Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number

Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number.     

An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium

Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD (171EDVSRFIKGKLLQKQQKIYKDLERF195) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues 48KKSYQDPEIIAHSRPRK64 that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to 48EF49 abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the 48EF49 construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.     

Cell type proportioning in Dictyostelium slugs: lack of regulation within a 2.5-fold tolerance range

The proportion of prestalk and prespore cells in Dictyostelium discoideum slugs is often cited as an example of "almost perfect" regulation. The pattern is similar over a very wide range of cell number; furthermore, removal of either of the cell types leads to compensatory transdifferentiation. Several studies of Dictyostelium fruiting bodies, however, have suggested that proportioning in Dictyostelium differs systematically from true constancy. We have confirmed this in the slug stage using a short-lived beta-galactosidase as a reporter of the prestalk specific ecmA gene expression: the prestalk proportion decreases from 24+/-5% in slugs of 10(3) cells to 10+/-3% when 10(5) cells are present. Regeneration experiments suggest that this difference is not due to a modulation of the proportioning set-point by size, as one might have expected; instead there appears to be a regulatory "tolerance zone" at all sizes. After amputation of the whole posterior region, transdifferentiation stops after the fraction of prestalk has been reduced from 100% to 28+/-20%, well above the initial value of 10+/-3%, while after anterior removal the transdifferentiation endpoint is about 10%. Most strikingly, we find no regulation at all after partial amputations of the prespore region. It seems that any prestalk proportion is stable between a approximately 10% lower threshold and a approximately 30% upper threshold. To explain this, we propose a regulation mechanism based on a negative feedback plus cell type bistability. In both intact and regenerating slugs we find that the slug morphology is regulated so that the length-to-width ratio of the anterior region is constant.     

Nucleolar localization and identification of nuclear/nucleolar localization signals of the calmodulin-binding protein nucleomorphin during growth and mitosis in <Emphasis Type="Italic">Dictyostelium</Emphasis>

The calmodulin-binding protein nucleomorphin isoform NumA1 is a nuclear number regulator in Dictyostelium that localizes to intra-nuclear patches adjacent to the nuclear envelope and to a lesser extent the nucleoplasm. Earlier studies have shown similar patches to be nucleoli but only three nucleolar proteins have been identified in Dictyostelium. Here, actinomycin-D treatment caused the loss of NumA1 localization, while calcium and calmodulin antagonists had no effect. In keeping with a nucleolar function, NumA1 moved out of the presumptive nucleoli during mitosis redistributing to areas within the nucleus, the spindle fibers, and centrosomal region before re-accumulating in the presumptive nucleoli at telophase. Together, these data verify NumA1 as a true nucleolar protein. Prior to this study, the dynamics of specific nucleolar proteins had not been determined during mitosis in Dictyostelium. FITC-conjugated peptides equivalent to presumptive nuclear localization signals within NumA1 localized to nucleoli indicating that they also act as nucleolar localization signals. To our knowledge, these represent the first precisely defined nucleolar localization signals as well as the first nuclear/nucleolar localization signals identified in Dictyostelium. Together, these results reveal that NumA1 is a true nucleolar protein and the only nucleolar calmodulin-binding protein identified in Dictyostelium. The possible use of nuclear/nucleolar localization signal-mediated drug targeting to nucleoli is discussed.     

RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes, and phagosomes

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64-69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP-RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP-RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP-RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.     

Purification and renaturation of Dictyostelium recombinant alkaline phosphatase by continuous elution electrophoresis

A 1583 bp fragment of Dictyostelium alp cDNA (94% of the gene) was cloned in pET32a+. The enzyme was expressed in an inactive form in the inclusion body of the expression host BL21-CodonPlus (DE3)-RIL. The recombinant ALP constituted more than 50% of the total protein in the inclusion body and 25-30% of the total protein in the expression host after 3 h induction with IPTG at 37 degrees C. A continuous elution polyacrylamide gel electrophoresis procedure was used to purify the recombinant enzyme. This technique yielded a homogeneous protein that retained enzymatic activity after dialysis without further treatment. A yield of 5mg per liter of culture broth was obtained with a specific activity of approximately 0.7 nmol/min/mg protein (0.7 mU/mg). Immunoinhibition studies using a polyclonal antibody produced against the recombinant protein showed complete inhibition of enzymatic activity when the enzyme was preincubated with the antibody at a 1:1000 dilution. The enzyme exhibited a pH optimum of approximately 9.0. The substrate specificity indicated that the Dictyostelium enzyme is a typical broad range alkaline phosphatase.     

Calmodulin-binding proteins in the model organism Dictyostelium: a complete & critical review

Calmodulin is an essential protein in the model organism Dictyostelium discoideum. As in other organisms, this small, calcium-regulated protein mediates a diversity of cellular events including chemotaxis, spore germination, and fertilization. Calmodulin works in a calcium-dependent or -independent manner by binding to and regulating the activity of target proteins called calmodulin-binding proteins. Profiling suggests that Dictyostelium has 60 or more calmodulin-binding proteins with specific subcellular localizations. In spite of the central importance of calmodulin, the study of these target proteins is still in its infancy. Here we critically review the history and state of the art of research into all of the identified and presumptive calmodulin-binding proteins of Dictyostelium detailing what is known about each one with suggestions for future research. Two individual calmodulin-binding proteins, the classic enzyme calcineurin A (CNA; protein phosphatase 2B) and the nuclear protein nucleomorphin (NumA), which is a regulator of nuclear number, have been particularly well studied. Research on the role of calmodulin in the function and regulation of the various myosins of Dictyostelium, especially during motility and chemotaxis, suggests that this is an area in which future active study would be particularly valuable. A general, hypothetical model for the role of calmodulin in myosin regulation is proposed.     

Metabolism of two Go alpha isoforms in neuronal cells during differentiation

We have previously shown that undifferentiated N1E-115 neuroblastoma cells express only one isoform of Go alpha (pI = 5.8), whereas differentiated neuroblastoma cells expressed, in addition to this isoform, another Go alpha with a more acidic pI (5.55). Moreover, primary cultures of cerebellar granule cells, which are extremely well differentiated cells yielding a high density of synapses, expressed only a single Go alpha isoform with a pI of 5.55 (Brabet, P., Pantaloni, C., Rodriguez Martinez, J., Bockaert, J., and Homburger, V. (1990) J. Neurochem. 54, 1310-1320). In this report, using biosynthetic labeling with [35S]methionine and specific quantitative immunoprecipitation with a polyclonal antibody raised against the purified Go alpha protein, we have determined 1) the degradation rate of total Go alpha (sum of the two isoforms) in differentiated as well as in undifferentiated neuroblastoma cells and in cerebellar granule cells, 2) the degradation rates of each isoform in differentiated neuroblastoma cells. The t 1/2 for total Go alpha protein degradation was very different in the three neuronal cell populations and was 28 +/- 5 h (n = 5), 58 +/- 9 h (n = 5), and 154 +/- 22 h (n = 6) in undifferentiated, differentiated neuroblastoma, and granule cells, respectively. Using two-dimensional gel analysis of immunoprecipitates, we have also determined the individual t 1/2 for degradation of each Go alpha isoform in differentiated neuroblastoma cells, in which the two Go alpha isoforms were expressed. Results indicated that the two Go alpha isoforms exhibit similar t1/2 for degradation (49 +/- 5 h, n = 3). Thus, the t1/2 for degradation of the more basic Go alpha isoform is higher in differentiated neuroblastoma cells (49 +/- 5 h, n = 3) than in undifferentiated neuroblastoma cells (28 +/- 5 h, n = 5) which expressed only the more basic Go alpha isoform. It can be concluded that the degradation rate of the more basic Go alpha isoform is not a characteristic of the protein itself but depends on the state of the cell differentiation. The comparison between the t1/2 for degradation of the more acidic Go alpha isoform is differentiated neuroblastoma cells (51 +/- 6 h, n = 3) with that of cerebellar granule cells (154 +/- 22 h, n = 6) suggests that there is also a decrease in the degradation rate of the more acidic Go alpha isoform during differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relative abundance of growth hormone receptor isoforms in rhesus monkey tissues and human transformed lymphocytes.

The growth hormone receptor (GHR) is expressed as one active, full-sequence isoform and one truncated, inactive one that lacks the intracellular signaling domain. The aim of this study was to investigate the variation in the tissue expression of the full and truncated mRNA and protein. Epstein-Barr virus-transformed human B lymphocyte lines were established from 9 normal individuals with a height standard deviation score (SDS) of - 0.1 +/- 1.1 (mean +/- SD). Tissues were also collected from 3 Rhesus monkeys, whose GHR has 94.1 % homology with the human molecule. mRNA quantitation was determined by Real Time Quantitative PCR. Growth hormone receptor expression in transformed lymphocytes was also studied by fluorescence-activated cell sorter analysis. Both isoforms were expressed in transformed lymphocytes, but individual variation in the relative mRNA expression was small (truncated isoform percentage of total receptor mRNA: 17.1 +/- 4.4, mean +/- SD). There was no correlation between donors' height SDS and the expression of either isoform or the ratio between them. Protein expression by FACS analysis showed wider variation among the subjects; however, the relative ratio was similar in all the subjects. In monkey tissues, the truncated receptor showed a tissue-specific distribution. In conclusion, the expression of both isoforms in transformed lymphocytes from normal subjects shows small differences at the RNA or protein levels, and does not correlate with height SDS. Growth hormone splice isoforms show tissue specificity, suggesting local regulation of splicing. Tissues with relatively high expression of the truncated isoform are likely to be more resistant to the effects of GH due to the dominant negative effect of this isoform. In addition, the differential tissue expression might influence the levels of growth hormone binding protein in the immediate milieu of each tissue.     

Targeted transgenic expression of beta(2)-adrenergic receptors to type II cells increases alveolar fluid clearance

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta-agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta(2)-adrenergic receptors (beta(2)-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta(2)-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta(2)-ARs than nontransgenic (NTG) mice (939 +/- 113 vs. 194 +/- 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was approximately 40% greater than that in untreated NTG mice (15 +/- 1.4 vs. 10.9 +/- 0.6%; P < 0.005) and approached that of NTG mice treated with the beta-agonist formoterol (19.8 +/- 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 +/- 0.5% but had no effect on NTG mice (11.5 +/- 1.0%). Na(+)-K(+)-ATPase alpha(1)-isoform expression was unchanged, whereas alpha(2)-isoform expression was approximately 80% greater in the TG mice. These findings show that beta(2)-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta(2)-ARs specifically expressed on ATII cells.     

The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F- actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.     

Cloning of the genomic locus of mouse SH2 containing inositol 5-phosphatase (SHIP) and a novel 110-kDa splice isoform, SHIPdelta

The SH2 domain containing inositol 5'-phosphatase (SHIP) was initially described as a 145-kDa protein phosphorylated on tyrosines upon growth factor and cytokine stimulation. It was shown to be phosphorylated after Fc and B cell receptor activation and plays a role in negative signaling. Different isoforms of the SHIP protein result from alternative mRNA splicing, proteolysis, or a combination of both. The expression of discrete SHIP isoforms changes with the potential developmental-dependent maturation state of myeloid cells, suggesting mechanisms for the regulation of SHIP interactions with other signaling molecules. A p135 (SHIPbeta) spliced isoform is known to be expressed in developing myeloid cells. Now we have identified a new SHIP isoform, SHIPdelta, which is the product of an out-of-frame splice with a deletion of 167 nucleotides in the C-terminal region, resulting in an approximately 110-kDa protein. Biochemically, SHIPdelta differs from SHIPalpha by exhibiting little or no tyrosine phosphorylation or association with the signaling protein Shc after M-CSF activation of FD-Fms cells. In addition, we have characterized the structure of the entire SHIP genomic locus, which provides a basis for understanding the alternative splicing events. SHIP is expressed in hematopoiesis and spermatogenesis, and we also describe the promoter for the SHIP gene, which has potential for explaining the tissue-specific expression pattern.     

Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 +/- 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Forty-eight hours after transfection rHFV antigen levels in the conditioned medium were 70 +/- 15 ng/mL. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 +/- 0.012 to 0.124 +/- 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 +/- 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1800 +/- 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1700-2000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component (approximately 330 kDa). Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. We have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. Immunoprecipitation of [35S]methionine-labeled rHFV-des-B811-1441 revealed a single-chain polypeptide with Mr approximately 230 kDa. This mutant constitutively expressed 38 +/- 7% of the activity of the RVV-V-activated protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase of brain-derived neurotrophic factor gene expression in NG108-15 cells by the nuclear isoforms of Ca2+/ calmodulin-dependent protein kinase II

We have reported that the delta3 isoform of Ca2+/ calmodulin-dependent protein kinase II (CaM kinase II) is abundant in the nucleus in cerebellar granule cells. To examine the possibility that the nuclear isoforms of CaM kinase II are involved in the expression of brain-derived neurotrophic factor (BDNF), we transiently overexpressed the delta3 isoform in NG108-15 cells. The quantitative RT-PCR analysis revealed that rat cerebellum and NG108-15 cells expressed the exon IV-containing mRNA of BDNF (exon IV-BDNF mRNA) more than the exon III-BDNF mRNA. Treatment of NG108-15 cells with Bay K 8644 increased both exon III- and exon IV-BDNF mRNAs, and overexpression of the 83 isoform potentiated the expression of the exon IV-BDNF mRNA. The potentiation was not observed in the cells that were overexpressed with either the 61 isoform, a nonnuclear isoform, or the inactive mutant of the delta3 isoform. We constructed the luciferase reporter gene following the promoter upstream of exon IV and confirmed that overexpression of the delta3 isoform increased luciferase gene expression. Double-immunostaining of NG108-15 cells with the antibodies to CaM kinase II and BDNF clearly showed that BDNF was highly expressed in the cells that were overexpressed with the delta3 isoform or the alphaB isoform, another nuclear isoform of CaM kinase II. These results suggest that the nuclear isoforms of CaM kinase II are involved in the expression of BDNF.