Proteomic analysis of bovine sperm YWHA binding partners identify proteins involved in signaling and metabolism

Abstract

Posttranslational modification of proteins by phosphorylation is involved in regulation of sperm function. Protein phosphatase 1 gamma isoform 2 (PPP1CC_v2) and protein YWHA (also known as 14-3-3) are likely to be key molecules in pathways involving sperm protein phosphorylation. We have shown that phosphorylated PPP1CC_v2 is bound to protein YWHAZ in spermatozoa. In somatic cells, protein YWHA is known to bind a number of phosphoproteins involved in signaling and energy metabolism. Thus, in addition to PPP1CC_v2, it is likely that sperm contain other YWHA-binding proteins. A goal of the present study was to identify these sperm YWHA-binding proteins. The binding proteins were isolated by affinity chromatography with GST-YWHAZ followed by elution with a peptide, R-11, which is known to disrupt YWHA complexes. The YWHA-binding proteins in sperm can be classified as those involved in fertilization, acrosome reaction, energy metabolism, protein folding, and ubiquitin-mediated proteolysis. A subset of these putative YWHA-binding proteins contain known amino acid consensus motifs, not only for YWHA binding but also for PPP1C binding. Identification of sperm PPP1CC_v2-binding proteins by microcystin-agarose chromatography confirmed that PPP1CC_v2 and YWHA interactomes contain several common proteins. These are metabolic enzymes phosphoglycerate kinase 2, hexokinase 1, and glucose phosphate isomerase; proteins involved in sperm-egg fusion; angiotensin-converting enzyme, sperm adhesion molecule, and chaperones; heat shock 70-kDa protein 5 (glucose-regulated protein 78 kDa; and heat shock 70-kDa protein 1-like. These proteins are likely to be phosphoproteins and potential PPP1CC_v2 substrates. Our data suggest that in addition to potential regulation of a number of important sperm functions, YWHA may act as an adaptor molecule for a subset of PPP1CC_v2 substrates.

FIG. 1.

Presence of YWHA-binding proteins in bovine caput and caudal epididymal spermatozoa. A) Caput and caudal sperm extracts (20 μl each with a sperm concentration corresponding to 1 × 10⁹/ml) were subjected to SDS-PAGE, followed by Western blot analysis with phosphoserine YWHA-binding motif antibody, which recognizes putative YWHA-binding proteins. B) Caput and caudal sperm lysates (20 μl each with sperm concentration corresponding to 1 × 10⁹/ml) were resolved by SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with GST-YWHAZ or GST alone. After washing, blots were probed with GST antibody.

FIG. 2.

A) The scheme for protein purification by GST-YWHAZ affinity column chromatography. B) Caudal epididymal sperm extracts were subjected to affinity chromatography using GST Sepharose and GST-YWHAZ Sepharose as described in Materials and Methods. Proteins specifically bound to YWHA were eluted by R-11 peptide and concentrated. Concentrated YWHAZ column eluate (40 μl) was resolved by SDS-PAGE and stained with colloidal Coomassie blue (right lane) and compared to proteins contained in the original sperm lysate. Protein bands in the R-11 lane from the stained gel were identified by LC-MS (Table 1).

FIG. 3.

Detection of YWHA-binding proteins eluted from the YWHAZ affinity column by Western blot analysis. Total sperm lysate (20 μg protein) and R-11 eluate from YWHAZ affinity chromatography (10 μl) were resolved by SDS-PAGE and transferred to PVDF membrane, followed by Western blot analysis. Membranes were probed with PPP1CC_v2 (A) and phosphoserine YWHA-binding motif antibody (B).

FIG. 4.

Western blot analysis of the YWHAZ affinity column eluate for the presence of selected YWHA-binding proteins. Total sperm lysate (20 μg protein) and R-11 eluate from YWHAZ affinity chromatography (20 μl) were resolved by SDS-PAGE and transferred to PVDF membrane. Membranes were probed with antibody against GSK3A/B (A), glycolytic enzyme PGK2 (B), or the flagellar protein RSPH1 (C).

FIG. 5.

ACE and PPP1CC_v2 coprecipitate with YWHA. YWHA protein complexes from caudal sperm extracts were immunoprecipitated using YWHAB antibody as described in Materials and Methods. Purified rabbit IgG was used as a control. Immunoprecipitated complexes captured on the protein A beads were eluted by boiling in 2× sample buffer and were analyzed by Western blot analysis. The PVDF membranes were probed with antibodies against ACE and YWHA (A) or antibodies against PPP1CC_v2 and YWHA (B). IP, Immunoprecipitation.

FIG. 6.

PGK2, YWHA and ACE coprecipitate with PPP1CC_v2. Protein complexes from caudal sperm extracts were immunoprecipitated using PPP1CC_v2 antibody or purified rabbit IgG (control), as described in Materials and Methods. Immunoprecipitaed complexes captured on the protein A beads were eluted by boiling in 2× sample buffer and were analyzed by Western blot analysis. A) PVDF membranes were probed first with PGK2 antibody, and later the same membrane was reprobed with PPP1CC_v2 and YWHA antibody). B) A separate set of immunoprecipitation experiments using control rabbit IgG and PPP1CC_v2 was done, followed by Western blot analysis with ACE and PPP1CC_v2 antibodies.