FSCB phosphorylation in mouse spermatozoa capacitation

Abstract

It is generally accepted that spermatozoa capacitation is associated with protein kinase A-mediated tyrosine phosphorylation. In our previous study, we identified the fibrous sheath CABYR binding protein (FSCB), which was phosphorylated by PKA. However, the phosphorylation status of FSCB protein during spermatozoa capacitation should be further investigated. To this aim, in this study, we found that phosphorylation of this 270-kDa protein occurred as early as 1 min after mouse spermatozoa capacitation, which increased over time and remained stable after 60 min. Immunoprecipitation assays demonstrated that the tyrosine and Ser/Thr phosphorylation of FSCB occurred during spermatozoa capacitation. The extent of phosphorylation and was closely associated with the PKA activity and spermatozoa motility characteristics. FSCB phosphorylation could be induced by PKA agonist DB-cAMP, but was blocked by PKA antagonist H-89.Therefore, FSCB contributes to spermatozoa capacitation in a tyrosine-phosphorylated format, which may help in further elucidating the molecular mechanism of spermatozoa capacitation. [BMB reports 2011; 44(8): 541-546]

Loss of R2D2 Proteins ROPN1 and ROPN1L Causes Defects in Murine Sperm Motility, Phosphorylation, and Fibrous Sheath Integrity1

“…4B). Combined with coimmunoprecipitation results presented by Liu et al [38] that identify FSCB as a PKA-phosphorylated substrate during sperm capacitation, our data strongly suggest that the identity of the p270 PKAphosphorylated bands we detected is FSCB (although immunoprecipitation followed by mass spectrometry analysis would be necessary to prove this point). Additionally, these data show that FSCB was expressed at normal levels in ROPN1L-knockoutsperm, indicating that the decrease in cross-reaction with the phospho-PKA antibody was due to a decrease in the level of phosphorylation (Fig.…”

“…FSCB is located primarily on the outer surface of the FS and is incorporated late in FS formation. Previously published studies indicate that ROPN1 and CABYR (which binds FSCB) are part of a shared complex in vivo, although whether the interaction between the two is direct or mediated through AKAPs is not certain [30,38,39]. Our data indicating that FSCB is lost in sperm lacking ROPN1 whereas expression of the major sperm AKAPs 3 and 4 is unchanged suggest that either ROPN1 binds directly to FSCB to tether it to the FS, or it binds to CABYR directly (perhaps through heterodimerization) to accomplish the same.…”

“…Recently, two separate groups have characterized a 270-kDa sperm protein that appears to be a direct target for PKA phosphorylation both in vitro and in vivo during capacitation, with one group identifying the protein as FSCB [30,35,38]. CABYR, like ROPN1, is an R2D2 protein located in the FS; during in vitro sperm capacitation it is phosphorylated on both tyrosine and serine/threonine residues [31,32,39,40].…”

“…4A). FSCB is a 270-kDa sperm protein localized in the FS that has recently been identified as a direct PKA substrate during capacitation [30,35,38]. To investigate whether the PKA substrate that is seen to be phosphorylated in Figure 4A could be FSCB, and also to determine whether the decrease in phospho-bands is due to decreased phosphorylation levels or, alternatively, due to decreased FSCB expression, we performed Western blot analyses with FSCB antibody.…”

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.…”