Regulation of sperm flagellar motility by calcium and cAMP-dependent phosphorylation

There is substantial evidence that cAMP-dependent phosphorylation is involved in the activation of motility of spermatozoa as they are released from storage in the male reproductive tract. This evidence includes observations that in vivo activation of motility can be inhibited by protein kinase inhibitors, can be reversed by protein phosphatase treatment of demembranated spermatozoa, and is associated with phosphorylation of sperm proteins, and observations that spermatozoa that have not been activated in vivo can be activated in vitro by cAMP-dependent phosphorylation. Activation in vivo can often be triggered by conditions that increase intracellular pH, but the relevance of this to in vivo activation under natural conditions and the steps between pH increase and cAMP increase have not been fully established. The relationships between changes in the protein substrates for cAMP-dependent phosphorylation and changes in axonemal function are still unknown. Sperm chemotaxis to egg secretions is widespread; in the sea urchin Arbacia, the egg jelly peptide resact has been identified as a chemoattractant. Response to chemoattractants involves changes in asymmetry of flagellar bending waves, and similar changes in asymmetry can be produced in vitro by increases in [Ca++]. Temporal changes in resact receptor occupancy might lead to transient changes in intracellular [Ca++] and the asymmetry of flagellar bending, but many links in this hypothetical sequence remain to be established. Both of these signalling systems offer immediate opportunities for investigations of biochemical pathways leading to easily assayable biological responses. However, complications resulting from interactions between these two systems need to be considered.     

Regulation of flagellar glycoprotein movements by protein phosphorylation

Cross-linking of surface exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. A number of observations suggest that active movements of flagellar membrane glycoproteins are associated with the processes of whole cell gliding motility and the early events of fertilization in Chlamydomonas. Protein redistribution is totally inhibited if the free calcium concentration in the medium is 10(-7) M or below or in the presence of a number of calcium channel blockers (Bloodgood, R. A., N. L. Salomonsky, J. Cell Sci. 96, 27-33 (1990]. The present report demonstrates that glycoprotein redistribution in vivo is inhibited reversibly by three different protein kinase inhibitors: H-7, H-8 and staurosporine. Taken together, these observations suggest that the flagellum uses a signaling pathway that involves calcium influx induced by glycoprotein cross-linking, calcium activation of a protein kinase and specific protein phosphorylation to initiate flagellar surface dynamics.     

Regulation of spermidine acetyltransferase activity by phosphorylation and dephosphorylation

Spermidine acetyltransferase activity is more than 10-fold higher in the pancreas of a 20-hr-fasted than in that of a fed chicken. The preparation of the fed bird inactivates the other. The effect is due to a thermolabile component of microsomes, and is also obtained with alkaline phosphatase. The inactivated preparation partially recovers its activity through phosphorylation catalyzed by a cAMP-dependent protein kinase. The results presented strongly suggest that spermidine acetyltransferase activity is regulated by phosphorylation and dephosphorylation.     

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.     

Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility

Using a selective inhibitor of cAMP-dependent protein kinase, N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the requirement for cAMP-dependent phosphoproteins in the initiation of dog sperm flagellar motility was examined. H-8 inhibited motility of live as well as reactivated sperm in a dose-dependent manner. The half-maximal inhibition of reactivated motility (32 microM) paralleled the inhibition of pure catalytic subunit of cAMP-dependent protein kinase (50 microM) measured under the same conditions. H-8 inhibited protein phosphorylation both in whole models and in isolated Nonidet P-40 (NP-40) extracts of sperm. Axokinin, the heat-stable NP-40-soluble protein whose phosphorylation is required for flagellar reactivation, represented 97% of the de novo phosphate incorporation in the NP-40 extract after stimulation by cAMP. 500 microM H-8 inhibited axokinin phosphorylation by 87%. When sperm were reactivated in the presence of up to 5 mM H-8 with NP-40 extract that had been prephosphorylated with cAMP-dependent protein kinase, then neither cAMP nor cAMP-dependent protein kinase activity was required for full flagellar reactivation. If sperm were rendered completely immotile by pretreatment with H-8, then the resulting model remained immotile in the continued presence of H-8 unless prephosphorylated axokinin was added. These results suggest that phosphorylated axokinin is not only required for flagellar reactivation but is sufficient as well.     

Regulation of a rat lung protein tyrosine kinase activity by reversible phosphorylation/dephosphorylation

Incubation of a partially purified protein tyrosine kinase from rat lung with Mg2+ and ATP resulted in about 10-15-fold activation of the enzyme activity as judged by the phosphorylation of poly(Glu:Tyr,4:1), an exogenous substrate. The activation was time dependent and was associated with the phosphorylation of a single protein band of 50 kDa. Phosphoamino acid analysis of the phosphorylated protein indicated that tyrosine was the amino acid being phosphorylated. Upon gel filtration on a Sephacryl S-200 column, the phosphorylated protein co-eluted with protein tyrosine kinase and ATP-binding activities, suggesting that all three activities are part of the same protein. In addition, pretreatment of the partially purified protein tyrosine kinase with alkaline phosphatase inhibited its enzyme activity which could be restored by reincubation with Mg2+ and ATP. These data suggest that a temporal relationship exists between the phosphorylation and the activation states of rat lung protein tyrosine kinase, and that the phospho- and dephospho- forms represent the active and inactive (or less active) forms, respectively, of the enzyme.     

Concerted regulation of protein phosphorylation and dephosphorylation by calmodulin

The multiple functions of calmodulin in brain bring to light an apparent paradox in the mechanism of action of this multifunctional regulatory protein: How can the simultaneous calmodulin stimulation of enzymes with opposing functions such as cyclic nucleotide phosphodiesterases and adenylate cyclase, which are responsible for the degradation and synthesis of cAMP, respectively, be physiologically significant? The same question applies to the simultaneous activation of protein kinases (in particular calmodulin kinase II) and a protein phosphatase (calcineurin). One could propose that the protein kinase(s) and the phosphatase may be located in different cells or in different cellular compartments, and are therefore not antagonizing each other. The same result could be achieved if the specific substrates of these enzymes have different cellular localizations. This does not seem to be the case. In many areas of the brain the two enzymes and their substrates coexist in the same cell. For example, the hippocampus is rich in calmodulin kinase II, calcineruin and substrates for the two enzymes. A more general scheme is presented here, based on different mechanisms of the calmodulin regulation of the two classes of enzyme, which helps to solve this apparent inconsistency in the mechanism of action of calmodulin.     

Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed.     

Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation

Acetyl CoA carboxylase, in a partially purified preparation, was inactivated by ATP in a time- and temperature-dependent reaction. Adenosine 3′,5′-monophosphate did not affect the inactivation. Further purification separated the carboxylase from a protein fraction which could greatly enhance the inactivation of the enzyme.Inactivation of the enzyme with [γ-³²P]ATP resulted in the incorporation of ³²P which copurified with the enzyme. No label was incorporated when [U-¹⁴C]ATP was used. When carboxylase inactivated by exposure to [γ-³²P]ATP was precipitated with antibody, isotope incorporation into the precipitate paralleled enzyme inactivation. The phosphate was bound to serine and threonine residues by an ester linkage.Sodium fluoride completely inhibited the activation of partially purified enzyme by magnesium ions. Activation by magnesium, accompanied by the release of protein-bound ³²P, was antagonistic to inactivation of the enzyme by ATP.The data presented in this communication are consistent with a mechanism for controlling acetyl CoA carboxylase activity by interconversion between phosphorylated and dephosphorylated forms. Phosphorylation of the enzyme by a portein kinase decreases enzyme activity, whereas dephosphorylation by a protein phosphatase reactivates the enzyme.     

Regulation of protein phosphorylation and motility of sperm by cyclic adenosine monophosphate and calcium

Motility and protein phosphorylation have been measured under identical experimental conditions in ejaculated dog sperm lysed with low concentrations of Triton X-100 and reactivated with [gamma-32P]ATP. Cyclic AMP stimulates motility and protein phosphorylation while calcium inhibits motility and the overall incorporation of phosphate into endogenous proteins. Analysis of 32P-labeled sperm proteins on 1- and 2-dimensional polyacrylamide gels demonstrates that an enhanced phosphorylation of a defined number of specific proteins is associated with cAMP-stimulated motility. A major axonemal proteins, namely tubulin, has been tentatively identified as a phosphoprotein subject to regulation by cAMP. The phosphorylation of tubulin is almost completely dependent upon cAMP and is not affected by microM calcium. On the other hand, the cAMP-dependent stimulated phosphorylation of the other sperm proteins still occurs, but in most instances at a reduced rate in the presence of calcium. Two high molecular weight (Mr) phosphoproteins (350,000 and 260,000 daltons) whose phosphorylation states are modified by cAMP and calcium also were identified. It is suggested that 1 or both these proteins may be high Mr subunits of dynein. The phosphorylation of 1 of these proteins is stimulated by cAMP, but not affected by calcium; the other is stimulated by cAMP and inhibited by calcium. Three major cAMP-independent phosphoproteins of Mr 98,000, 43,000 and 26,000 have been identified. The phosphorylation of the 98,000 Mr protein is markedly reduced by micromolar calcium and not restored by cAMP. Using anticalmodulin drugs to inhibit motility, we suggest that the inhibitory effects of calcium on flagellar motility may be mediated in part by calmodulin. We conclude that the regulation of flagellar motility in cAMP and calcium includes mechanisms involving the control of the phosphorylation state of sperm proteins, some of which may be axonemal components.     

Regulation through phosphorylation/dephosphorylation cascade systems

The cyclic interconversion of enzymes between phosphorylated and unphosphorylated forms comprises a major mechanism of cellular regulation. A theoretical analysis of reversible covalent modification systems (Stadtman, E.R., and Chock, P.B. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2761-2765) revealed that they are endowed with extraordinary regulatory capacities; they may exhibit smooth, flexible responses to changes in single and multiple metabolite levels, signal amplification, and apparent positive cooperativity. To test qualitatively and quantitatively the theories and equations involved in this analysis, a model in vitro phosphorylation/dephosphorylation cyclic cascade was developed in which the converter enzymes catalyzing the covalent modifications were cAMP-dependent protein kinase (EC 2.7.1.37; type II) and phosphoprotein phosphatase (EC 3.1.3.16; Mr = 38,000), both purified to near homogeneity from bovine heart. The kinetic constants for both enzymes were fully characterized using the nanopeptide Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu as the interconvertible substrate, cAMP as an activator for the kinase, and Pi as an inhibitor for the phosphatase. In the presence of a nearly constant concentration of ATP, a steady-state level of phosphorylation of the peptide was attained which was determined by the relative concentrations of the kinase, phosphatase, and effectors. As predicted by the cyclic cascade model, this monocyclic cascade exhibited both signal amplification and an increase in sensitivity to variations in multiple effector concentrations. In addition, the data show that the steady-state level of phosphorylation obtained in the presence of an activator of the kinase (e.g. cAMP) and an inhibitor of the phosphatase (e.g. Pi) is a function of the product of the relative effector concentrations. Finally, the results reveal that when the concentration of enzyme-substrate complex is not negligible, cyclic cascades are potentially more sensitive to variations in effector concentrations and can achieve even greater signal amplification than predicted previously.     

Peptide-induced hyperactivation-like vigorous flagellar movement in starfish sperm

Asterosap, a sperm-activating peptide (SAP) from the starfish egg jelly coat, is diffusible and controls a cGMP-signalling pathway in starfish sperm in the same manner as resact, a potent chemoattracting SAP in sea urchins. This fact suggests that asterosap may serve as a chemoattractant like resact at concentrations with appropriate gradients. Since asterosap is one of three egg jelly components, which in concert induce the acrosome reaction, it is still worthwhile to evaluate how asterosap modulates sperm motility prior to this reaction. We analysed the flagellar movement of sperm of the starfish Aphelasterias japonica in artificial seawater (ASW) containing the asterosap isoform P15 at 1 micromol l(-1). We found that sperm swim straighter with more symmetrical flagellar movement in P15 than in ASW, but without any significant difference in the flagellar beat frequency and the swimming velocity. The flagellar movement is, however, dramatically different between sperm firmly attached to the solid surface by the head in P15 and those attached in ASW: in P15 the flagellum bends to a greater extent, with higher curvature and with higher shear angle up to a right angle to the flagellar wave axis, and beats at an increased frequency. The vigorous flagellar movement of sperm, which can be activated when sperm are placed in high-load circumstances just as entering into a jelly layer, may increase propulsive forces and hydrodynamic resistances, allowing sperm to undergo the acrosome reaction as effectively as possible.     

Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation

Fructose-1,6-bisphosphatase was precipitated with purified rabbit antiserum from extracts of ³²P-orthophosphate labelled yeast cells, submitted to SDS polyacrylamide gel electrophoresis, extracted from the gels and counted for radioactivity due to ³²P incorporation. Fructose-1,6-bisphosphatase from glucose starved yeast cells contained a very low ³²P label. During 3 min treatment of the glucose starved cells with glucose the ³²P-label increased drastically. Subsequent incubation of the cells in an acetate containing, glucose-free medium led to a label which was again low. Analysis for phosphorylated amino acids in the immunpprecipitated fructose-1,6-bisphosphatase protein from the 3 min glucose-inactivated cells exhibited phospho-serine as the only labelled phosphoamino acid. These data demonstrate a phosphorylation of a serine residue of fructose-1,6-bisphosphatase during this 3 min glucose treatment of glucose starved cells. A concomitant about 60 % inactivation of the enzyme had been shown to occur. The data in addition show a release of the esterified phosphate from the enzyme upon incubation of cells in a glucose-free medium, a treatment which leads to peactivation of enzyme activity. A protein kinase and a protein phosphatase catalysing this metabolic interconversion of fructose-1,6-bisphosphatase are postulated. It is assumed that metabolites accumulating after the addition of glucose exert a positive effect on the kinase activity and/or have a negative effect on the phosphatase activity. A role of the enzymic phosphorylation of fructose-1,6-bisphosphatase in the initiation of complete proteolysis of the enzyme during “catabolite inactivation” is discussed.     

A lithium-sensitive regulator of sperm flagellar oscillation is activated by cAMP-dependent phosphorylation

Specific effects of both in vivo activation and in vitro activation by cAMP-dependent phosphorylation on bending wave parameters of demembranated, reactivated, tunicate (Ciona intestinalis) and sea urchin (Lytechinus pictus) sperm flagella can be reversed by exposure to protein phosphatase. The effects of protein phosphatase incubation can be imitated by inclusion of LiCl in the reactivation solutions. The primary effect of cAMP-dependent phosphorylation appears to be activation of a regulatory mechanism controlling flagellar oscillation, rather than activation of the active sliding mechanism. Lithium appears to act on the same regulatory mechanism.     

Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein

Cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata, is mediated by a specialized viral movement protein (MP). In vivo studies using transgenic tobacco plants showed that MP is phosphorylated at its C-terminus at amino acid residues Ser258, Thr261 and Ser265. When MP phosphorylation was mimicked by negatively charged amino acid substitutions, MP lost its ability to gate plasmodesmata. This effect on MP-plasmodesmata interactions was specific because other activities of MP, such as RNA binding and interaction with pectin methylesterases, were not affected. Furthermore, TMV encoding the MP mutant mimicking phosphorylation was unable to spread from cell to cell in inoculated tobacco plants. The regulatory effect of MP phosphorylation on plasmodesmal permeability was host dependent, occurring in tobacco but not in a more promiscuous Nicotiana benthamiana host. Thus, phosphorylation may represent a regulatory mechanism for controlling the TMV MP-plasmodesmata interactions in a host-dependent fashion.     

Regulation of adrenocortical pregnenolone-binding protein activity by phosphorylation/dephosphorylation. Phosphatase-mediated inactivation is reversed by cytosolic kinase

The pregnenolone-binding protein (PBP) in guinea pig adrenocortical cytosol is inactivated (converted to a nonsteroid-binding form) by incubation with calf intestinal alkaline phosphatase at pH 9. Previously bound pregnenolone does not prevent this inactivation, and dephosphorylation causes dissociation of bound ligand from the protein. Cytosolic PBP, partially purified PBP, and highly purified PBP are equally susceptible to alkaline phosphatase-mediated inactivation. No change in apparent molecular weight or immunoreactivity is evident by Western blot analysis. Loss of pregnenolone-binding capacity of cytosolic PBP (but not partially purified PBP) could be reversed by inhibiting the phosphatase, lowering the pH to approximately 7, and adding ATP to the incubation. Reactivation is absolutely and specifically dependent upon ATP, which restores binding capacity in a concentration-dependent manner. Other nucleoside triphosphates, including the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate, as well as cAMP and cGMP are ineffectual as cofactors for reactivation. These data strongly implicate a cytosolic kinase which is apparently inactivated or separated from PBP during purification. Preliminary investigations indicate that the reactivating kinase is not cAMP-dependent, but may have a requirement for calcium and/or calmodulin. The identification of phosphorylation/dephosphorylation as the regulatory mechanism for steroid binding should prove pivital in elucidating the functional role of PBP.     

Regulation of neuronal plasticity in the central nervous system by phosphorylation and dephosphorylation

Neuronal plasticity can be defined as adaptive changes in structure and function of the nervous system, an obvious example of which is the capacity to remember and learn. Long-term potentiation and long-term depression are the experimental models of memory in the central nervous system (CNS), and have been frequently utilized for the analysis of the molecular mechanisms of memory formation. Extensive studies have demonstrated that various kinases and phosphatases regulate neuronal plasticity by phosphorylating and dephosphorylating proteins essential to the basic processes of adaptive changes in the CNS. These proteins include receptors, ion channels, synaptic vesicle proteins, and nuclear proteins. Multifunctional kinases (cAMP-dependent protein kinase, Ca²⁺/phospholipid-dependent protein kinase, and Ca²⁺/calmodulin-dependent protein kinases) and phosphatases (calcineurin, protein phosphatases 1, and 2A) that specifically modulate the phosphorylation status of neuronal-signaling proteins have been shown to be required for neuronal plasticity. In general, kinases are involved in upregulation of the activity of target substrates, and phosphatases downregulate them. Although this rule is applicable in most of the cases studied, there are also a number of exceptions. A variety of regulation mechanisms via phosphorylation and dephosphorylation mediated by multiple kinases and phosphatases are discussed.