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-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 Ropporin. 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 RII-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 RII-like domain. This is the first demonstration that CABYR variants form a complex not only with the scaffolding protein AKAP3 but also with another RII-like domain-containing protein in the human sperm FS.
Department of Urology, Daping Hospital, Institute of Surgery Research, Third Military Medical University, Chongqing 400042, China. lyf1000@yahoo.com.cn
Yan-Feng
YF
Li
Wei
W
He
Arabinda
A
Mandal
Young-Hwan
YH
Kim
Laura
L
Digilio
Ken
K
Klotz
Charles J
CJ
Flickinger
John C
JC
Herr
John C
JC
Herr
D054758:A Kinase Anchor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013082:Sperm Tail / Q000378:metabolism* / Q000648:ultrastructure; D020798:Two-Hybrid System Techniques; D020741:rho GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D054758:A Kinase Anchor Proteins; C119530:AKAP3 protein, human; C448922:CABYR protein, human; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D010750:Phosphoproteins; C404565:ROPN1 protein, human; D011993:Recombinant Fusion Proteins; D020741:rho GTP-Binding Proteins
Mol Biol Cell. 1998 Feb;9(2):263-76
9450953
Biol Reprod. 2005 Jan;72(1):22-32
15342355
Microsc Res Tech. 2003 May 1;61(1):103-15
12672126
J Mol Biol. 2003 Mar 28;327(3):609-18
12634056
Reproduction. 2002 Dec;124(6):767-74
12530914
Biol Reprod. 1992 May;46(5):869-78
1375514
Mol Reprod Dev. 1996 Jan;43(1):82-93
8720117
J Cell Sci. 2000 Jan;113 ( Pt 1):103-12
10591629
Cell Motil Cytoskeleton. 2008 Jul;65(7):539-52
18421703
Reprod Biol Endocrinol. 2004 Jul 16;2:57
15257753
Reprod Biol Endocrinol. 2010 Aug 23;8:101
20731842
Eur J Biochem. 1997 Jun 1;246(2):425-32
9208934
Biol Reprod. 2001 Jul;65(1):277-87
11420250
Biol Reprod. 1995 Sep;53(3):579-90
7578682
Dev Biol. 2005 Oct 1;286(1):46-56
16139264
Dev Biol. 2002 Feb 15;242(2):236-54
11820818
Gamete Res. 1989 Feb;22(2):205-17
2707727
Biol Reprod. 1998 Mar;58(3):834-41
9510974
J Biol Chem. 2007 Nov 23;282(47):34104-19
17855365
Mol Endocrinol. 1999 May;13(5):705-17
10319321
Biol Reprod. 2006 Aug;75(2):270-8
16687649
Dev Biol. 1994 Sep;165(1):272-84
8088444
Biol Reprod. 2006 Jan;74(1):109-18
16177223
J Biol Chem. 2001 May 18;276(20):17332-8
11278869
Fertil Steril. 1990 Sep;54(3):527-9
2397797
Dev Biol. 1975 Jun;44(2):394-436
805734
J Biol Chem. 2003 Mar 28;278(13):11579-89
12509440
Biol Reprod. 2003 Jun;68(6):2241-8
12606363
Mol Reprod Dev. 1995 Dec;42(4):415-24
8607970
Mol Reprod Dev. 1998 Apr;49(4):374-85
9508088
Dev Biol. 2007 Feb 15;302(2):463-76
17137571
Biochem Biophys Res Commun. 2005 Apr 15;329(3):1108-17
15752768
Biol Reprod. 2008 Jan;78(1):184-92
17928627
Curr Biol. 2001 Oct 30;11(21):1686-90
11696326
Biol Reprod. 1993 Aug;49(2):191-203
8396993
Biol Reprod. 1999 Nov;61(5):1184-97
10529264