CASK interacts with PMCA4b and JAM‐A on the mouse sperm flagellum to regulate Ca²⁺ homeostasis and motility

Abstract

Deletion of the highly conserved gene for the major Ca2+ 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 Ca2+ concentration ([Ca2+]c) and ~10-fold higher mitochondrial sequestration, indicating Ca2+ overload. Investigating the mechanism involved, we used coimmunoprecipitation studies to show that CASK (Ca2+/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 Ca2+ 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 Ca2+ homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions.

Plasma membrane calcium ATPase 4 (PMCA4) co‐ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity

“…Thus, when WT and nulls were compared, UNCAP and CAP, the median fluorescence units for each increased ∼2-fold for nulls versus WT-sperm (Figure 5a,b). 4 | DISCUSSION 4.1 | PMCA4 and the NOSs co-localize and intimately interact in murine sperm in a Ca 2+ -dependent manner While PMCA4 (Aravindan et al, 2012;Wennemuth et al, 2003) and nNOS (Herrero, Perez Martinez, Viggiano, Polak, & Gimeno, 1996) have been localized in murine sperm on the head, over the acrosome, Figure 1a, we show that nNOS is more abundant and more widely distributed on the sperm, being found in the distal principal piece where eNOS and PMCA4 do not reside. In the present study, the pre-and post-bleaching images for the FRET data revealed the close interaction of PMCA4 and the NOSs on the sperm head, neck (Figures 2 and 3), and on the proximal principal piece of the flagellum, reflecting their co-localization.…”

“…There was a greater that twofold increase in fluorescence units for the median of each null population studied (Figure 4a). Jam-A null sperm were also studied for NOS activity since they have significantly reduced PMCA4 activity (Aravindan et al, 2012). In WT-sperm, NOS activity was shown to be tightly regulated at high [Ca 2+ ] c in CAP sperm, with the mean fluorescence units being <5% greater than that in UNCAP sperm.…”

“…Sperm protein extracts were used for co-immunoprecipitation assays as previously described from our Lab (Aravindan et al, 2012;Modelski et al, 2014). Briefly, 50 μl of PureProteome Protein G Magnetic Beads (Millipore Corp, Billerica, MA) were washed twice with 1 ml PBS + 0.01% Tween 20 by vortexing, and recovered by centrifugation at 2,500g for 5 min.…”

“…This down-regulation is necessary since the NOSs are rapidly activated in the presence of elevated [Ca 2+ ] c (Knowles & Moncada, 1994). To assess the importance of the PMCA4-NOS interaction on NO regulation, NOS levels were determined in Pmca4 null and Jam-A null sperm where PMCA4 activity is significantly decreased (Aravindan et al, 2012). The Figure 4 show NOS activity levels that are elevated ∼2-fold for both CAP and UNCAP Pmca4 null sperm versus WT, while for Jam-A nulls the elevation was seen only in CAP sperm, indicating PMCA4's role in regulating NOS activity and NO levels in sperm.…”

“…lacking Jam-A (Shao, Ghosh, Cooke, Naik, & Martin-DeLeon, 2008) and have shown that it is associated with decreased activity of PMCA4 (Aravindan et al, 2012).…”