FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis

Abstract

We report characterization of a novel testis-and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.Hyperactivated motility along with changes in the sperm head that confer the capacity to fertilize an oocyte result from a series of time-dependent processes collectively referred to as capacitation (1, 2). Although the molecular mechanisms underlying capacitation, including hyperactivation, are incompletely understood, some fundamental processes related to capacitation have been elucidated.The presence of a protein source such as albumin, bicarbonate, and Ca 2ϩ and an energy substrate such as glucose, pyruvate, or lactate are essential to achieve in vitro capacitation (3). Capacitation is also marked by an increase in tyrosine phosphorylation through a unique signal transduction cascade involving a sperm-specific soluble adenylyl cyclase, protein kinase A (PKA), 3 and tyrosine kinase(s). A variety of factors, including Ca 2ϩ, HCO 3 Ϫ , and H 2 O 2 stimulate soluble adenylyl cyclase leading to increased cytosolic levels of cAMP (4). This increase in cAMP then activates PKA, and the consequence is a significant increase in tyrosine phosphorylation of protein substrates localized in the flagellum such as AKAP3, AKAP4, CABYR, hsp-90, ODF2, and tubulin (3,(5)(6)(7)(8)(9)(10)(11)(12).The fact that PKA is a key regulator of capacitation-associated changes such as protein phosphorylation and hyperactivation has been well established. Pharmacological stimulants that elevate intracellular cAMP, such as the phosphodiesterase inhibitors, caffeine, and pentoxifylline, enhance hyperactivated motility of sperm (13). cAMP agonists can accelerate protein tyrosine phosphorylation in sperm, whereas antagonists of PKA inhibit tyrosine phosphorylation and capacitation (14). Mice in which sperm-specific PKA ␣ is knocked out are infertile, owing to the complete absence of normal sperm movement...

CABYR is essential for fibrous sheath integrity and progressive motility in mouse spermatozoa

“…As the name suggests, FSCB binds directly to CABYR (Li et al, 2007), and research has shown that it is phosphorylated by PKA and that it binds to Ca 2+ , placing it firmly in the centre of sperm motility pathways (Liu et al, 2011;Zhang et al, 2016). In light of the findings of this research, FSCB would be a very promising candidate for future research, and with the speed and ease of the CRISPR-Cas9 system, it should not take too long to discover the function in vivo.…”

FSCB phosphorylation in mouse spermatozoa capacitation

“…Herein we describe findings that indicate FSCB is a novel protein expressed specifically on the surface of the fibrous sheath of the mouse spermatozoa principal piece (6). FSCB is a calcium-binding protein, which can be phosphorylated by PKA in vitro.…”

“…We reported previously that FSCB is phosphorylated in vitro by the PKA catalytic subunit, and that it possesses multiple phosphorylation sites (6). To further investigate whether FSCB is phosphorylated during spermatozoa capacitation, spermatozoa before and at 2 h after capacitation were lysed and analyzed by Western blotting.…”

“…Spermatozoa (approximately 2 × 10 8 cells/ml) were cultured in pre-equilibrated HTF and M2 media in a CO2 incubator at 37 o C for 2 h and lysed in 200 μl Celis buffer (6). FSCB is a high molecular weight protein with low solubility.…”

The protein and transcript profiles of human semen

“…A total of 30 of the 322 specific RNAs retained in sperm were not identified as proteins during proteomic analyses (reviewed in Amaral et al 2014), but have been identified in sperm by western blot or in situ hybridization. This is best exemplified by PRM1 and FSCB (Li et al 2007;Jodar and Oliva 2014). Of the RNAs that remain as sperm-specific yet protein-absent, approximately 20 % correspond to non-coding RNAs including pseudogenes, intronic elements and long non-coding RNAs.…”