CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility.
Deletion of the highly conserved gene for the major Ca(2+) efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.001) greater cytosolic Ca(2+) concentration ([Ca(2+) ](c)) and ∼10-fold higher mitochondrial sequestration, indicating Ca(2+) overload. Investigating the mechanism involved, we used co-immunoprecipitation studies to show that CASK (Ca(2+) /calmodulin-dependent serine kinase), identified for the first time on the sperm flagellum where it co-localizes with both PMCA4b and JAM-A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non-synergistically with each of these molecules via its single PDZ (PDS-95/Dlg/ZO-1) domain to either inhibit or promote efflux. In the absence of CASK-JAM-A interaction in Jam-A null sperm, CASK-PMCA4b interaction is increased, resulting in inhibition of PMCA4b's enzymatic activity, consequent Ca(2+) accumulation, and a ∼6-fold over-expression of constitutively ATP-utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM-A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca(2+) homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions.
Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA. pdeleon@udel.edu
Rolands G
RG
Aravindan
Victor P
VP
Fomin
Ulhas P
UP
Naik
Mark J
MJ
Modelski
Meghna U
MU
Naik
Deni S
DS
Galileo
Randall L
RL
Duncan
Patricia A
PA
Martin-Deleon
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D051528:Guanylate Kinases / Q000378:metabolism*; D007246:Infertility / Q000235:genetics* / Q000378:metabolism; D008297:Male; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008810:Mice, Inbred C57BL; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism*; D059010:Single-Cell Analysis; D013081:Sperm Motility / Q000235:genetics; D013082:Sperm Tail / Q000378:metabolism; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism
D015220:Calcium Channels; C488642:Catsper1 protein, mouse; D015815:Cell Adhesion Molecules; C498686:F11r protein, mouse; D011956:Receptors, Cell Surface; D000255:Adenosine Triphosphate; C106868:CASK kinases; D051528:Guanylate Kinases; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium