CABYR binds to AKAP3 and Ropporin in the human sperm fibrous sheath

Calcium-binding tyrosine phosphorylation-regulated protein （CABYR） is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath （FS） protein involved in capacitation. A putative domain （amino acids 12-48） homologous to the regulatory subunit of type II cAMP-d...

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Bibliographic Details
Published in:Asian journal of andrology 2011-03, Vol.13 (2), p.266-274
Main Authors: Li, Yan-Feng, He, Wei, Mandal, Arabinda, Kim, Young-Hwan, Digilio, Laura, Klotz, Ken, Flickinger, Charles J, Herr, John C
Format: Article
Language:English
Subjects:
A Kinase Anchor Proteins - chemistry
A Kinase Anchor Proteins - genetics
A Kinase Anchor Proteins - metabolism
Amino Acid Sequence
Calcium-Binding Proteins - chemistry
Calcium-Binding Proteins - genetics
Calcium-Binding Proteins - metabolism
Genetic Variation
Humans
Immunoprecipitation
Male
Membrane Proteins - chemistry
Membrane Proteins - genetics
Membrane Proteins - metabolism
Molecular Sequence Data
Multiprotein Complexes - chemistry
Original
Phosphoproteins - chemistry
Phosphoproteins - genetics
Phosphoproteins - metabolism
Protein Binding
Protein Interaction Domains and Motifs
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
rho GTP-Binding Proteins - chemistry
rho GTP-Binding Proteins - genetics
rho GTP-Binding Proteins - metabolism
Sperm Tail - metabolism
Sperm Tail - ultrastructure
Two-Hybrid System Techniques
免疫共沉淀
多克隆抗体
蛋白激酶A
调节蛋白
酪氨酸磷酸化
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Summary:Calcium-binding tyrosine phosphorylation-regulated protein （CABYR） is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath （FS） protein involved in capacitation. A putative domain （amino acids 12-48） homologous to the regulatory subunit of type II cAMP-dependent protein kinase A （RII） dimerisation and A kinase-anchoring protein （AKAP）-binding domains of protein kinase A at the N-terminus suggests that CABYR may self-assemble and bind to AKAPs. Moreover, there is evidence that CABYR has limited interaction with AKAPs. However, further evidence and new relationships between CABYR and other FS proteins, including AKAPs, will be helpful in understanding the basic physiology of FS. In this study, a new strategy for co-immunoprecipitation of insoluble proteins, as well as the standard co-immunoprecipitation method in combination with mass spectrometry and western blot, was employed to explore the relationship between CABYR, AKAP3 and Ropperin. The results showed that AKAP3 was co.immunoprecipitated with CABYR by the anti-CABYR-A polyclonal antibody, and, conversely, CABYR was also co.immunoprecipitated with AKAP3 by the anti-AKAP3 polyclonal antibody. Another RIl-like domain containing protein, Ropporin, was also co-immunoprecipitated with CABYR, indicating that Ropporin is one of CABYR＇s binding partners. The interactions between CABYR, AKAP3 and Ropporin were confirmed by yeast two-hybrid assays. Further analysis showed that CABYR not only binds to AKAP3 by its RII domain but binds to Ropporin through other regions besides the RIl-like domain. This is the first demonstration that CABYR variants form a complex not only with the scaffolding protein AKAP3 but also with another Rll-like domain-containing protein in the human sperm FS.
ISSN:1008-682X
1745-7262
DOI:10.1038/aja.2010.149