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

Yan-Feng Li,Wei He,Kula N Jha,Ken Klotz,Young-Hwan Kim,Arabinda Mandal,Silvia Pulido,Laura Digilio,Charles J Flickinger,John C Herr

doi:10.1074/jbc.m702238200

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.

Highlights

The presence of a protein source such as albumin, bicarbonate, and Ca2ϩ and an energy substrate such as glucose, pyruvate, or lactate are essential to achieve in vitro capacitation [3]
Capacitation is 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)
This increase in cAMP 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]

Summary

EXPERIMENTAL PROCEDURES

Immunoprecipitation and Mass Spectrometric Analysis of CABYR-binding Proteins—Immunoprecipitations were performed with an immunoprecipitation kit (Roche Applied Science) using 2 ϫ 108 sperm for each tube and 20 ␮l of polyclonal anti-CABYR CR-A (CABYR coding region A protein) or preimmune serum following instructions from the manufacturer. Immune complexes from a duplicate set of the immunoprecipitations (including anti-CABYR CR-A and pre-immune serum) were sent for mass spectrometry These samples were washed three times with digestion buffer to remove any remaining Nonidet P-40, reduced with DTT and alkylated with iodoacetamide before adding 1 ␮g of Promega-modified trypsin (Promega, Madison, WI) overnight at room temperature. The specimens were incubated with anti-FSCB serum or pre-immune serum (1:400 dilution) in PBS-T containing 1% BSA (PBS-T-BSA) for 1 h at room temperature or overnight at 4 °C, washed 3 ϫ 5 min in PBS-T, incubated with fluorescein isothiocyanate-labeled goat antiguinea pig IgG (1:400 dilution, Jackson ImmunoResearch) in PBS-T-BSA for 1 h, washed 3 ؋ 5 min in PBS-T, and mounted with Slow Fade (Molecular Probes, Eugene, OR).

RESULTS

DISCUSSION

Phosphorylated amino acid residue and location

DDQISTFK VLNLPTDLFNSVMNVGRQVCIPPELPELLKLIIHADEL