Protein kinase C is an important signaling mediator associated with motility of intact sea urchin spermatozoa

Urology Research Laboratory, Royal Victoria Hospital, McGill University Health Center and Faculty of Medicine, McGill University, Montréal, H3A 1A1, Canada.
Journal of Experimental Biology (Impact Factor: 2.9). 12/2007; 210(Pt 22):4053-64. DOI: 10.1242/jeb.007013
Source: PubMed


Numerous kinases and phosphatases are most likely implicated in sperm motility initiation and maintenance. Data on these signaling molecules were mostly obtained from studies conducted on in vitro demembranated-reactivated sperm models but are not necessarily representative of the in vivo situation. We therefore investigated the effect of a variety of cell-permeable chemicals, mostly kinase inhibitors, on the motility initiation and maintenance of intact sea urchin spermatozoa. Among the 20 substances tested, the protein kinase C (PKC) inhibitor chelerythrine was the most potent, arresting motility at concentrations starting from 1.5-2 mumol l(-1). Motility was also inhibited by two other PKC inhibitors as well as staurosporine. Furthermore, these inhibitors prevented the motility-associated increase in phosphorylation of at least four PKC substrates. These phospho-PKC target proteins, as assessed with an antibody specific to phosphorylated motifs of PKC substrates, were found to be associated with the flagellum, either in the Triton X-100 soluble portion or the axoneme (Triton X-100 insoluble). A phosphorylated PKC-like enzyme was also detected by immunoblotting in the flagellum, as well as a significant 50 kDa PKC cleavage product. Taken together, the data strongly indicate for the first time that, in vivo, which means on intact spermatozoa, PKC is a key signaling mediator associated with the maintenance of sea urchin sperm motility.

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Available from: Eve de Lamirande, Apr 13, 2014
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    • "It has also been reported that PKC is essential for the maintenance of sea urchin sperm motility, although other kinases such as PKA and tyrosine kinase are present, but not key for motility (White et al., 2007). Intriguingly, this article also reported the presence of PKM, the nonmembrane-bound catalytic subunit of PKC in the sea urchin sperm. "
    Meiosis - Molecular Mechanisms and Cytogenetic Diversity, 02/2012; , ISBN: 978-953-51-0118-5
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    • "Finally, one of the interesting aspects of our study is that it parallels earlier work on the role of PKC in flagellar motility of mammalian sperm and spermatozoa of aquatic vertebrates (Rotem et al. 1990; White et al. 2007). Our findings suggest that the requirement of PKC activity may be a common mechanism for maintaining motility of all these cross-kingdom specialized cells. "
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    ABSTRACT: The motility of zoospores is critical in the disease cycles of Peronosporomycetes that cause devastating diseases in plants, fishes, vertebrates, and microbes. In the course of screening for secondary metabolites, we found that ethyl acetate extracts of a marine Streptomyces sp. strain B5136 rapidly impaired the motility of zoospores of the grapevine downy mildew pathogen Plasmopara viticola at 0.1 μg/ml. The active principle in the extracts was identified as staurosporine, a known broad-spectrum inhibitor of protein kinases, including protein kinase C (PKC). In the presence of staurosporine (2 nM), zoospores moved very slowly in their axis or spun in tight circles, instead of displaying straight swimming in a helical fashion. Compounds such as K-252a, K-252b, and K-252c structurally related to staurosporine also impaired the motility of zoospores in a similar manner but at varying doses. Among the 22 known kinase inhibitors tested, the PKC inhibitor chelerythrine was the most potent to arrest the motility of zoospores at concentrations starting from 5 nM. Inhibitors that targeted kinase pathways other than PKC pathways did not practically show any activity in impairing zoospore motility. Interestingly, both staurosporine (5 nM) and chelerythrine (10 nM) also inhibited the release of zoospores from the P. viticola sporangia in a dose-dependent manner. In addition, staurosporine completely suppressed downy mildew disease in grapevine leaves at 2 μM, suggesting the potential of small-molecule PKC inhibitors for the control of peronosporomycete phytopathogens. Taken together, these results suggest that PKC is likely to be a key signaling mediator associated with zoosporogenesis and the maintenance of flagellar motility in peronosporomycete zoospores.
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    • "In order to test this hypothesis, we examined mitochondrial lysates by Western blotting with an antibody that recognises specifically the motif that conventional PKCs (cPKC) phosphorylate [40]. This phospho-(serine) PKC substrate antibody has been shown to detect endogenous levels of known and unknown proteins, when phosphorylated at serine residues surrounded by arginine or lysine at the − 2 and + 2 positions and a hydrophobic residue at the + 1 position [41]. We used mitochondrial lysates from MCF-7 cells that had been transfected with pOMML-PLCδPHd (70% of cells were GFP positive as judged by fluorescence microscopy), pOMML-EGFP control cells, chelerythrine chloride (a selective inhibitor of group A and B PKC isoforms) [42] treated cells and amino acid starved cells. "
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