Certain Strongylocentrotus purpuratus sperm mitochondrial proteins co-purify with low density detergent-insoluble membranes and are PKA or PKC-substrates possibly involved in sperm motility regulation.
BACKGROUND
Sea urchin sperm motility is regulated by Speract, a sperm-activating peptide (SAP) secreted from the outer egg coat. Upon binding to its receptor in the sperm flagellum, Speract induces a series of ionic and metabolic changes in Strongylocentrotus purpuratus spermatozoa that regulate their motility. Among these events, protein phosphorylation is one of the most relevant and evidence indicates that some proteins of the Speract signaling cascade localize in low density detergent-insoluble membranes (LD-DIM).


METHODS
LD-DIM-derived proteins from immotile, motile or Speract-stimulated S. purpuratus sperm were resolved in 2-D gels and the PKA and PKC substrates detected with specific antibodies were identified by LC-MS/MS.


RESULTS
Differential PKA and PKC substrate phosphorylation levels among the LD-DIM isolated from sperm in different motility conditions were found and identified by mass spectrometry as: ATP synthase, creatine kinase, NADH dehydrogenase (ubiquinone) flavoprotein 2, succinyl-CoA ligase and the voltage-dependent anion channel 2 (VDAC2), which are mitochondrial proteins, as well as, the cAMP-dependent protein kinase type II regulatory (PKA RII) subunit, Tubulin β chain and Actin Cy I changed their phosphorylation state.


CONCLUSIONS
Some mitochondrial proteins regulated by PKA or PKC may influence sea urchin sperm motility.


CONCLUSIONS
The fact that a high percentage (66%) of the PKA or PKC substrates identified in LD-DIM are mitochondrial proteins suggests that the phosphorylation of these proteins modulates sea urchin sperm motility via Speract stimulation by providing sufficient energy to sperm physiology. Those mitochondrial proteins are indeed PKA- or PKC-substrates in the sea urchin spermatozoa.
Departamentos de Genética del Desarrollo y Fisiología Molecular, Universidad Nacional Autónoma de México, Cuernavaca, Morelos CP 62210, Mexico.
Arlet
A
Loza-Huerta
Rosario
R
Vera-Estrella
Alberto
A
Darszon
Carmen
C
Beltrán
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D008297:Male; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D012617:Sea Urchins; D015398:Signal Transduction; D013081:Sperm Motility / Q000502:physiology*; D013082:Sperm Tail / Q000378:metabolism / Q000502:physiology; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D047270:Strongylocentrotus purpuratus / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
D003902:Detergents; D024101:Mitochondrial Proteins; D009842:Oligopeptides; C028364:speract; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C
ASW; Artificial sea water; CK; LC–MS/MS; LD-DIM; Mitochondria; PKA; PKC; Protein phosphorylation; SAP; Sea urchin sperm; Speract; Sperm motility; Sperm-Activating Peptide; VDAC; cAMP-dependent protein kinase; creatine kinase; low density detergent-insoluble membranes; protein kinase C; tandem mass spectrometry; voltage-dependent anion channel