Fertilization, a crucial event for species preservation, in sea urchins, as in many other organisms, requires sperm motility regulation. In Strongylocentrotus purpuratus sea urchins, speract, a sperm chemoattractant component released to seawater from the outer egg layer, attracts sperm after binding to its receptor in the sperm flagellum. Previous experiments performed in demembranated sperm indicated that motility regulation in these cells involved protein phosphorylation mainly due to the cAMP-dependent protein kinase (PKA). However, little information is known about the involvement of protein kinase C (PKC) in this process. In this work, using intact S. purpuratus sea urchin sperm, we show that: (i) the levels of both phosphorylated PKA (PKA substrates) and PKC (PKC substrates) substrates change between immotile, motile and speract-stimulated sperm, and (ii) the non-competitive PKA (H89) and PKC (chelerythrine) inhibitors diminish the circular velocity of sperm and alter the phosphorylation levels of PKA substrates and PKC substrates, while the competitive inhibitors Rp-cAMP and bisindolylmaleimide (BIM) do not. Altogether, our results show that both PKA and PKC participate in sperm motility regulation through a crosstalk in the signalling pathway. These results contribute to a better understanding of the mechanisms that govern motility in sea urchin sperm.

The cellular basis of motor learning in the cerebellum has been attributed mostly to long-term depression (LTD) at excitatory parallel fiber (PF)-Purkinje cell (PC) synapses. LTD is induced when PFs are activated in conjunction with a climbing fiber (CF), the other excitatory input to PCs. Recently, by using whole-cell patch-clamp recording from PCs in cerebellar slices, a new form of synaptic plasticity was discovered. Stimulation of excitatory CFs induced a long-lasting (usually longer than 30 min) “rebound potentiation (RP)” of γ-amino-butyric acid A (GABAa)-receptor mediated inhibitory postsynaptic, currents (IPSCs). As in LTD, induction of RP requires transient elevation of intracellular calcium concentration ([Ca2+]1) due to activation of voltage-gated Ca2+ channels. Activity of inhibitory synapses seems also to be necessary for RP to occur. RP is mainly due to up-regulation of postsynaptic GABAa receptor function, since PC response to bath-applied exogenous GABA is also potentiated with a time course similar to RP. The difference in the time scale between the Ca2+ transients (10–30 sec) and the durations of RP (>30 min) strongly suggests that some intracellular biochemical machinery is involved. Pharmacological evidence suggests that protein kinases are involved in RP of inhibitory synapses and LTD of excitatory PF synapses. Besides the well-described LTD, RP could be a cellular mechanism that plays an important role in motor learning.

Autophosphorylation by protein kinases has been implicated as an important control mechanism in signal transduction and growth regulatory pathways in mammalian cells. We have set out to investigate whether any such autophosphorylating protein kinase activities can be found in Trypanosoma brucei. In order to do this, we have developed a system for characterizing such protein kinase activities using an in vitro assay. This assay was carried out by fractionation of trypanosome lysates using isoelectric focusing gel electrophoresis followed by incubation of the gel in γ32P-labelled nucleotide triphosphate and subsequent autoradiography. We have identified two classes of autophosphorylating protein kinase activities. In the first class all were dependent on ATP as the phosphate donor substrate and were all found to have a molecular size of 60 kDa. Differences in the activity of these protein kinases were observed between the bloodstream and procyclic life-cyle stages. Furthermore, the addition of mammalian epidermal growth factor to bloodstream stage lysates stimulated an additional activity. The second class of autophosphorylating protein kinases utilized GTP as the phosphate donor and were all found to be 90 kDa in size. Stage-specific differences were also observed in the activity of these protein kinases.

The dependence of pig oocyte activation (both nuclear activation and cortical granule exocytosis) induced by staurosporine on intracellular Ca2+ rise and spindle assembly was studied. Nuclear activation was evaluated by pronuclear (PN) formation, cleavage and their developmental ability, and cortical granule (CG) exocytosis was assessed by electron microscopy and laser confocal microscopy of oocytes labelled with fluorescein isothiocyanate-peanut agglutinin. Exposure of pig oocytes of 0.3 and 3μM protein kinase inhibitor staurosporine for 30 min resulted in the nuclear activation in 71.8% and 85.7% of the oocytes, respectively. The pronuclei in activated oocytes contained several compact nucleoli. When the cleaved 2-cell oocytes were further cultured in vitro, 93.5% developed beyond the 4-cell stage, and 12.9% developed to the morula stage after 4 days of culture. Of the oocytes treated with 3μM staurosporine, 62.5% and 9.4% released their CGs partially and completely, respectively. The nuclear activation induced by staurosporine was overcome by the prior treatment of oocytes with okadaic acid, resulting in only 33.3% of the oocytes undergoing nuclear activation. However, when oocytes were exposed first to 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethanal ester), a cell permeate calcium chelator, or Colcemid, a meiotic spindle disrupter, and then to staurosporine, nuclear activation was observed in 74.2% and 82.3% of the oocytes, espectively. These data were the same as those in oocytes treated only with staurosporine (85.7%). The present study indicates that pig oocytes can be activated by the inhibition of staurosporine-sensitive protein kinase(s), and that this activation is dependent upon mitogen-activated protein kinase but independent of the intracellular Ca2+ rise and spindle integrity.

Staurosporine, an inhibitor of protein kinase activity, causes premature intercellular flattening of blastomeres but does not induce their premature polarisation. The flattening induced is calcium dependent, is reversed transiently at mitosis and requires the continuing presence of the drug. Staurosporine also blocks the decompacting effect of phorbol ester on 8-cell embryos.

An abscisic acid (ABA)-induced cDNA fragment encoding a putative protein kinase (PK) was obtained by differential screening of a cDNA library from Fagus sylvatica seeds. The full-length clone, named FsPK1, was produced by 5′ rapid amplification of cDNA ends (RACE) extension. This clone contained the 11 catalytic domains present in all protein kinases, but displayed unusual characteristics found only in a few plant PKs. FsPK1 exhibits features of both serine/threonine and tyrosine protein kinases within the catalytic domain, a putative nuclear localization signal within the regulatory domain and the consensus sequence involved in binding of 14-3-3 proteins. The catalytic domain, expressed in Escherichia coli as a fusion protein, showed Ca2+-dependent in vitro kinase activity and dual serine/threonine and tyrosine specificity. Transcription of the FsPK1 gene was reduced by seed stratification at 4°C, and clearly increased when seeds were treated with 0.1 mM ABA, correlating with the inhibition of germination. Interestingly, FsPK1 transcripts were enhanced when ABA (0.1 mM) and calcium (1 mM) were added together, while the addition of EGTA (calcium chelator) and 3,4,5,-trimethoxibenzoic acid 8-(diethylamino) octyl ester (TMB-8, a calcium antagonist) decreased its expression. Furthermore, FsPK1 transcript expression was tissue specific and accumulated only in ABA-treated seeds, but not in any ABA-treated vegetative tissues examined. These results suggest that the expression of the corresponding protein could be related to the inhibition of germination mediated by ABA in a calcium-dependent pathway.

Low catalytic efficiency of basal-state protein kinases often depends on activation loop residues blocking substrate access to the catalytic cleft. Using the recombinant soluble form of the insulin receptor's kinase domain (IRKD) in its unphosphorylated state, activation loop conformation was analyzed by limited proteolysis. The rate of activation loop cleavage by trypsin is slow in the apo-IRKD. Bound Mg-adenine nucleoside di- and triphosphates increased the cleavage rate with half-maximal effects observed at 0.4–0.9 mM nucleotide. Adenosine monophosphate at concentrations up to 10 mM was not bound appreciably by the IRKD and had virtually no impact on activation loop cleavage. Amino-terminal and carboxy-terminal core-flanking regions of the IRKD had no statistically significant impact on the ligand-dependent or -independent activation loop cleavages. Furthermore, the core-flanking regions did not change the inherent conformational stability of the active site or the global stability of the IRKD, as determined by guanidinium chloride-induced denaturation. These measurements indicate that the intrasterically inhibitory conformation encompasses ≥90% of the ligand-free basal state kinase. However, normal intracellular concentrations of Mg-adenine nucleotides, which are in the millimolar range, would favor a basal-state conformation of the activation loop that is more accessible.

Protein kinase activity of intact, motile sperm was assessed by measuring the transfer of the terminal phosphate from [32P]ATP to tricholoroacetic acid (TCA)-precipitable casein. The action of TEST (TES and Tris) yolk buffer (TYB) treatment on phosphorylation of sperm and TYB proteins was studied by detecting labelled phosphoproteins by autoradiography of polyacrylamide gel electrophoresis (PAGE). Results demonstrate that intact, forward-motile sperm have cell surface protein kinase activities. Although the difference between the kinase activity of freshly ejaculated sperm and sperm incubated in TYB was not significant, the protein phosphorylation during incubation in TYB showed that: (i) specific sperm surface proteins were phosphorylated to different degrees during the course of treatment; (ii) TYB proteins were phosphorylated by sperm during incubation; (iii) solubilised [32P]-labelled surface proteins were similar in molecular weight to TYB-labelled proteins. Taking into account that specific proteins on the human sperm surface undergo phosphorylation during incubation in TYB and that the sperm enzyme also acts specifically on some TYB proteins that become attached to the surface of the sperm, working hypotheses are proposed that suggest some correlation between the preservation of semen in TYB and the phosphorylation of proteins by intact human sperm.