T0 T5 T18 Time (hr) Figure 1. Effect of different concentrations of the protein kinase A inhibitor H89 on the chymotrypsin-like activity of human sperm proteasome. The control group is significantly higher than all the other groups. Protein phosphorylation and proteasome activity To determine the relationship between proteasome activity and protein phosphorylation, we proceeded to evaluate the pattern of protein phosphorylation during sperm capacitation and P- induced acrosome reaction in the presence of the proteasome inhibitor epoxomicin (Fig. 2). Aliquots of highly motile sperm were divided in six groups: non-capacitated (0); capacitated for 5 h (5); capacitated for 18 h (18); capacitated for 18 h in the presence of 10 µm epoxomicin (18+E); capacitated for 18 h and incubated with 7 µm P for 15 min (P); and capacitated for 18 h and then incubated with 10 µm epoxomicin for 30 min followed by incubation with P for 15 min (P+E). Then, a SDS-PAGE followed by a Western blot was performed. The bands were revealed with an anti phosphoserine antibody (Fig. 2). The densitometric analysis of the bands revealed an increase in protein phosphorylation in serine residues during capacitation, that is between the 0 and the 18 h of incubation. This increase was detected in seven bands, whose molecular weights were 155, 107, 86, 70, 28, 26 and 10 kda, approximately (lanes 0, 5, and 18). In addition, sperm treated with epoxomicin during capacitation exhibited a diminution in the degree of phosphorylation in serine residues of those bands (lane 18+E). On the other hand, a band of 26 kda increased its degree of phosphorylation in serine when the sperm were treated with P (lane P). This increase was not observed when the sperm were previously treated with epoxomicin (lane P+E). When the phosphorylation pattern in threonine residues was analysed during capacitation, a gradual increase in phosphorylation of bands of molecular weight in the range from 38.5 to 53 kda was observed (data not shown). This increase was not reverted by incubating the sperm with epoxomicin. Sperm treated with P did not show a phosphorylation pattern different from sperm capacitated for 18 h, with the exception of a band of approximately 23 kda. There was a small increase in the degree of phosphorylation of this band after P treatment. There was a 3
4 P. Morales et al. further increase in the phosphorylation of this band after the sperm were treated with epoxomicin and P. Figure 2. Western blot analysis of proteins phosphorylated in serine residues during human sperm capacitation (0, 5, and 18 h) and progesterone-induced acrosome reaction (P). Other sperm aliquots, capacitated for 18 h, were incubated with the proteasome inhibitor epoxomicin (18+E) or with progesterone and epoxomicin (P+E). The panel on the right shows the densitometric analysis of the phosphorylated bands. An increase in the pattern of protein phosphorylation in tyrosine residues was observed during capacitation in bands of an approximate molecular weight from 16 to 111 kda. When the sperm were incubated in the presence of epoxomicin, a slight increase in the phosphorylation degree of the band corresponding to an approximate molecular weight of 26 kda was observed. Conclusions On the basis of the results obtained, we suggest that the activity of the sperm proteasome is directly or indirectly modulated by PKA. In turn, the proteasome is able to modulate not only the activity of protein kinases but also of phosphatases. Acknowledgements This work was financed by FONDECYT 1040295 and DIRINV 1322-06 and 1316-06 Bibliography Baldi, E., Luconi, M., Bonaccorsi, L. and Forti, G. (2002). Signal transduction pathways in human spermatozoa. Journal of Reproductive Immunology 53: 121-131 Ciechanover, A. (1998). The ubiquitin-proteasome pathway: on protein death and cell life. The Embo Journal 17: 7151-7160 Coux, O., Tanaka, K. and Goldberg, A.L. (1996). Structure and function of the 20S and 26S proteasomes. Annual Review of Biochemistry 65: 801-847 Hunter, T. (2000). Signaling 2000 and beyond. Cell 100: 113-127 Jha, K. and Shivaji, S. (2002). Protein serine and threonine phosphorylation, hyperactivation and acrosome reaction in in vitro capacitated hamster spermatozoa. Molecular Reproduction and Development 63: 119-130 Lewis, B. and Aitken, R. (2001). Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited by rat spermatozoa. Biology of Reproduction 64: 1545-1556 4
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