Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm.

Abstract

Abstract. Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca 2÷ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KC1 from isolated flagella from dog, pig, and sea urchin sperm. All saltextractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.T rlE apparent requirement of cAMP-dependent protein phosphorylation for flagellar motility (Tash et al., 1984(Tash et al., , 1986) is contrasted by the inhibitory and waveform modulating effects of Ca 2+ (Hyams and Borisy, 1978;Gibbons, 1980). Considerable effort has been exerted by a number of laboratories concerning possible mechanisms of action of Ca 2÷ on the regulation of flagellar movement (Satir, 1985;Gibbons, 1982;Brokaw et al., 1974;Bessen et al., 1980). More recent attention has concentrated on calmodulin as the potential primary signal transducer in Ca 2+-regulated flagellar motility. However, results have been contradictory, which may result from differences in species as well as the techniques used (Gitelman and Witman, 1980;Blum et al., 1980). Perhaps the most compelling evidence to date to suggest an involvement of calmodulin in this regulation comes from the observations of Brokaw and Nagayama (1985). The asymmetry-inducing effects of calmodulin-conraining flagellar extracts were eliminated by passing the extracts through trifluoperazine columns. Furthermore, they found that purified calmodulin reverses the symmetric swimming patterns of sperm demembranated in the presence of Ca 2+ to produce asymmetric flagellar bending. While evidence suggesting an involvement of calmodulin in the regulation of flagellar motility is increasing, a defined calmodulindependent biochemical pathway, which could explain the effects of Ca2+-calmodulin on motility, remains to be elucidated and characterized, The present study reports the identification of calmodulindependent protein phosp...

The radial spokes and central apparatus: Mechano‐chemical transducers that regulate flagellar motility

“…Motility changes mediated by these second messengers include inducing quiescence in beating flagella, activating motility, increasing beat frequency, reversing the direction of the effective stroke, or changing waveform. The effect of second messengers is mediated at least in part through second-messenger-dependent kinases and phosphatases, such as cAMP-dependent protein kinase A (PKA), cGMP-dependent protein kinase (PKG), calmodulin dependent kinase, and calcineurin (PP2B) [Tash and Means, 1983;Tash et al, 1988;Chaudhry et al, 1995;San Agustin et al, 1998;Smith, 2002a]. It is possible that some second messengers affect motility by additional mechanisms that are independent of phosphorylation.…”

Regulation of organelle transport in melanophores by calcineurin.

“…3 and 4}, CN may be the phosphatase required for aggregation. If so, then specific inhibition of CN activity by anti-CN (Tash et al, 1988;Klee, C. B., personal communication) should result in loss of the melanophores' ability to aggregate pigment.…”

“…Taxol was the generous gift of Dr. M. Suffness, National Institutes of Health (NIH). Bovine brain CN and anti-CN (a rabbit polyclonal antibody used either as an ammonium sulfate-precipitated IgG fraction or as a CN affinity column-purified fraction) were the generous gift of Claude B. Klee, NIH; the anti-CN has been characterized previously (Tash et al, 1988). Bovine brain CN was also purchased from Sigma Chemical Co. (St. Louis, MO); all other chemicals were purchased from Sigma Chemical Co.…”

“…CN was originally isolated from brain (Klee et al, 1979), is identical to phosphatase 2B (Stewart et al, 1982;Ingebritsen and Cohen, 1983a,b), and has a wide phylogenetic and tissue distribution in eukaryotes (Tallant and Cheung, 1983;Klee et al, 1988). It is possible that the regulatory role of Ca 2÷ in axonemes is mediated via CN-induced protein dephosphorylation (Tash et al, 1988). Potentially, in organelle transport in melanophores, the cAMP-dependent phosphorylation events required for dispersion are reversed by Ca 2÷ activation of CN to generate protein dephosphorylation and, thus, aggregation.…”

“…Recently, it has been demonstrated that wave form can be affected by the Ca2÷/calmodulin (CaM)l-stimulated phosphatase, calcineurin (CN) (Tash et al, 1988). CN was originally isolated from brain (Klee et al, 1979), is identical to phosphatase 2B (Stewart et al, 1982;Ingebritsen and Cohen, 1983a,b), and has a wide phylogenetic and tissue distribution in eukaryotes (Tallant and Cheung, 1983;Klee et al, 1988).…”