A sperm component, HSD-3.8 (SPAG1), interacts with G-protein beta 1 subunit and activates extracellular signal-regulated kinases (ERK).
HSD-3.8 cDNA (accession number AF311312) encodes a human sperm component. A 0.7 kb fragment (HSD-0.7) containing three immunological epitopes of HSD-3.8 cDNA was prepared and expressed in E. coli. Immunization of female rats with the recombinant HSD-0.7 proteins induced infertility. A cDNA fragment encoding the C-terminal 144 amino acids of human G-protein beta l subunit (Gbeta1-C144) was screened by yeast two-hybrid, when HSD-0.7 segment was used as a bait. Recombinant His6-tagged-Gbeta1-C144 protein was expressed in E. coli BL21 and Anti-Gbeta1 serum was raised with purified Gbeta1-C144. HA-tagged HSD-0.7 and FLAG-tagged Gbeta1 plasmids were constructed and co-transfected into human embryonal kidney 293 cells. Two proteins were localized at superimposable sites in the cytoplasm, and they formed a complex when 500 micromol/L GDP existed. Overexpression of HSD-0.7 activated the G-protein-mediated extracellular signal-regulated kinases (ERK1/2); however, the truncated fragments of HSD-0.7, which lacked either TPR domain or P-loop, lost the ability to activate the ERK1/2 pathway. Further study revealed that the activation of ERK1/2 was protein kinase C (PKC) rather than Ras dependent. These results provide evidence that HSD-3.8 present in spermatocytes and sperm may participate in spermatogenesis and fertilization process by activating the PKC-dependent ERK1/2 signal transduction pathway.
Liu
Ning
N
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing, 100005, People Republic of China.
Qiao
Yuan
Y
Cai
Congli
C
Lin
Wen
W
Zhang
Jianchao
J
Miao
Shiying
S
Zong
Shudong
S
Koide
S S
SS
Wang
Linfang
L
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism / Q000502:physiology*; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005260:Female; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D020935:MAP Kinase Signaling System; D008297:Male; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010053:Ovary / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013091:Spermatogenesis; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection; D020798:Two-Hybrid System Techniques
D000954:Antigens, Surface; D018076:DNA, Complementary; D044387:GTP-Binding Protein beta Subunits; D011994:Recombinant Proteins; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019204:GTP-Binding Proteins; C483942:SPAG1 protein, human