Molecular Cloning and Expression of Starfish Protein Kinase C Isoforms

To investigate the roles of protein kinase C (PKC) isoforms in Echinoderms, we cloned starfish cDNAs for novel, atypical, and conventional PKCs. They showed highest homology with PKCδ, ι, and α isoforms respectively. It was predicted from the whole genome sequence and by RT-PCR that sea urchin has only one isoform of each PKC subgroups. It is thus likely that these isoforms are the prototypes or ancestors of the PKC subgroups. The phylogenetic tree suggests that atypical PKC was first formed by evolution from the common prototype of AGC protein kinase family, and novel and conventional PKCs next. RT-PCR analysis indicated that novel and atypical PKC mRNAs are expressed ubiquitously in all tissues of adult starfish, whereas conventional PKC mRNA is expressed mainly in the ovary and oocytes, and only slightly in the tube foot and stomach. Upon heterologous expression, only atypical PKC was expressed in the functional form in insect cells.     

Pinocytosis of Ferritin from the Gut Lumen in Larvae of a Sea Star (Patiria miniata) and a Sea Urchin (Lytechinus pictus)

Pinocytosis of macromolecules from the gut lumen is demonstrated for the first time in larval stages of invertebrates. Developing sea stars ( Patiria miniata ) and sea urchins ( Lytechinus pictus ) were incubated in seawater containing ferritin, which was detected in cell organelles by transmission electron microscopy. Pinocytotic uptake of ferritin by gut cells of Patiria could be detected as soon as the larval mouth opened before the esophagus, stomach and intestine could be distinguished from one another; in contrast, no pinocytosis was detected at the comparable developmental stage (the prism larva) of Lytechinus . Pinocytosis was first detected in developing Lytechinus in pluteus larvae, especially in the stomach and intestine. In gut cells of both kinds of echinoderm larvae, the ferritin progressed rapidly from coated pits at the luminal cell membrane to secondary lysosomes (e.g. this progression took only about 10 min in stomach cells of Patiria larvae). Phagocytosis from the gut lumen was never observed after latex beads or starch granules were fed to larvae of Patiria and Lytechinus . Moreover, there was no evidence of pinocytosis of ferritin or phagocytosis of large particles by epidermal cells of larvae of either species.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

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.     

一个小麦丝氨酸—苏氨酸蛋白激酶基因的克隆和分析

用mRNA差异显示技术在含有抗白粉病基因Pm2 1的小麦 (TriticumaestivumL .)_簇毛麦 (Haynaldiavillosa)6VS/ 6AL易位系 92R137中分离与抗白粉病相关的基因 ,获得一个命名为TaPK1的全长cDNA克隆。序列分析表明 ,它与大豆 (Glycinemax (L .)Merr.)蛋白激酶基因GmPK6高度同源。经推测 ,TaPK1编码 416个氨基酸的多肽 ,属丝氨酸_苏氨酸蛋白激酶家族 ,并具酪氨酸激酶特性。TaPK1是从小麦中分离的新基因。     

一个小麦丝氨酸-苏氨酸蛋白激酶基因的克隆和分析

用mRNA差异显示技术在含有抗白粉病基因Pm21的小麦(Tri ticum aestivum L.) -簇毛麦(Haynaldia villosa) 6VS /6AL易位系92R137中分离与抗白粉病相关的基因,获得一个命名为TaPK1的全长cDNA克隆.序列分析表明,它与大豆(Glycine max (L.) Merr.)蛋白激酶基因GmPK6高度同源.经推测,TaPK1 编码416个氨基酸的多肽,属丝氨酸-苏氨酸蛋白激酶家族,并具酪氨酸激酶特性.TaPK1是从小麦中分离的新基因.     

Molecular Cloning and Characterization of a Radial Spoke Head Protein of Sea Urchin Sperm Axonemes: Involvement of the Protein in the Regulation of Sperm Motility

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40°C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form α-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.     

Purification and Characterization of Echinonectin, a Carbohydrate-Binding Protein from Sea Urchin Eggs

A galactose-specific carbohydrate binding protein has been identified in eggs and embryos of the sea urchin Lytechinus variegatus . This protein, named echinonectin (Alliegro et al., 1988, J. Cell Biol. 107; 2319–2327) has been described as a cell-substrate adhesion protein functioning during embryonic development. The purified protein has an apparent molecular weight of 220 kDa and exists as a dimer of apparently identical 110 kDa subunits. The carbohytrate specificity of the purified protein was examined through the use of competition assays. The protein has a marked specificity for galactose and fucose and a higher affinity for polymers of galactose or galactose sulfate such as carrageenan.     

蛋白激酶C的分离纯化及其性质研究

 本文由兔脑细胞质可溶部分分离纯化了蛋白激酶C,测得该酶分子量为79.2kD,最适pH为6.5,最适反应温度为20℃,热不稳定,即使在4℃下,24h就丧失活力50％,同时观察了蛋白激酶C的抑制剂H_7对酶活力的影响。     

Purification and Characterization of Sperm Creatine Kinase and Guanylate Cyclase of the Sea Urchin Hemicentrotus pulcherrimus

Creatine kinase and guanylate cyclase were purified from Hemicentrotus pulcherrimus spermatozoa. The molecular weight of the purified sperm tail creatine kinase was estimated to be 137,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sperm tail guanylate cyclase was purified by chromatography on a WGA-Sepharose column connected to a Concanavalin A-Sepharose column, and a Superose 12 HR column. The molecular weight of the tail guanylate cyclase was estimated to be 128,000 by SDS-PAGE. The specific activity of the purified enzyme was 8.25 μmol of cGMP formed/min/mg protein. Sperm-activating peptide I (SAP-I) causes an electrophoretic mobility shift of H. pulcherrimus sperm guanylate cyclase from 131 kDa to 128 kDa. The 131 kDa form of guanylate cyclase was co-purified with a 76 kDa protein, whose molecular mass is similar to that of a SAP-I receptor. The purified 131 kDa form of guanylate cyclase had higher activity than the 128 kDa form. The 131 kDa and 128 kDa forms of guanylate cyclase contained 23.83 ± 0.65 and 4.16 ± 0.45 moles of phosphate per mol protein (mean ± S.D.; n = 3), respectively. The activities of guanylate cyclase and creatine kinase increased during the testis development. During spermatogenesis, sperm tail creatine kinase was detected immunohistochemically only in mature spermatozoa.     

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.     

Protein and Molecular Characterization of Hippocampal Protein Kinase C in C57BL/6 and DBA/2 Mice

Abstract: Measures of protein kinase C (PKC) in C57BL/6 and DBA/2 mice using [³H]phorbol 12,13-dibutyrate binding to tissue homogenates and brain slices demonstrated that levels of activated, membrane-bound PKC were greater in C57BL hippocampus than in DBA hippocampus. Western analysis of α-, β_I-, β_II-, γ-, δ-, and ɛ-PKC using isozyme-specific antibodies indicated that the increase observed in C57BL hippocampus was due primarily to the γ-PKC protein, whose immunoreactivity was greater in the membrane-bound fraction in C57BL mice. Characterization of α-, β_I,II-, and γ-PKC hippocampal mRNA using northern analysis and isozyme-specific nucleic acid probes did not reveal differences between the strains in levels of gene expression. Restriction fragment length polymorphisms (RFLP) were found in the α- and γ-, but not β-PKC genomic DNA. The RFLPs appeared to be located in noncoding, nonregulatory regions of the gene. These findings suggest that the γ-PKC isozyme is largely responsible for the PKC activity difference in C57BL and DBA hippocampus that has been reported previously and may be closely associated with differences in learning ability observed in these strains.     

Receptor-mediated responses of plasma membranes isolated from Lytechinus pictus spermatozoa

Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), a peptide obtained from eggs, has been shown to bind to a plasma membrane receptor of Lytechinus pictus spermatozoa. Here, we show that the addition of speract to intact cells caused the appearance of a new protein-staining band (Mr = 140,000) on sodium dodecyl sulfate (SDS) polyacrylamide gels; concomitantly, a protein of apparent molecular weight (Mr) 150,000 disappeared. Guanylate cyclase activity also decreased approximately 50% after the addition of speract to intact cells. Plasma membranes were subsequently prepared from spermatozoa in the presence of fluoride at pH 6.0, conditions that resulted in retention of the speract receptor and the Mr 150,000 protein. Addition of speract to the membranes resulted in a disappearance of the Mr 150,000 protein and the appearance of a Mr 140,000 protein. Coincident with the apparent change in molecular weight, guanylate cyclase activity decreased 30% at maximal speract concentrations. A physiological event that occurs in the intact cell in response to speract can now be reproduced in isolated plasma membranes; it should, therefore, now be possible to define the molecular events that occur as a result of speract: receptor interaction.     

Molecular Cloning and Characterization of a Novel Retinoblastoma-Binding Protein

We describe the isolation and characterization of a novel cDNA encoding a polypeptide that interacts in a yeast two-hybrid system as well as in mammalian cells with the retinoblastoma (RB) protein. This new protein, which we call Rim, consists of 897 amino acids, has two leucine zipper motifs, and has a LECEE sequence previously identified as an RB-binding domain. Rim also has an E1A/CtBP-binding motif and four putative nuclear localization signals.RimmRNA is expressed ubiquitously at low levels in all human adult tissues tested and at much higher levels in several tumor cell lines. TheRimgene (HGMW-approved symbol RBBP8) is localized on human chromosome 18q11.2.     

Characterization of Protein Kinase C in Photoreceptor Outer Segments

Abstract: Protein kinase C (PKC) has been implicated in regulating several proteins involved in phototransduction. This contribution characterizes the biochemical and immunological properties of PKC isozyme(s) in the photoreceptor outer segment. Activity measurements revealed that at least 85% of the PKC in this specialized compartment belongs to the subfamily of Ca²⁺-regulated (conventional) PKCs. Of the known Ca²⁺-dependent PKCs, only PKCα was immunodetected by western blot analysis of rod outer segment proteins. However, the ratio of immunoreactivity to enzyme activity for rod outer segment PKC was no more than 40% of that for brain PKC, using antibodies against conventional PKCs. Therefore, at least half the Ca²⁺/lipid-stimulated activity in rod outer segment preparations cannot be accounted for by the known isozymes, suggesting the presence of a previously uncharacterized isozyme. Despite extensive tests using a variety of antibodies against different domains of PKCα, PKCα could not be detected in rod outer segments by immunofluorescence of retinal sections. In summary, our data reveal that most of the PKC in photoreceptor outer segments is of the conventional type and that most, if not all, of this conventional PKC activity comes from a novel isozyme(s).     

海胆主要卵黄蛋白研究进展

海胆的主要卵黄蛋白(major yolk protein,MYP)大量存在于海胆卵中,同时存在于海胆精巢和卵巢中,为配子发生及胚胎发育提供营养.经比对发现,海胆MYP cDNA序列与其他物种卵黄蛋白原并无明显相似性,而与铁传递蛋白具有更高的相似性,有研究认为MYP为铁传递蛋白.据其存在场所和合成时期的差异,MYP可分为为CFMYP(存在于体腔内,受精后合成)和EGMYP(存在于卵中,母源性),二者均由同一基因编码.就CFMYP和EGMYP合成时期、场所和功能作用等加以比较;并对海胆MYP与其他物种卵黄蛋白(原)发生、结构与分子量和功能等方面进行了综述.     

Functional Characterization of Protein Kinase MAC-V with the Use of Two-Hybrid Cloning in Yeast

Identification of interaction partners opens a way to direct functional characterization of proteins. Several cDNAs coding for potential partners of protein kinase MAK-V/Hunk were isolated using two-hybrid cloning in yeast. Based on the partner properties, MAK-V/Hunk was assumed to play a role in tumorigenesis and tumor progression. With the previous results of two-hybrid cloning, MAK-V/Hunk was shown to participate in vesicular transport.     

Effects of Inhibitors of Myosin Light Chain Kinase and Other Protein Kinases on the First Cell Division of Sea Urchin Eggs

We investigated effects of protein kinase inhibitors on the first cell division in sea urchin eggs on the assumption that phosphorylation of myosin is requisite for the formation and/or the contraction of the contractile ring. ML-7 or ML-9, which inhibits myosin light chain kinase (MLCK), inhibited cytokinesis with a half maximal inhibition at 0.1–0.2 mM. The nuclear division was accomplished normally at 0.2–0.25 mM where the cytokinesis was completely blocked. Fluorescent staining of actin filaments with rhodamine-labeled phalloidin revealed that the contractile ring was not formed in the cleavage-inhibited eggs. H-7 which inhibits cAMP-dependent protein kinase, cGMP-dependent protein kinase and protein kinase C arrested the process of the division at mid-cleavage at 0.25–0.3 mM and at metaphase or anaphase at 0.5 mM. H-8 and HA1004, which inhibit cAMP-dependent and cGMP-dependent protein kinases did not show significant effect at millimolar order. In the presence of micromolar concentrations of staurosporine which preferentially inhibits protein kinase C and MLCK small mitotic apparatuses were formed, in which chromosomes did not form the metaphase plate. The role of phosphorylation in the cell division is discussed.     

Changes in the Distribution of Calcium-Sequestering Membranes during the First Cell Cycle of the Sea Urchin, Lytechinus variegatus

Chlortetracycline (CTC) has been used to study sequential changes in the distribution of calcium-sequestering membranes during the first cell cycle of fertilized sea urchin eggs CTC staining patterns first appear as a diffuse ring around the centered zygote nucleus at the time of syngamy. As development proceeds, the ring becomes brighter and then elongates concurrently with the formation of the streak apparatus. Fluorescence subsequently accumulates in the centrospheres of the developing mitotic apparatus and is present in mitotic asters throughout mitosis. When the mitotic apparatus disappears, the fluorescence associated with each aster condenses into a bright ring surrounding each daughter nucleus. Ultrastructural studies show that CTC-fluorescent areas are rich in membranes while experiments with rhodamine 123, a mitochondrion-specific laser dye, indicate that mitochondria are excluded from areas in which membranes accumulate. Microtubule inhibitors prevent the initial accumulation of fluorescence around the zygote nucleus and arrest the development of existing fluorescence patterns when applied at later stages. In contrast, changes in fluorescence patterns are unaffected by the microfilament inhibitor cytochalasin D. These observations show that calcium-sequestering membranes are associated consecutively with the sperm aster, streak, and mitotic apparatus and that the continual reorganization of these membranes during the first cell cycle depends on the assembly and disassembly of microtubules.