Participation of the Human Sperm Proteasome in the Capacitation Process and Its Regulation by Protein Kinase A and Tyrosine Kinase
Participation of the Human Sperm Proteasome in the Capacitation Process and Its Regulation by Protein Kinase A and Tyrosine Kinase
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DOI：
10.1095/biolreprod.108.073924
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发表时间：
2009-05-01
影响因子：
3.6
通讯作者：
Morales, Patricio
中科院分区：
文献类型：
--
作者：
Kong, Milene;Diaz, Emilce S.;Morales, Patricio
蛋白酶体是存在于人类精子中的多催化性细胞复合物，在哺乳动物受精的几个步骤中起重要作用。在这里，我们提出的证据表明，蛋白酶体参与人类精子获能。将高活动性精子的等分试样与蛋白酶体抑制剂MG 132或环氧霉素一起孵育。分别用盐酸金霉素法、荧光底物法、cAMP酶免疫试剂盒和Western blot法测定获能精子百分率、蛋白酶体糜蛋白酶样活性、cAMP含量和蛋白磷酸化模式。我们的研究结果表明，用蛋白酶体抑制剂处理精子阻断获能过程，不改变cAMP浓度，并改变蛋白磷酸化的模式。为了阐明蛋白酶体活性在获能过程中是如何调节的，将精子与以下物质一起孵育：1）酪氨酸激酶（TK）抑制剂（染料木黄酮或除莠霉素A）; 2）蛋白激酶（PK）A抑制剂或激活剂（分别为H89和Rp-cAMPS和8-Br-cAMP）;或3）PKC抑制剂（他莫昔芬或星形孢菌素）在不同获能时间。然后测定胰凝乳蛋白酶样活性和蛋白酶体的磷酸化程度。结果表明，精子处理TK和PKA抑制剂显着降低获能过程中的蛋白酶体胰凝乳蛋白酶样活性。免疫沉淀和蛋白质印迹结果表明，蛋白酶体在获能过程中的TK和PKA依赖性途径磷酸化。总之，我们认为精子蛋白酶体参与获能过程，其活性受PKs调节。
The proteasome is a multicatalytic cellular complex present in human sperm that plays a significant role during several steps of mammalian fertilization. Here, we present evidence that the proteasome is involved in human sperm capacitation. Aliquots of highly motile sperm were incubated with proteasome inhibitors MG132 or epoxomicin. The percentage of capacitated sperm, the chymotrypsin-like activity of the proteasome, cAMP content, and the pattern of protein phosphorylation were assayed by using the chlortetracycline hydrochloride assay, a fluorogenic substrate, the cAMP enzyme immunoassay kit, and Western blot analysis, respectively. Our results indicate that treatment of sperm with proteasome inhibitors blocks the capacitation process, does not alter cAMP concentration, and changes the pattern of protein phosphorylation. To elucidate how proteasome activity is regulated during capacitation, sperm were incubated with: 1) tyrosine kinase (TK) inhibitors (genistein or herbimycin A); 2) protein kinase (PK) A inhibitors or activators (H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKC inhibitors (tamoxifen or staurosporin) at different capacitation times. The chymotrypsin-like activity and degree of phosphorylation of the proteasome were then assayed. The results indicate that sperm treatment with TK and PKA inhibitors significantly decreases the chymotrypsin-like activity of the proteasome during capacitation. Immunoprecipitation and Western blot results suggest that the proteasome is phosphorylated during capacitation in a TK- and PKA-dependent pathway. In conclusion, we suggest that the sperm proteasome participates in the capacitation process, and that its activity is modulated by PKs.