Abstract

The acidic proteins 14-3-3 are a family of ubiquitously expressed polypeptides, highly conserved from yeast to mammals. 14-3-3 proteins play an important role in multiple signaling pathways like cell cycle regulation, apoptosis, synaptic vesicle dynamics etc. 14-3-3 proteins have a regulatory role mediated mostly if not exclusively, by their ability to bind to signaling proteins and enzymes with phosphoserine and phosphothrenonine residues with RSXpSXP and RX(Y/F)XpSXP motifs. We showed for the first time that 14-3-3 zeta is expressed in spermatozoa and that it binds to protein phosphatase PP1 gamma2, a key enzyme involved in sperm morphogenesis and motility. Far western analysis with recombinant 14-3-3 and western blot analysis with 14-3-3 binding motif antibodies showed that in addition to PP1 gamma2 there are other sperm 14-3-3 binding proteins. The purpose of this study was to isolate and identify the 14-3-3 binding proteins in bovine caudal epididymal spermatozoa. Affinity chromatography with recombinant GST- 14-3-3 covalently coupled to sepharose was used. Caudal sperm extracts precleared with GST were passed through the GST 14-3-3 sepharose column. After extensive low and high salt washings; bound proteins were eluted with R-11, a peptide known to disrupt 14-3-3 complexes. Strong immunoreactivity with phosphoserine 14-3-3 binding motif and PP1 gamma2 antibodies confirmed the validity of the approach. Eluted proteins were concentrated, subjected to SDS-gel electrophoresis following identification by tandem mass spectroscopy. Micro sequencing revealed various metabolic enzymes such phosphoglycerate kinase, mitochondrial Fo- ATPase, citrate synthase, malate dehydrogenase, nucleoside diphosphate kinase etc. Various acrosomal proteins like PH-20, acrosin, acrosin binding protein, sperm acrosomal membrane protein were also identified as 14-3-3 interacting proteins. Other proteins known to be important for sperm function like glycogen synthase kinase-3, angiotensin converting enzyme, heat shock protein 70, kelch like 10 protein were also identified. These 14-3-3 binding sperm proteins are likely to be phosphoproteins. Our data suggest that 14-3-3 zeta might have direct or regulatory role in metabolism and signaling mechanisms in mammalian spermatozoa. (poster)

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