Henk B. Nielander

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.

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

Abstract: 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 and 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.

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.

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

Abstract: To investigate a possible function of the nervous tissuespecific protein kinase C substrate B‐50/GAP‐43 in regulati of the dynamics of the submembranous cytoskeleton. we studii the interaction between purified 6–50 and actin. Both the phosphorylated and dephosphorylated forms of 8–50 cosedi‐mented with filamentous actin (F‐actin) in a Ca2+‐independent manner. Neither 6–50 nor phospho‐6–50 had any effect on the kinetics of actin polymerization and on the critical concentration at steady state, as measured using pyrenylated actin. tight scattering of F‐actin samples was not increased in the presence of 550, suggesting that 550 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 550 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 6–50, We conclude that 6–50 is an actin‐binding protein that does not directly affect actin dynamics.

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.

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 withS. 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 eitherN-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 from32P-labeled B-50, indicating that these fragments areN-terminal. Relative mobilities of theN-terminal B-50 fragments of known length were used as internal standards for the calculation of the length of SAP and phospho fragments. Comparing the35S- and32P-labeled products, four SAP sites at Glu12, Glu28, Glu65, and Glu132 could be deduced. The latter two sites are in accordance with sequence data ofC-terminal fragments from the literature. All available data could be fitted into one scheme.

The Iron-Uptake System of the Plant-Growth-Stimulating Pseudomonas Putida WCS358: Genetic Analysis and Properties and Structure Analysis of Its Siderophore

Treatment of potato seed tubers with certain root-colonizing Pseudomonos putida and Pseudomonas fluorescens strains protects the plants from deleterious microorganisms which are present in the root environment during narrow rotation cropping, resulting in an increase of tuber yield in these soils (Schippers et al 1985). The plant-growth-stimulating activity is thought to be the consequence of their very efficient iron (III)-binding capacity. Under iron-limiting conditions which exist in most soils, the pseudomonads produce and excrete large quantities of yellow-green, fluorescent siderophores which chelate iron (III) with high affinity. The pseudomonads take up the iron(III)-siderophore complex specifically and with high efficiency. The deleterious microorganisms, however, are not able to do so and thus are prevented to grow efficiently due to iron (III)-depletion.