Tandem mass spectrometry identifies proteins phosphorylated by cyclic AMP-dependent protein kinase when sea urchin sperm undergo the acrosome reaction.
The exocytotic acrosome reaction (AR), which is required for fertilization, occurs when sea urchin sperm contact the egg jelly (EJ) layer. Among other physiological changes, increases in adenylyl cyclase activity, cAMP and cAMP-dependent protein kinase (PKA) activity occur coincident with the AR. By using inhibitors of PKA, a permeable analog of cAMP and the phosphodiesterase inhibitor IBMX, we show that PKA activity is required for AR induction by EJ. A minimum of six sperm proteins are phosphorylated by PKA upon exposure to EJ, as detected by a PKA substrate-specific antibody. The phosphorylation of these proteins and the percentage of acrosome reacted sperm can be regulated by PKA modulators. The fucose sulfate polymer (FSP), a major component of EJ, is the molecule that triggers sperm PKA activation. Extracellular Ca(2+) is required for PKA activation. Six sperm proteins phosphorylated by PKA were identified by tandem mass spectrometry (MS/MS) utilizing the emerging sea urchin genome. Based on their identities and localizations in sperm head and flagellum, the putative functions of these proteins in sperm physiology and AR induction are discussed.
Su
Yi-Hsien
YH
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California-San Diego, La Jolla, CA 92093-0202, USA. yhsu@ucsd.edu
Chen
Sheng-Hong
SH
Zhou
Huilin
H
Vacquier
Victor D
VD
D020101:Acrosome Reaction; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D020013:Calcium Signaling; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005260:Female; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011134:Polysaccharides / Q000494:pharmacology; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D047270:Strongylocentrotus purpuratus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D010750:Phosphoproteins; D011134:Polysaccharides; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium