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M Affolter

Publications and source records attributed to M Affolter.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular and biochemical characterization of the aaNAT1 (Dat) locus in Drosophila melanogaster: differential expression of two gene products.

In insects, arylalkylamine N-acetyltransferases (AANATs) have been implicated in several physiological processes, including sclerotization, inactivation of certain neurotransmitters, and, similar to the function in vertebrates, catalysis of the rate-limiting step in melatonin biosynthesis. Here, we report an extensive biochemical and functional analysis of the products of the aaNAT1 gene of Drosophila melanogaster. The aaNAT1 gene generates two transcripts through alternative first-exon usage. These transcripts are under tissue-specific and developmental control and encode proteins which differ in their N-terminus with respect to their starting methionine. The more abundant isoform, AANATlb, is first expressed during late embryogenesis in the brain, the ventral nerve cord, and the midgut; in adults, AANATlb is still detectable in the brain and midgut. The less abundant isoform, AANATla, appears only during late pupal stages and in adults is found predominantly in the brain. We demonstrate that the mutation Dat(lo) represents a hypomorphic allele of aaNAT1b, in which an insertion of two transposable elements, MDG412 and blastopia, has occurred within the first intron of the gene. Using a deficiency which removes the aaNAT1 gene, we provide evidence that aaNAT1 is not essential for the process of sclerotization. Furthermore, neither of the two enzyme isoforms shows circadian regulation of RNA or protein levels. The differing levels of abundance and distinct developmental control of AANAT1a and AANAT1b suggest different in vivo functions for these two enzymes.

Alternative Splicing↗
PubMed1998

Characterization of the prolyl dipeptidyl peptidase gene (dppIV) from the koji mold Aspergillus oryzae.

The koji mold Aspergillus oryzae secretes a prolyl dipeptidyl peptidase (DPPIV) when the fungus is cultivated in a medium containing wheat gluten as the sole nitrogen and carbon source (MMWG). We cloned and sequenced the DPPIV gene from an A. oryzae library by using the A. fumigatus dppIV gene as a probe. Reverse transcriptase PCR experiments showed that the A. oryzae dppIV gene consists of two exons, the first of which is only 6 bp long. The gene encodes an 87.2-kDa polypeptide chain which is secreted into the medium as a 95-kDa glycoprotein. Introduction of this gene into A. oryzae leads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG. The dppIV null mutants did not exhibit any change in phenotype other than the absence of prolyl dipeptidyl peptidase activity, suggesting that this activity is not essential. This loss of activity diminished the number of dipeptides and increased the number of larger peptides present in the MMWG culture broth. These effects were reversed by the addition of purified, recombinant DPPIV from the methylotrophic yeast expression vector Pichia pastoris. Our results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.

Aspergillus oryzae↗
PubMed1998

The transcription factors KNIRPS and KNIRPS RELATED control cell migration and branch morphogenesis during Drosophila tracheal development.

Cell migration during embryonic tracheal system development in Drosophila requires DPP and EGF signaling to generate the archetypal branching pattern. We show that two genes encoding the transcription factors KNIRPS and KNIRPS RELATED possess multiple and redundant functions during tracheal development. knirps/knirps related activity is necessary to mediate DPP signaling which is required for tracheal cell migration and formation of the dorsal and ventral branches. Ectopic knirps or knirps related expression in lateral tracheal cells respecifies their anteroposterior to a dorsoventral migration behavior, similar to that observed in the case of ectopic DPP expression. In dorsal tracheal cells knirps/knirps related activity represses the transcription factor SPALT; this repression is essential for secondary and terminal branch formation. However, in cells of the dorsal trunk, spalt expression is required for normal anteroposterior cell migration and morphogenesis. spalt expression is maintained by the EGF receptor pathway and, hence, some of the opposing activities of the EGF and DPP signaling pathways are mediated by spalt and knirps/knirps related. Furthermore, we provide evidence that the border between cells acquiring dorsal branch and dorsal trunk identity is established by the direct interaction of KNIRPS with a spalt cis-regulatory element.

Animals↗
PubMed1997

Synergistic activation of a Drosophila enhancer by HOM/EXD and DPP signaling.

The homeotic proteins encoded by the genes of the Drosophila HOM and the vertebrate HOX complexes do not bind divergent DNA sequences with a high selectivity. In vitro, HOM (HOX) specificity can be increased by the formation of heterodimers with Extradenticle (EXD) or PBX homeodomain proteins. We have identified a single essential Labial (LAB)/EXD-binding site in a Decapentaplegic (DPP)-responsive enhancer of the homeotic gene lab which drives expression in the developing midgut. We show that LAB and EXD bind cooperatively to the site in vitro, and that the expression of the enhancer in vivo requires exd and lab function. In addition, point mutations in either the EXD or the LAB subsite compromise enhancer function, strongly suggesting that EXD and LAB bind to this site in vivo. Interestingly, we found that the activity of the enhancer is only stimulated by DPP signaling significantly upon binding of LAB and EXD. Thus, the enhancer appears to integrate positional information via the homeotic gene lab, and spatiotemporal information via DPP signaling; only when these inputs act in concert in an endodermal cell is the enhancer fully active. Our results illustrate how a tissue-specific response to DPP can be generated through synergistic effects on an enhancer carrying both DPP- and HOX-responsive sequences.

Animals↗
PubMed1997

Identification of the autophosphorylation sites of the Xenopus laevis Pim-1 proto-oncogene-encoded protein kinase.

Pim-1 is an oncogene-encoded serine/threonine kinase expressed primarily in cells of the hematopoietic and germ line lineages. Previously identified only in mammals, pim-1 cDNA was cloned and sequenced from the African clawed frog Xenopus laevis. The coding region of Xenopus pim-1 encoded a protein of 324 residues, which exhibited 64% amino acid identity with the full-length human cognate. Xenopus Pim-1 was expressed in bacteria as a glutathione S-transferase (GST) fusion protein and in COS cells. Phosphoamino acid analysis revealed that recombinant Pim-1 autophosphorylated on serine and threonine and to a more limited extent on tyrosine. Electrospray ionization mass spectroscopy was undertaken to locate these phosphorylation sites, and the primary autophosphorylation site of GST-Pim-1 was identified as Ser-190 with Thr-205 and Ser-4 being minor sites. Ser-190, which immediately follows the high conserved Asp-Phe-Gly motif in catalytic subdomain VII, is also featured in more than 20 other protein kinases. To evaluate the importance of the Ser-190 site on the phosphotransferase activity of Pim-1, Ser-190 was mutated to either alanine or glutamic acid, and the constructs were expressed in bacteria as GST fusion proteins and in COS cells. These mutants confirmed that Ser-190 is a major autophosphorylation site of Pim-1 and indicated that phosphorylation of Pim-1 on the Ser-190 residue may serve to activate this kinase.

Amino Acid Sequence↗
PubMed1997

DPP controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo.

We report that DPP signaling is required for directed tracheal cell migration during Drosophila embryogenesis. The failure of tracheal cells to receive the DPP signal from adjacent dorsal and ventral cells results in the absence of dorsal and ventral migrations. Ectopic DPP signaling can reprogram cells in the center of the placode to adopt a dorsoventral migration behavior. The effects observed in response to ectopic DPP signaling are also observed upon the tracheal-specific expression of a constitutive active DPP type I receptor (TKV(Q253D)), indicating that the DPP signal is received and transmitted in tracheal cells to control their migration behavior. DPP signaling determines localized gene expression patterns in the developing tracheal placode, and is also required for the dorsal expression of the recently identified BRANCHLESS (BNL) guidance molecule, the ligand of the BREATHLESS (BTL) receptor. Thus, DPP plays a dual role during tracheal cell migration. It is required to control the dorsal expression of the BNL ligand; in addition, the DPP signal recruits groups of dorsal and ventral tracheal cells and programs them to migrate in dorsal and ventral directions.

Activin Receptors↗
PubMed1997

The cramped gene of Drosophila is a member of the Polycomb-group, and interacts with mus209, the gene encoding Proliferating Cell Nuclear Antigen.

We have isolated and molecularly characterized the cramped (crm) gene of Drosophila melanogaster, and show that it can be classified as a Polycomb-group (Pc-G) gene. crm mutants exhibit typical Pc-G mutant phenotypes, reminiscent of ectopic homeotic gene expression, with additional sex comb teeth found on mesothoracic and metathoracic legs, and proximodistal transformations of the tarsal segments. crm encodes an 693 amino acids protein, with no significant homology to known proteins. We used polyclonal antibodies raised against bacterially expressed truncated CRM protein to show that the crm gene product is localized to the nucleus during embryogenesis. This nuclear localization appears to be restricted to S-phase nuclei, as CRM immunostaining disappears at mitosis. We found that this cell-cycle-dependent staining pattern was identical to that of Proliferating Cell Nuclear Antigen (PCNA). Furthermore, we provide evidence for co-localization of CRM and PCNA proteins in salivary gland polytene nuclei, and for a genetic interaction between crm and mus209, the Drosophila gene encoding PCNA. Together, our data suggest that these two proteins are involved in a common regulatory pathway and highlight possible interactions between Pc-G-mediated silencing and DNA replication in Drosophila.

Amino Acid Sequence↗
PubMed1996

Recombinant gene expression and 1H NMR characteristics of the kringle (2 + 3) supermodule: spectroscopic/functional individuality of plasminogen kringle domains.

The plasminogen kringle 2 (K2HPg) and kringle 3 (K3HPg) modules occur in tandem within the polypeptide segment that affords the heavy chain of plasmin. The K2HPg and K3HPg are unique among the plasminogen kringle domains in that they also are linked to each other via the Cys169-Cys297 (Cys4 of K2HPg to Cys43 of K3HPg, kringle numbering convention) disulfide bridge, thus generating a K2HPg-K3HPg "supermodule". The kringle (2 + 3) sequence of human plasminogen (r-EE[K2HPgK3HPg]DS) was expressed in Escherichia coli, using an expression vector containing the phage T5 promoter/operator N250PSN250P29 and the codons for an N-terminal hexahistidine tag to ensure the isolation of the recombinant protein by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose under denaturing and reducing conditions. Kringle (2 + 3) was refolded in the presence of glutathione redox buffer. By taking advantage of the lysine affinity of kringle 2, the protein was purified by affinity chromatography on lysine-Bio-Gel. Recombinant kringle (2 + 3) was identified by amino acid composition, N-terminal sequence and mass determination. The 1H NMR spectrum shows that the intact r-K2HPgK3HPg is properly folded. By reference to spectra of the individual kringles, r-K2HPg and r-K3HPg, resonances of the K2HPg and K3HPg components in the spectrum of the intact r-K2HPgK3HPg can be readily distinguished. The strictly conserved Leu46 residue (kringle residue number convention) yields delta-methyl signals that are characteristic for K2HPg and K3HPg, exhibiting chemical shifts of -0.87 and -0.94 ppm, respectively, which are distinct from those of K1HPg, K4HPg, and K5HPg, (-1.04 to -1.05 ppm). Thus, the high-field Leu46 signals from K2HPg and K3HPg are well resolved from those of other kringles and can be identified unambiguously in spectra of the K1HPgK2HPgK3HPg elastolytic fragment of plasminogen as well as in spectra of Glu-plasminogen. Overall, r-K2HPgK3HPg exhibits broader resonance line widths than does the K1HPg component, consistent with a lesser mobility of the K2HPgK3HPg segment within the K1HPgK2HPgK3HPg fragment, a reflection of the extra structural constraint imposed by the disulfide bridge linking K2HPg to K3HPg. The ligand 6-aminohexanoic acid (6-AHA), which is known to interact with r-K2HPg but not with r-K3HPg, selectively perturbs K2 aromatic signals in the intact r-K2HPgK3HPg spectrum while leaving K3 resonances largely unaffected. Association constant (K(a)) values for 6-AHA determined from 1H NMR ligand titration experiments yield K(a) approximately 2.2 +/- 0.3 mM(-1) for the intact r-K2HPgK3HPg, comparable to K(a) approximately 2.3 +/- 0.2 mM(-1) determined for the isolated r-K2HPg, which demonstrates that the interactions of 6-AHA with the K2HPg ligand-binding site are not significantly affected by the neighboring K3HPg domain within the intact r-K2HPgK3HPg supermodule.

Amino Acid Sequence↗
PubMed1996

The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system.

We identified a Drosophila gene, pruned, that regulates formation of the terminal branches of the tracheal (respiratory) system. These branches arise by extension of long cytoplasmic processes from terminal tracheal cells towards oxygen-starved tissues, followed by formation of a lumen within the processes. The pruned gene is expressed in terminal cells throughout the period of terminal branching. pruned encodes the Drosophila homologue of serum response factor (SRF), which functions with an ETS domain ternary complex factor as a growth-factor-activated transcription complex in mammalian cells. In pruned loss of function mutants, terminal cells fail to extend cytoplasmic projections. A constitutively activated SRF drives formation of extra projections that grow out in an unregulated fashion. An activated ternary complex factor has a similar effect. We propose that the Drosophila SRF functions like mammalian SRF in an inducible transcription complex, and that activation of this complex by signals from target tissues induces expression of genes involved in cytoplasmic outgrowth.

Animals↗
PubMed1996

The Drosophila Serum Response Factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered.

The adult Drosophila wing is formed by an epithelial sheet, which differentiates into two non-neural tissues, vein or intervein. A large number of genes, many of them encoding components of an EGF-receptor signaling pathway, have previously been shown to be required for differentiation of vein tissue. Much less is known about the molecular control of intervein differentiation. Here we report that the Drosophila homolog of the mammalian Serum Response Factor gene (DSRF), which encodes a MADS-box containing transcriptional regulator, is expressed in the future intervein tissue of wing imaginal discs. In adult flies carrying only one functional copy of the DSRF gene, additional vein tissue develops in the wing, indicating that DSRF is required to spatially restrict the formation of veins. In mitotic clones lacking DSRF, intervein tissue fails to differentiate and becomes vein-like in appearance. Genetic and molecular evidence demonstrates that DSRF is encoded by the blistered locus, which produces ectopic veins and blistered wings when mutant. Our results show that DSRF plays a dual role during wing differentiation. It acts in a dosage-dependent [correction of dosage-dependant] manner to suppress the formation of wing veins and is required cell-autonomously to promote the development of intervein cells. We propose that DSRF acts at a key step between regulatory genes that define the early positional values in the developing wing disc and the subsequent localized expression of intervein-specific structural genes.

Alleles↗
PubMed1995

Comparison of the specificity of bacterially expressed cytoplasmic protein-tyrosine phosphatases SHP and SH-PTP2 towards synthetic phosphopeptide substrates.

SHP and SH-PTP2 are related cytoplasmic protein-tyrosine phosphatases having two tandem amino-terminal src homology 2 domains linked to a single catalytic domain. There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways. However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known. To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases. As seen previously, the catalytic activities of bacterially expressed SHP and SH-PTP2 were regulated by the presence of the linked src homology 2 domains. In addition, we characterized a cryptic thrombin cleavage site within the carboxy-terminus of SHP that led to a striking increase in the activity of the catalytic domain. Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH-PTP2 demonstrated a similar specificity pattern. Similar to SH-PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over approximately 40-1.6 x 10(3) M-1 s-1 towards other substrates. In contrast, the PTP-1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease. The overall similarity demonstrated by the catalytic domains of SHP and SH-PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard.

Amino Acid Sequence↗
PubMed1995

Schnurri is required for Drosophila Dpp signaling and encodes a zinc finger protein similar to the mammalian transcription factor PRDII-BF1.

Cytokines of the TGF beta superfamily regulate many aspects of cellular function by activating receptor complexes consisting of two distantly related serine/threonine kinases. Previous studies have indicated that Drosophila dpp uses similar signaling complexes and strictly requires the punt and thick veins receptors to transduce the signal across the membrane. Here, we show that the schnurri (shn) gene is required for many aspects of dpp signaling. Genetic epistasis experiments indicate that shn functions downstream of the dpp signal and its receptors. The shn gene encodes a large protein similar to a family of mammalian zinc finger transcription factors. The shn protein might therefore act as a nuclear target in the dpp signaling pathway directly regulating the expression of dpp-responsive genes.

Activin Receptors↗
PubMed1995

An absolute requirement for both the type II and type I receptors, punt and thick veins, for dpp signaling in vivo.

TGF beta elicits diverse cellular responses by signaling through receptor complexes formed by two distantly related transmembrane serine/threonine kinases called type II and type I receptors. Previous studies have indicated that the product of the Drosophila thick veins (tkv) gene is a type I receptor for decapentaplegic (dpp). Here, we show that the Drosophila gene punt encodes a homolog of a vertebrate type II receptor, and we demonstrate that punt, like tkv, is essential in vivo for dpp-dependent patterning processes. Because no dpp-related signalling is apparent in the absence of either the punt or tkv receptor, we infer that both receptors act in concert to transduce the dpp signal and that their functions cannot be replaced by the other extant type II and I receptors.

Activin Receptors↗
PubMed1994

Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70.

We have developed a rapid and sensitive two capillary-column chromatography and mass spectrometry-based method for the determination of protein phosphorylation sites following recovery of individual phosphopeptides from two-dimensional phosphopeptide maps. With a standard phosphopeptide, we demonstrate detection sensitivity of at least 250 fmol for this system. We applied this technique to the analysis of in vitro sites of tyrosine phosphorylation induced on the T cell-specific protein tyrosine kinase ZAP-70 in the absence and presence of p56lck. We show that ZAP-70 has a primary autophosphorylation site at Tyr-292, with a secondary site at Tyr-126. We also show additional phosphorylation at Tyr-69, Tyr-178, Tyr-492, and Tyr-493 upon the addition of the protein tyrosine kinase, p56lck. By comparative two-dimensional phosphopeptide mapping, we show that ZAP-70 isolated from Jurkat T cells also autophosphorylates at Tyr-292 and Tyr-126. Similar analysis of 32P-labeled Jurkat cells stimulated with anti-T cell receptor antibodies reveals Tyr-492 and Tyr-493 as the principal sites of T cell antigen receptor-induced tyrosine phosphorylation, with additional phosphorylation at the Tyr-292, but not the Tyr-126 autophosphorylation site. The high degree of sensitivity achieved with this technology should greatly facilitate the direct biochemical determination of inducible protein phosphorylation events, an experimental strategy that until now has been both time consuming and difficult.

Amino Acid Sequence↗
PubMed1994

Evaluation of two-dimensional phosphopeptide maps by electrospray ionization mass spectrometry of recovered peptides.

Intracellular signaling pathways are to a large extent regulated by reversible protein phosphorylation of pathway components. To fully investigate the regulation of these pathways, it is often necessary to identify the sites of protein phosphorylation induced on individual components. The low abundance of many of these molecules and the potentially low stoichiometry of phosphorylation means that conventional analytical techniques are incapable of identifying specific sites of modification inducible in vivo. The most common technique used is two-dimensional (2D) phosphopeptide mapping (electrophoresis, thin-layer chromatography) of peptides derived by proteolysis of a phosphoprotein. The number of spots detected is commonly interpreted as the number of sites of phosphorylation. Here we have achieved positive identification of phosphorylation sites by capillary high-performance liquid chromatography, with on-line mass spectrometric detection, of phosphopeptides recovered from 2D phosphopeptide maps. We demonstrate that the chemical composition of phosphopeptides is not altered during the 2D mapping procedure. By detailed analysis of the sites of phosphorylation induced in vitro on CD3-zeta by p56lck we demonstrate that interpretation of the sites of phosphorylation based on 2D phosphopeptide mapping alone is difficult. To minimize over- or misinterpretation of 2D phosphopeptide maps we therefore postulate rules that should be applied generally in cases in which protein phosphorylation sites are being evaluated by 2D phosphopeptide patterns alone.

Amino Acid Sequence↗
PubMed1994

Activation and serine phosphorylation of the p56lck protein tyrosine kinase in response to antigen receptor cross-linking in B lymphocytes.

We show that cross-linking the B cell AgR with anti-Ig Abs activates p56lck (Lck) in both the immature B cell line WEHI-231 and mature resting B cells from mouse spleen. Anti-Ig-stimulated Lck activity peaked after 1 to 2 min, but remained elevated for at least 15 min. Consistent with the proposed role for src family tyrosine kinases in AgR signaling, we found that Lck could phosphorylate the cytoplasmic tails of the Ig-alpha and Ig-beta components of the B cell AgR in vitro. Lck phosphorylated both of the tyrosines in the Ig-beta AgR homology motif and one of the two tyrosines in the Ig-alpha AgR homology motif. Finally, we show that AgR ligation in B cells caused a significant portion of the Lck to migrate with an apparent molecular mass of 60 kDa on SDS-PAGE gels. Conversion of p56lck to p60lck was maximal at 5 to 15 min, at which times Lck activity in the cells was decreasing. This Lck "band shift" has been observed previously in activated T cells and correlates with phosphorylation of Lck at serine 59. We show that the 60-kDa form of Lck induced by AgR cross-linking in B cells is also phosphorylated at serine 59. Phosphorylation of Lck at this site in vitro decreases its activity. Thus, in B cells, AgR cross-linking activates Lck and subsequently activates a kinase that phosphorylates Lck at serine 59, a potential negative regulatory site.

Amino Acid Sequence↗
PubMed1994

Receptor serine/threonine kinases implicated in the control of Drosophila body pattern by decapentaplegic.

Members of the TGF beta superfamily of secreted signaling molecules regulate growth and cellular patterning during development and interact with specific type I and type II membrane receptors possessing a cytoplasmic serine/threonine kinase domain. We describe two members of the type I receptor family in Drosophila and demonstrate that they are encoded by the genes saxophone (sax) and thick veins (tkv). Further, we show that mutations that abolish sax or tkv activity cause phenotypes similar to partial or complete loss of activity, respectively, of the TGF beta homolog decapentaplegic (dpp). We propose that specification of distinct cell fates in response to different concentrations of dpp may be achieved combinatorially by the sax and tkv receptors.

Amino Acid Sequence↗