Phosphorylation-dependent effects of synapsin IIa on actin polymerization and network formation.

The synapsins are a family of synaptic vesicle phosphoproteins which play a key role in the regulation of neurotransmitter release and synapse formation. In the case of synapsin I, these biological properties have been attributed to its ability to interact with both synaptic vesicles and the actin-based cytoskeleton. Although synapsin II shares some of the biological properties of synapsin I, much less is known of its molecular properties. We have investigated the interactions of recombinant rat synapsin Ila with monomeric and filamentous actin and the sensitivity of those interactions to phosphorylation, and found that: i) dephosphorylated synapsin II stimulates actin polymerization by binding to actin monomers and forming actively elongating nuclei and by facilitating the spontaneous nucleation/elongation processes; ii) dephosphorylated synapsin II induces the formation of thick and ordered bundles of actin filaments with greater potency than synapsin I; iii) phosphorylation by protein kinase A markedly inhibits the ability of synapsin II to interact with both actin monomers and filaments. The results indicate that the interactions of synapsin II with actin are similar but not identical to those of synapsin I and suggest that synapsin II may play a major structural role in mature and developing nerve terminals, which is only partially overlapping with the role played by synapsin I.

Phosphorylation of VAMP/synaptobrevin in synaptic vesicles by endogenous protein kinases.

VAMP/synaptobrevin (SYB), an integral membrane protein of small synaptic vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G is thought to play an important role in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca2+/calmodulin-dependent protein kinase II (CaMKII) associated with synaptic vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with synaptic vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaMKII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane.

Expression levels of B-50/GAP-43 in PC12 cells are decisive for the complexity of their neurites and growth cones.

To study the role of the protein B-50/GAP-43 in NGF-induced neurite outgrowth, a number of stable PC12 subclones with either very low or considerably enhanced expression levels of the protein were selected. Cell bodies of subclones with suppressed B-50 expression (-B2, -B5, or -B12) possessed a relative small spherical shape and, on NGF-treatment for 7 d, developed processes that were virtually devoid of branches and that mostly bore short or blunt-ended growth cones. Cells of subclones with overexpression of B-50 (+B3, +B4, or +B11), on NGF treatment, acquired a flattened, spiky appearance with highly branched neurites possessing extended and complex growth cones. Confocal microscopy with immunofluorescence for B-50 and F-actin revealed that in neurites and growth cones of the B-50-deficient subclone -B2, no detectable B-50 and reduced amounts of filamentous F-actin were present, whereas in overexpressing +B3 cells, cell membranes, neurites, and complex growth cones were intensively stained for B-50 and exhibited numerous spikes, in which B-50 was strikingly colocalized with F-actin. These data suggest that, under normal conditions of neuritogenesis, the expression level of B-50 in PC12 cells is decisive for the complexity of neurites and growth cones.

Identification of two promoter regions in the rat B-50/GAP-43 gene.

To determine cis-acting elements controlling the rat B-50/GAP-43 gene expression, the genomic DNA encoding exon 1 and the 5' flanking sequence was isolated. Sequence analysis of 1 kb 5' untranslated region (UTR) revealed the presence of a (GA)-repeat and a (GT)-repeat. The size of the (GA)-repeat varied due to both an instability of phage lambda lambda DNA in E. coli and genomic variation between rats. Transcription initiation sites were mapped in 8-day-old rat brain poly(A)+ mRNA. Primer extension indicated multiple transcription start sites at -159 and -339/-342 nt upstream of the translation start site; reverse transcriptase coupled PCR showed that the most 5' transcription start site is located between -465 and -440. Northern blotting demonstrated that approximately 90% of the B-50 mRNAs initiates at approximately -50. Promoter analysis by transient transfection assays in undifferentiated and retinoic acid-differentiated P19-EC cells revealed that the rat B-50 gene contains two promoters. P1 (located between -750 and -407) contains commonly observed promoter elements such as a TATA box and CCAAT boxes. P2 (located between -233 and -1) neither contains TATA boxes, CCAAT boxes nor consensus sequences of house-keeping gene promoters like GC-boxes. The activity of P1 is inhibited at neuroectodermal differentiation of P19-EC cells whereas the activity of P2 is stimulated. In 8 day old rat brain the majority of the B-50 mRNA transcripts are derived from P2. It is concluded that at this developmental stage P2 is the most important promoter.

Spontaneous morphological changes by overexpression of the growth-associated protein B-50/GAP-43 in a PC12 cell line.

In order to study the direct effects of B-50 on neural cell morphology, rat B-50 cDNA was transfected into a PC12 cell line (PC-B2) exhibiting neurite outgrowth independent of the expression of endogenous B-50. The morphological changes were visualized by confocal scanning laser microscopy using fluorescence labelling for B-50 and for F-actin. The transfected cells exhibited filopodia and/or blebs on the plasma membrane, containing most of the B-50 immunoreactivity. No spontaneous neurite outgrowth was observed. Following NGF treatment transfected and nontransfected PC-B2 cells extended F-actin positive filopodia and neurites with a striking colocalisation of B-50 and F-actin. Our data show that the presence of B-50 can influence cell surface morphology independent of the presence of NGF. The colocalisation of B-50 and F-actin in the filopodial protrusions but not in the blebs might be indicative for a role of B-50 in actin polymerization and depolymerization.

B-50/GAP-43 binds to actin filaments without affecting actin polymerization and filament organization.

To investigate a possible function of the nervous tissue-specific protein kinase C substrate B-50/GAP-43 in regulation of the dynamics of the submembranous cytoskeleton, we studied the interaction between purified B-50 and actin. Both the phosphorylated and dephosphorylated forms of B-50 cosedimented with filamentous actin (F-actin) in a Ca(2+)-independent manner. Neither B-50 nor phospho-B-50 had any effect on the kinetics of actin polymerization and on the critical concentration at steady state, as measured using pyrenylated actin. Light scattering of F-actin samples was not increased in the presence of B-50, suggesting that B-50 does not bundle actin filaments. The number of actin filaments, determined by [3H]cytochalasin B binding, was not affected by either phospho- or dephospho-B-50, indicating that B-50 has neither a severing nor a capping effect. These observations were confirmed by electron microscopic evaluation of negatively stained F-actin samples, which did not reveal any structural changes in the actin meshwork on addition of B-50. We conclude that B-50 is an actin-binding protein that does not directly affect actin dynamics.

Structure of the human gene for the neural phosphoprotein B-50 (GAP-43).

The genomic DNA encoding the exons for the human neural phosphoprotein B-50 (GAP-43) was isolated using rat-based cDNA probes and oligonucleotides. Exons 2 and 3 were isolated from a genomic library, exon 1 was amplified by PCR on total genomic DNA. The gene consists of 3 exons and 2 large introns. The first exon encodes the N-terminal 10 amino acids of B-50 involved in membrane association of the protein. Exon 2 encodes the main part of the protein with the sites for protein kinase C-mediated phosphorylation and calmodulin binding, and includes a 10 amino acid residue insert not found in rodents. Exon 3 encodes the last 29 amino acid residues. The reported sequence extends the known cDNA structure to both the 5' and 3' ends. The 358 bp region upstream of the translational initiation codon, containing the main transcription starts, is purine-rich and does not include TATA or GC boxes. At the 3' end potential polyadenylation signals were found 510 bp and 584 bp downstream of the stopcodon in exon 3. The 5' end of the mRNA is heterogeneous in length, with primer extension products corresponding to a 5' untranslated region of 159 and 343 bases. Northern hybridizations, however, indicate that the majority of B-50 mRNA has a shorter 5' untranslated region, as was reported for the rat (Schrama et al., Soc. Neurosci. Abstr., 18 (1992) 333.4). The structural organization of the human gene is similar to that described for the rat (Grabczyk et al., Eur. J. Neurosci. 2 (1990) 822-827), and both translated and untranslated regions show a high degree of sequence homology to the rat gene.

Phosphoprotein B-50: localization of proteolytic sites for S. aureus V8 protease using truncated cRNAs for cell-free translation.

B-50 (= GAP-43, F1, and P-57 or neuromodulin) is a nervous tissue-specific, growth-associated protein, localized in the presynaptic membrane. Phosphorylation by protein kinase C at Ser41 appears to play a role in B-50/calmodulin interaction and neurotransmitter release. Previous studies have shown that digestion of the phosphorylated protein with S. aureus V8 protease (SAP) resulted consecutively in 28- and 15-kDa phospho fragments, the latter containing all incorporated phosphate. These proteolytic products of digestion with SAP have frequently been used to identify B-50 in various systems. Therefore we were interested to find out the location of these fragments in the rat B-50 molecule. For this purpose, the rat cDNA for B-50 was used to generate full-length and truncated cRNAs for cell-free translation. B-50 and B-50 peptides were either N-terminally labeled with [35S]methionine (residues 1 and 5) as a tracer, or they were phosphorylated in vitro by protein kinase C. SAP digestion of the immunoprecipitated, 35S-labeled translation products produced similar 28- and 15-kDa fragments as were obtained from 32P-labeled B-50, indicating that these fragments are N-terminal. Relative mobilities of the N-terminal B-50 fragments of known length were used as internal standards for the calculation of the length of SAP and phospho fragments. Comparing the 35S- and 32P-labeled products, four SAP sites at Glu12, Glu28, Glu65, and Glu132 could be deduced. The latter two sites are in accordance with sequence data of C-terminal fragments from the literature. All available data could be fitted into one scheme.

Role of the growth-associated protein B-50/GAP-43 in neuronal plasticity.

The neuronal phosphoprotein B-50/GAP-43 has been implicated in neuritogenesis during developmental stages of the nervous system and in regenerative processes and neuronal plasticity in the adult. The protein appears to be a member of a family of acidic substrates of protein kinase C (PKC) that bind calmodulin at low calcium concentrations. Two of these substrates, B-50 and neurogranin, share the primary sequence coding for the phospho- and calmodulin-binding sites and might exert similar functions in axonal and dendritic processes, respectively. In the adult brain, B-50 is exclusively located at the presynaptic membrane. During neuritogenesis in cell culture, the protein is translocated to the growth cones, i.e., into the filopodia. In view of many positive correlations between B-50 expression and neurite outgrowth and the specific localization of B-50, a role in growth cone function has been proposed. Its phosphorylation state may regulate the local intracellular free calmodulin and calcium concentrations or vice versa. Both views link the B-50 protein to processes of signal transduction and transmitter release.

Mutation of serine 41 in the neuron-specific protein B-50 (GAP-43) prohibits phosphorylation by protein kinase C.

The neuron-specific, calmodulin-binding protein B-50 (also known as GAP-43, F1, or neuromodulin) is an endogenous substrate of protein kinase C (PKC). PKC exclusively phosphorylates Ser residues in B-50. As potential phosphorylation sites for PKC, Ser41, Ser110, and Ser122 were indicated, of which Ser41 is contained in the sequence ASF, which matches with the sequence of a synthetic PKC substrate. N-terminally 35S-labeled B-50, produced from cDNA, was subjected to digestion with Staphylococcus aureus V8 protease (SAP). Consecutively, 35S-labeled 28- and 15-kDa fragments were formed, similar to those after digestion of 32P-labeled B-50. In a previous study, we showed that the 32P-labeled 15-kDa SAP fragment contains all 32P radioactivity. The present data indicate that it contains the N-terminus of B-50 as well. The 15-kDa fragment, with a calculated length ranging from amino acid residue 1 to 65, contains only one potential PKC phosphorylation site, at Ser41. Mutagenesis of Ser41 into Thr or Ala resulted in recombinant B-50 products with mobilities on two-dimensional electrophoresis similar to those of the nonmutated recombinant B-50 and the rat brain B-50. Only [Ser41]B-50 was phosphorylated by PKC, whereas [Thr41]- or [Ala41]B-50 did not show any phosphorylation at the positions indicated on the immunoblots. This leads us to the conclusion that Ser41 is the sole phosphorylation site for PKC in vitro.

Microheterogeneity of the growth-associated neuronal protein B-50 (GAP-43). Contribution of phosphorylation by protein kinase Ca.

The neuron-specific, growth-associated protein B-50, also known as GAP-43. F1 and neuromodulin, shows a striking heterogeneous behaviour in many chromatographic and electrophoretic systems. A modulatory function has been proposed for the protein in receptor-mediated processes in the presynaptic membrane. Fatty acid acylation, calmodulin binding and phosphorylation appear to be tools in this respect. At least three discrete isoforms were present in separations made by reversed-phase fast protein liquid chromatography (FPLC) of the phosphorylated protein. In anion-exchange FPLC chromatography a conglomerate of eight peaks was eluted, which migrated as eight parallel curves in electrophoretic mobility studies. After dephosphorylation of the protein this number was reduced to two. Under non-reducing conditions, the phosphoprotein was eluted from an FPLC gel filtration column at Mr = 270 kDa, i.e. 8-12 times the size of the monomer (m = 23.6 kDa.) In sodium dodecyl sulphate polyacrylamide gel electrophoresis all isoforms showed only B-50 at Mr of 48 kDa and its breakdown product (Mr = 40 kDa) in a constant ratio. It was concluded that phosphorylation by protein kinase C of a single serine residue is only one factor in the microheterogeneity of B-50. Multimeric forms may also add to the heterogeneous behaviour of phosphorylated B-50.

Expression of growth-associated protein B-50 (GAP43) in dorsal root ganglia and sciatic nerve during regenerative sprouting.

Recently it has been shown that B-50 is identical to the neuron-specific, growth-associated protein GAP43. The present study reports on the fate of B-50/GAP43 mRNA and B-50/GAP43 protein, determined by radioimmunoassay, in a rat model of peripheral nerve regeneration (sciatic nerve crush) over a period of 37 and 312 d, respectively. Moreover, the effects of repeated subcutaneous injection of the neurotrophic peptide Org.2766 (an ACTH4-9 analog) and of a conditioning lesion on B-50/GAP43 protein levels in the regenerating nerve and dorsal root ganglia (DRG) were investigated. Both treatments enhanced the functional recovery as evidenced by a foot-flick withdrawal test. Immunocytochemical analysis using antineurofilament antibodies revealed a peptide-induced increase in the number of outgrowing sprouts in the sciatic nerve. Both the peptide and the conditioning lesion amplified the crush lesion-induced increase in B-50 protein content in the nerve as determined by radioimmunoassay. B-50 protein levels seem to correlate proportionally with the number of sprouts. In the DRG of the crushed sciatic nerve, the time course of B-50 expression was studied. B-50 mRNA was quantified from Northern blots. A linear increase up to 10 times the basal level of B-50 mRNA was observed 2 d postsurgery, followed by a gradual decline to normal levels at day 37. The first significant rise in B-50 mRNA level became apparent between 8 and 16 hr after placement of the crush lesion. The first significant rise in B-50 protein level occurred 40 hr after the crush lesion, reaching a plateau of 3 times the basal level between day 6 and 20. B-50 protein levels in DRG cell bodies remained elevated up to 60 d after crush, a period much longer than that observed for B-50 mRNA. Thus, during a later phase of peripheral axonal regeneration, the presence of B-50 appears to be prolonged, probably by an increase in half-life and not so much by enhanced transcription. Treatment with Org.2766 did not affect the B-50/GAP43 levels in DRG cell bodies during the first 6 d following crush. Conditioning lesion resulted in a DRG B-50/GAP43 protein amount at the same level as in rats 14 d after the test lesion. B-50/GAP43 levels in DRG are probably influenced by the rapid axonal transport of the protein, as has been reported by others.

Cloning and characterization of a gene encoding an outer membrane protein required for siderophore-mediated uptake of Fe3+ in Pseudomonas putida WCS358.

In iron-limited environments plant-growth-stimulating Pseudomonas putida WCS358 produces a yellow-green fluorescent siderophore called pseudobactin 358. Ferric pseudobactin 358 is efficiently taken up by cells of WCS358 but not by cells of another rhizophere-colonizing strain, Pseudomonas fluorescens WCS374. A gene bank containing partial Sau3A DNA fragments from WCS358 was constructed in a derivative of the broad-host-range cosmid pLAFR1. By mobilization of this gene bank to strain WCS374 a cosmid clone, pMR, which made WCS374 competent for the utilization of pseudobactin 358 was identified. By subcloning of the 29.4-kilobase (kb) insert of pMR the essential genetic information was localized on a BglII fragment of 5.3 kb. Tn5 mutagenesis limited the responsible gene to a region of approximately 2.5 kb within this fragment. Since the gene encodes an outer membrane protein with a predicted molecular mass of 90,000 daltons, it probably functions as the receptor for ferric pseudobactin 358. The gene is flanked by pseudobactin 358 biosynthesis genes on both sides and is on a separate transcriptional unit. WCS374 cells carrying pMR derivatives with Tn5 insertions in the putative receptor gene did not produce the 90,000-dalton protein anymore and were unable to take up Fe3+ via pseudobactin 358. In WCS358 cells as well as in WCS374 cells the gene is expressed only under iron-limited conditions.

Genetic organization and transcriptional analysis of a major gene cluster involved in siderophore biosynthesis in Pseudomonas putida WCS358.

In iron-limited environments, the plant-growth-stimulating Pseudomonas putida WCS358 produces a yellow-green fluorescent siderophore called pseudobactin 358. The transcriptional organization and the iron-regulated expression of a major gene cluster involved in the biosynthesis and transport of pseudobactin 358 were analyzed in detail. The cluster comprises a region with a minimum length of 33.5 kilobases and contains at least five transcriptional units, of which some are relatively large. The directions of transcription of four transcriptional units were determined by RNA-RNA hybridization and by analysis in Escherichia coli minicells. The latter also demonstrated that large polypeptides were encoded by these transcriptional units. The results allowed us to localize several promoter regions on the DNA. The iron-dependent expression of at least two genes within this cluster appears to be regulated at the transcriptional level.