A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.
Spermatozoa require the process of capacitation to enable them to fertilize an egg. PKA is crucial to capacitation and the development of hyperactivated motility. Sperm PKA is activated by cAMP generated by the germ cell-enriched adenylyl cyclase encoded by Adcy10 Male mice lacking Adcy10 are sterile, because their spermatozoa are immotile. The current study was designed to identify binding partners of the sperm-specific (Cα2) catalytic subunit of PKA (PRKACA) by using it as the "bait" in a yeast 2-hybrid system. This approach was used to identify a novel germ cell-enriched protein, sperm PKA interacting factor (SPIF), in 25% of the positive clones. Homozygous Spif-null mice were embryonically lethal. SPIF was coexpressed and coregulated with PRKACA and with t-complex protein (TCP)-11, a protein associated with PKA signaling. We established that these 3 proteins form part of a novel complex in mouse spermatozoa. Upon capacitation, the SPIF protein becomes tyrosine phosphorylated in >95% of sperm. An apparent molecular rearrangement in the complex occurs, bringing PRKACA and TCP11 into proximity. Taken together, these results suggest a role for the novel complex of SPIF, PRKACA, and TCP11 during sperm capacitation, fertilization, and embryogenesis.-Stanger, S. J., Law, E. A., Jamsai, D., O'Bryan, M. K., Nixon, B., McLaughlin, E. A., Aitken, R. J., Roman, S. D. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.
Centre for Chemical Biology, University of Newcastle, Callaghan, New South Wales, Australia; Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, New South Wales, Australia; School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia; Australian Research Council Centre of Excellence in Biotechnology and Development, University of Newcastle, Callaghan, New South Wales, Australia; and.
Simone J
SJ
Stanger
Centre for Chemical Biology, University of Newcastle, Callaghan, New South Wales, Australia;
Estelle A
EA
Law
Australian Research Council Centre of Excellence in Biotechnology and Development, University of Newcastle, Callaghan, New South Wales, Australia; and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia.
Duangporn
D
Jamsai
Australian Research Council Centre of Excellence in Biotechnology and Development, University of Newcastle, Callaghan, New South Wales, Australia; and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, Australia.
Moira K
MK
O'Bryan
Centre for Chemical Biology, University of Newcastle, Callaghan, New South Wales, Australia; Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, New South Wales, Australia; School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia; Australian Research Council Centre of Excellence in Biotechnology and Development, University of Newcastle, Callaghan, New South Wales, Australia; and.
Brett
B
Nixon
Centre for Chemical Biology, University of Newcastle, Callaghan, New South Wales, Australia; Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, New South Wales, Australia; School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia; Australian Research Council Centre of Excellence in Biotechnology and Development, University of Newcastle, Callaghan, New South Wales, Australia; and.
Eileen A
EA
McLaughlin
School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia; Australian Research Council Centre of Excellence in Biotechnology and Development, University of Newcastle, Callaghan, New South Wales, Australia; and john.aitken@newcastle.edu.au.
R John
RJ
Aitken
Centre for Chemical Biology, University of Newcastle, Callaghan, New South Wales, Australia; Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, New South Wales, Australia; School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia; Australian Research Council Centre of Excellence in Biotechnology and Development, University of Newcastle, Callaghan, New South Wales, Australia; and shaun.roman@newcastle.edu.au.
Shaun D
SD
Roman
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D006579:Heterozygote; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011487:Protein Conformation; D020033:Protein Isoforms; D021122:Protein Subunits; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012744:Sex Ratio; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D021122:Protein Subunits; D012333:RNA, Messenger; C533400:Tcp11 protein, mouse; C000622006:sperm PKA interacting factor, mouse; D017868:Cyclic AMP-Dependent Protein Kinases
protein complex; protein kinase A; sperm capacitation; tyrosine kinase