Role of the Fyn kinase in calcium release during fertilization of the sea urchin egg

Protein tyrosine kinase activity has been implicated as part of the signaling mechanism leading to the sperm-induced calcium transient following fertilization. In the present study, we have tested the role of the Fyn kinase in triggering the calcium transient by microinjecting domain-specific fusion proteins encoding regions of Fyn sequence as inhibitors of Fyn function in vivo. A fusion protein encoding the SH2 domain of Fyn caused an increase in the latent period between sperm-egg fusion and the beginning of the calcium transient and reduced the amplitude of the calcium signal. A fusion protein encoding the U + SH3 domains also caused a small increase in the latent period. Microscopic examination revealed that a large percentage of eggs injected with the U+SH3 or SH2 domains became polyspermic as a result of the delayed block to polyspermy. Affinity experiments demonstrated that the U+SH3 and SH2 domains of Fyn were capable of forming a stable complex with phospholipase Cgamma from the sea urchin egg. The results suggest that the Fyn kinase participates in the signaling events leading up to the calcium transient and may directly regulate phospholipase Cgamma activity at fertilization.     

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.     

Quantitation of a src-like tyrosine protein kinase during fertilization of the sea urchin egg

Fertilization of the sea urchin egg is known to involve an increase in overall protein tyrosine kinase activity which precede the first cell division. In order to determine the types of tyrosine kinases that are involved in fertilization, we have used immunological and other criteria to identify a c-src related protein kinase in eggs of the sea urchin L. variegatus. Using an immune complex assay, we have measured the level of this c-src related protein kinase during fertilization and early embryonic development. Fertilization results in a decrease in the c-src kinase detectable by this technique suggesting that c-src does not contribute to the fertilization induced increase in protein tyrosine kinase activity.     

Anti-peptide Antibody Identifies a 57 kDa Protein Tyrosine Kinase in the Sea Urchin Egg Cortex

The egg plasma membrane and cortical structures are highly enriched in protein tyrosine kinase activity which is thought to play an important role in the fertilization process. In order to identify the tyrosine protein kinases in the egg cortex, a site directed polyclonal antibody was produced against a peptide duplicating a conserved region of the catalytic domain of the sea urchin c-abl gene product. The region chosen as an antigen had a high degree of homology (57%) to other protein tyrosine kinases. The antibody was found to bind with a high degree of specificity to a 57 kDa protein tyrosine kinase in S. purpuratus eggs. The antibody was capable of immunoprecipitating the enzyme as a 57 kDa phosphoprotein from purified egg cortex fractions solubilized in NP-40. Immunoprecipitation was completely inhibited by prior incubation of the antibody with the synthetic peptide used as an antigen. Binding of the antibody completely inhibited kinase activity. However, the immunoprecipitated kinase activity could be eluted from the Sepharose-coupled antibody and was shown to have catalytic activity towards a tyrosine containing peptide substrate. The enzyme also underwent autophosphorylation on tyrosine in vitro. Ultrastructural localization of the kinase by immuno-electron microscopy revealed that the enzyme was primarily restricted to the egg plasma membrane.     

Motility and centrosomal organization during sea urchin and mouse fertilization

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.     

Protein tyrosine kinase activity of eggs of the sea urchin Strongylocentrotus purpuratus: the regulation of its increase after fertilization

Protein tyrosine kinase activity in eggs of the sea urchin, Strongylocentrotus purpuratus, increased two- to fourfold as early as several min after fertilization at 8-10 degrees C. Artificial activation of eggs with the divalent cation ionophore, A23187, or with butyric acid induced the increase in enzyme activity. The transfer of eggs to seawater containing either no Na+ or 50 mM Na+ and 10(-4) M amiloride immediately after fertilization did not block the increases in enzyme activity. When eggs were activated with seawater containing NH4OH, enzyme activity did not increase at 1 hr after activation, although the increased activity was detected at 3 hr after activation. Increased enzyme activity also was observed in enucleated egg fragments activated with butyric acid. Puromycin and emetine, inhibitors of protein synthesis, also did not inhibit the initial increases of enzyme activity after fertilization. These results demonstrated that the increased protein tyrosine kinase activity observed after fertilization of S. purpuratus eggs can be initiated independent of various other known events such as fusion with sperm cells and protein and DNA synthesis.     

Prolonged incubation in seawater induces a DNA-dependent protein phosphorylation activity in Arbacia punctulata eggs

Various protein kinases are activated in eggs in response to fertilization. We have previously shown that the induction of DNA-dependent protein phosphorylation activity in the sea urchin eggs is triggered by fertilization. The present study demonstrates that the activation of a DNA-dependent serine/threonine kinase in unfertilized eggs of Arbacia punctulata can be achieved without fertilization. Prolonged incubation in seawater resulted in the activation of the eggs with concomitant induction of DNA-dependent protein phosphorylation activity. The activated eggs when fertilized show a slight increase in the phosphorylation activity 10-min post-insemination. The activity gradually declines as the first and second cleavages proceed. The cytoplasmic extracts of the blastulae, gastrulae, and plutei lack the enzyme activity. These findings reveal that not only fertilization but also egg activation serves as a signal for the induction of a DNA-dependent protein phosphorylation activity in sea urchin eggs suggesting that sperm-entry is not required for the induction of the enzyme activity.     

Regulation of tyrosine-specific kinase activity at fertilization

The sea urchin egg contains a protein kinase which phosphorylates tyrosine residues of endogenous membrane proteins as well as synthetic peptide substrates. Fertilization results in an increase in tyrosine kinase activity which first becomes apparent 20–30 min postinsemination and continues throughout the early cleavage stages. This effect can be duplicated by treating unfertilized eggs with the calcium ionophore A23187. The kinase activity begins to increase about 20 min after addition of the ionophore and continues to increase for at least 1 hr. Both the time course and the extent of kinase activity in ionophore treated eggs closely resemble the effects of fertilization. The concentration of ionophore necessary to induce the increase in enzyme activity (2–5 μM) is also effective in inducing the cortical reaction. Neither A23187 nor calcium has a significant effect on the kinase activity of egg homogenates solubilized in NP40, suggesting that the ionophore affects tyrosine phosphorylation indirectly, possibly acting through other calcium-sensitive enzymes.     

Identification of myosin II kinase from sea urchin eggs as protein kinase CK2

Here we purified and identified a myosin II kinase from sea urchin eggs. The activity of this myosin II kinase in the egg extract was not significantly affected by Ca(2+)/calmodulin (CaM). Using sequential column chromatographies, we purified the myosin II kinase from the egg extract as a complex composed of 36- (p36) and 28-kDa (p28) proteins. Partial amino acid sequences of these two components were highly coincident with those of the alpha and beta subunits of protein kinase CK2 (formerly casein kinase II) in sea urchin eggs, respectively. To confirm that the purified myosin II kinase was CK2, we obtained a cDNA which encodes p36 from a cDNA library of sea urchin eggs. The amino acid sequence derived from the obtained cDNA showed over 70% homology to CK2 from various eukaryotes. Furthermore, recombinant p36, as well as the purified myosin II kinase, phosphorylated MRLC. One dimensional phosphopeptide mapping revealed that the phosphorylation site(s) of MRLC by both recombinant p36 and the purified myosin II kinase was identical. These clearly showed that the Ca(2+)/CaM-independent myosin II kinase activity in sea urchin eggs was identical to CK2.     

Evidence that Src-type tyrosine kinase activity is necessary for initiation of calcium release at fertilization in sea urchin eggs

The initiation of Ca(2+) release from internal stores in the egg is a hallmark of egg activation. In sea urchins, PLCgamma activity is necessary for the production of IP(3), which leads to the initial rise in Ca(2+). To examine the possible function of a tyrosine kinase in activating PLCgamma at fertilization, sea urchin eggs were treated with the specific Src kinase inhibitor PP1 or microinjected with recombinant Src-family SH2-domain proteins, which act as dominant interfering inhibitors of Src-family kinase function. Both modes of inhibiting Src-family kinases resulted in a specific and dose-dependent delay in the onset of Ca(2+) release from the endoplasmic reticulum at fertilization. The rise in cytoplasmic pH at fertilization also was inhibited by microinjection of Src-family SH2-domain proteins. Further, an antibody directed against Src-type kinases recognized a protein of ca. M(r) 57K that was enriched in the membrane fraction of eggs. The kinase activity of this protein was stimulated rapidly and transiently at fertilization, as measured by autophosphorylation and by phosphorylation of an exogenous substrate. Together, these data indicate that a Src-type tyrosine kinase is necessary for the initiation of Ca(2+) release from the egg ER at fertilization and identify a Src-type p57 protein as a candidate in the signaling pathway leading to this Ca(2+) release.     

Cloning and characterization of a phospholipase C-beta isoform from the sea urchin Lytechinus pictus

Calcium is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes including fertilization and egg activation. The mechanism for triggering intracellular Ca(2+) release in sea urchin eggs during fertilization is the generation of inositol-1,4,5-trisphosphate by phospholipase C (PLC) hydrolysis of phosphatidylinositol-4,5-bisphosphate. Of the five PLC isoforms identified in mammals (beta, gamma, delta, epsilon and zeta), only PLCgamma and PLCdelta have been detected in echinoderms. Here, we provide direct evidence of the presence of a PLCbeta isoform, named suPLCbeta, within sea urchin eggs. The coding sequence was cloned from eggs of Lytechinus pictus and determined to have the greatest degree of homology and identity with the mammalian PLCbeta4. The presence of suPLCbeta within the egg was verified using a specifically generated antibody. The majority of the enzyme is localized in the non-soluble fraction, presumably the plasma membrane of the unfertilized egg. This distribution remains unchanged 1 min postfertilization. Unlike PLCbeta4, suPLCbeta is activated by G protein betagamma subunits, and this activity is Ca(2+)-dependent. In contrast to all known PLCbeta enzymes, suPLCbeta is not activated by Galphaq-GTPgammaS subunit suggesting other protein regulators may be present in sea urchin eggs.     

Protein tyrosine phosphorylation in response to fertilization

The sea urchin egg contains one or more protein tyrosine kinases which are active during the response of the egg to fertilization. In the present study, we have used an antibody specific for phosphotyrosine to determine which egg proteins are phosphorylated on tyrosine in response to fertilization. Analysis of immunoblots prepared from fertilized and unfertilized eggs revealed that fertilization results in a major increase in the phosphotyrosine content of a 350-kDa egg protein. Increased phosphorylation of this protein was detected as early as 1 min after fertilization, at which time it represented the most prominent phosphotyrosine containing protein in the egg. Tyrosine phosphorylation of this protein was transient however, and after 5 min post-insemination, the protein was dephosphorylated or otherwise degraded. Egg membrane proteins of approximately 40, 75, and 145 kDa were also found to act as substrates for protein tyrosine kinases in vitro, but did not exhibit significant changes in phosphotyrosine content during egg activation.     

Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown

The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in nuclear envelope breakdown (NEB) was investigated in sea urchin eggs. The eggs contain a 56-kD polypeptide which appears to be a homologue of neuronal CaM kinase. For example, it undergoes Ca2+/calmodulin-dependent autophosphorylation that converts it to a Ca2(+)-independent species, a hallmark of multifunctional CaM kinase. It is homologous to the alpha subunit of rat brain CaM kinase. Autophosphorylation and substrate phosphorylation by the sea urchin egg kinase are inhibited in vitro by CaMK(273-302), a synthetic peptide corresponding to the autoinhibitory domain of the neuronal CaM kinase. This peptide inhibited NEB when microinjected into sea urchin eggs. Only one mAb to the neuronal enzyme immunoprecipitated the 56-kD polypeptide. Only this antibody blocked or significantly delayed NEB when microinjected into sea urchin eggs. These results suggest that sea urchin eggs contain multifunctional CaM kinase, and that this enzyme is involved in the control of NEB during mitotic division.     

Activation of Src-family PTK activity at fertilization: role of the SH2 domain

The role of Src-family protein tyrosine kinases (SFKs) in egg activation has been established, in large part, by the observation that GST fusion proteins encoding the SH2 domain of Src or Fyn suppress the sperm-induced calcium transient and cause polyspermy in marine invertebrate eggs. These fusion proteins are thought to act as dominant-negative inhibitors of SFK function; however, the mechanism by which they work is not known. The objective of the present study was to test the hypothesis that fusion proteins containing the above SH2 domains prevent activation of SFKs in response to fertilization. A single cell assay was developed that allows estimation of SFK activity in eggs injected with the GST-Fyn-SH2 fusion protein. The results demonstrate that the GST-Fyn-SH2 fusion protein prevents fertilization induced stimulation of SFK activity at concentrations that also suppress the sperm-induced calcium transient in zebrafish eggs.     

Phorbol ester treatment stimulates tyrosine phosphorylation of a sea urchin egg cortex protein

Fertilization of the sea urchin egg results in the phosphorylation, on tyrosine, of a high molecular weight protein localized in the egg cortex. In the present study, treatment of unfertilized eggs with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulated tyrosine phosphorylation of the high molecular weight cortical protein to levels three- to fivefold higher than that occurring in response to fertilization. Experiments using agents that inhibit the egg Na+/H+ exchange system or mimic the fertilization-induced shift in cytoplasmic pHi, suggest a signal transduction pathway in which protein kinase C activates the egg Na+/H+ exchange system and the resultant cytoplasmic pHi shift promotes tyrosine phosphorylation of the high molecular weight cortical protein.     

Detection of phospholipase Cγ in sea urchin eggs

Phosphorylation on tyrosine and turnover of polyphosphoinositide metabolism are rapidly stimulated after fertilization. However, the interconnection between these pathways remains to be determined. In the present paper it is demonstrated that eggs of two different sea urchin species contain tyrosine phosphorylated proteins with calcium-sensitive phospholipase C activity. We have investigated whether phospholipase Cγ (PLCγ), characteristic of tyrosine kinase receptors, could be responsible for this activity. Western blot and immunocytochemistry performed with antibodies directed against PLCγ revealed the presence of this protein in cortical regions. It was also observed that PLCγ displayed calcium-sensitive activity. The present results suggest that PLCγ may be part of the cascade of events leading to the calcium signal responsible for egg activation at fertilization.     

Role of PTPase(s) in regulating Fyn kinase at fertilization of the zebrafish egg

Fertilization involves the activation of Src-family protein kinases which play a role at multiple stages of the egg activation process. The objective of the present study was to determine the mechanism by which one of these kinases, the Fyn kinase, is activated in response to fertilization of the zebrafish egg. Inhibitor studies demonstrated that many aspects of egg activation, including Fyn activation, require phosphotyrosyl phosphatase activity. A phosphotyrosyl phosphatase was found to be tightly associated with Fyn kinase and this interaction was mapped to the SH2 domain of Fyn. Coimmunoprecipitation studies identified rPTPalpha as a phosphatase that is complexed with Fyn in the egg, raising the possibility that rPTPalpha is part of the regulatory mechanism responsible for activating Fyn at fertilization.     

Inhibition of mitosis in fertilized sea urchin eggs by inhibition of the cyclic AMP-dependent protein kinase

Inhibition of cAMP-dependent protein kinase activity by microinjection of a specific physiologic protein inhibitor into sea urchin eggs inhibits the first cleavage after fertilization. Inhibition apparently occurs at some time prior to or during formation of the mitotic spindle. Measurement of the total protein kinase activity of sea urchin egg homogenates after fertilization showed that cAMP-dependent phosphorylation increases after fertilization and then declines prior to or at the time of the first cleavage. It is concluded that a cAMP-dependent phosphorylation plays a significant role in events leading to regulation of mitotic spindle assembly.     

Sea urchin egg-cortical granule protease has arginyl proteolytic specificity

Sea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83–88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP-HPLC analysis with a spectrophotometric Nα-benzoyl-L-arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin-like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.     

Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization

Egg activation at fertilization requires the release of Ca(2+) from the egg's endoplasmic reticulum, and recent evidence has indicated that a Src family kinase (SFK) may function in initiating this signaling pathway in echinoderm eggs. Here, we identify and characterize a SFK from the sea urchin Strongylocentrotus purpuratus, SpSFK1. SpSFK1 RNA is present in eggs, and an antibody made against a SpSFK1 peptide recognizes an approximately 58-kDa egg membrane-associated protein in eggs of S. purpuratus as well as another sea urchin Lytechinus variegatus. Injection of both species of sea urchin eggs with dominant-interfering Src homology 2 domains of SpSFK1 delays and reduces the release of Ca(2+) at fertilization. Injection of an antibody against SpSFK1 into S. purpuratus eggs also causes a small increase in the delay between sperm-egg fusion and Ca(2+) release. In contrast, when injected into eggs of L. variegatus, this same antibody has a dramatic stimulatory effect: it causes PLCgamma-dependent Ca(2+) release like that occurring at fertilization. Correspondingly, in lysates of L. variegatus eggs, but not S. purpuratus eggs, the antibody stimulates SFK activity. Injection of L. variegatus eggs with another antibody that recognizes the L. variegatus egg SFK also causes PLCgamma-dependent Ca(2+) release like that at fertilization. These results indicate that activation of a Src family kinase present in sea urchin eggs is necessary to cause Ca(2+) release at fertilization and is capable of stimulating Ca(2+) release in the unfertilized egg via PLCgamma, as at fertilization.