Matthias Mann

Phosphorylation and inactivation of BAD by mitochondria-anchored protein kinase A

Signaling pathways between cell surface receptors and the BCL-2 family of proteins regulate cell death. Survival factors induce the phosphorylation and inactivation of BAD, a proapoptotic member. Purification of BAD kinase(s) identified membrane-based cAMP-dependent protein kinase (PKA) as a BAD Ser-112 (S112) site-specific kinase. PKA-specific inhibitors blocked the IL-3-induced phosphorylation on S112 of endogenous BAD as well as mitochondria-based BAD S112 kinase activity. A blocking peptide that disrupts type II PKA holoenzyme association with A-kinase-anchoring proteins (AKAPs) also inhibited BAD phosphorylation and eliminated the BAD S112 kinase activity at mitochondria. Thus, the anchoring of PKA to mitochondria represents a focused subcellular kinase/substrate interaction that inactivates BAD at its target organelle in response to a survival factor.

Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells

Mutation of the XRCC4 gene in mammalian cells prevents the formation of the signal and coding joints in the V(D)J recombination reaction, which is necessary for production of a functional immunoglobulin gene, and renders the cells highly sensitive to ionizing radiation. However, XRCC4 shares no sequence homology with other proteins, nor does it have a biochemical activity to indicate what its function might be. Here we show that DNA ligase IV (ref. 5) co-immunoprecipitates with XRCC4 and that these two proteins specifically interact with one another in a yeast two-hybrid system. Ligation of DNA double-strand breaks in a cell-free system by DNA ligase IV is increased fivefold by purified XRCC4 and seven- to eightfold when XRCC4 is co-expressed with DNA ligase IV. We conclude that the biological consequences of mutating XRCC4 are primarily due to the loss of its stimulatory effect on DNA ligase IV: the function of the XRCC4–DNA ligase IV complex may be to carry out the final steps of V(D)J recombination and joining of DNA ends.

Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1

The actin cytoskeleton is regulated by GTP-hydrolysing proteins, the Rho GTPases,, which act as molecular switches in diverse signal-transduction processes. Various bacterial toxins can inactivate Rho GTPases by ADP-ribosylation or glucosylation. Previous research has identified Rho proteins as putative targets for Escherichia coli cytotoxic necrotizing factors 1 and 2 (CNF1 and 2),. These toxins induce actin assembly and multinucleation in culture cells. Here we show that treatment of RhoA with CNF1 inhibits the intrinsic GTPase activity of RhoA and completely blocks GTPase activity stimulated by the Rho-GTPase-activating protein (rhoGAP). Analysis by mass spectrometry and amino-acid sequencing of proteolytic peptides derived from CNF1-treated RhoA indicate that CNF1 induces deamidation of a glutamine residue at position 63 (Gln 63) to give constitutively active Rho protein.

In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho‐associated coiled‐coil kinase, the Rac‐associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co‐localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.

Delayed Extraction Improves Specificity in Database Searches by Matrix-assisted Laser Desorption/Ionization Peptide Maps

Peptide mass maps obtained by matrix-assisted laser desorption ionization (MALDI) are an attractive means to identify proteins by searches in sequence databases. Here we demonstrate that the recently introduced delayed ion-extraction technique, when coupled to reflectron MALDI time-of-flight mass spectrometry, leads to dramatically improved search specificity. Routine resolution in the range of 6,000 to 12,000 allows assignment of monoisotopic masses throughout the peptide mass range. Database searches can be performed with high precision by use of a mass accuracy which is currently better than 30 ppm over a wide mass range and better than 5 ppm for a narrow mass range. This high performance makes it possible to identify proteins with fewer peptide masses than before. Additional low intensity peaks can be assigned after a search because of the improved signal-to-noise ratio of delayed-extraction peptide mass spectra, increasing sequence coverage of matched proteins. The improvements in database search specificity can be used to identify the components of simple protein mixtures. In combination with advanced sample preparation and automation techniques, delayed-extraction MALDI time-of-flight mass spectrometry is now an extremely powerful tool for the database identification of proteins.

SH2 Signaling in a Lower Eukaryote: A STAT Protein That Regulates Stalk Cell Differentiation in Dictyostelium

The TTGA-binding factor is a transcriptional regulator activated by DIF, the chlorinated hexaphenone that induces prestalk cell differentiation in Dictyostelium. The same activity also functions as a repressor, controlling stalk cell differentiation. We show that the TTGA-binding factor is a STAT protein. Like the metazoan STATs, it functions via the reciprocal interaction of a phosphotyrosine residue on one molecule with an SH2 domain on a dimerizing partner. Furthermore, it will bind specifically to a mammalian interferon-stimulated response element. In Saccharomyces cerevisiae, where the entire genomic sequence is known, SH2 domains have not been identified. It would seem, therefore, that SH2 signaling pathways arose very early in the evolution of multicellular organisms, perhaps to facilitate intercellular comunication.

Electrospray mass spectrometry for protein characterization

Mass spectrometry is a venerable analytical tool that has been used for some time in biochemistry for the analysis of small molecules, such as steroids. More recently, physicists have solved the problems associated with vaporizing and ionizing proteins and peptides, thereby allowing mass spectrometry to take on new roles in investigating protein sequences, structures and modifications.

Mtr10p functions as a nuclear import receptor for the mRNA‐binding protein Npl3p

MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)‐tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA‐binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)‐containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non‐repetitive C‐terminal tail. Mtr10p interacts in vitro with Gsp1p‐GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran‐GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran‐GTP and newly synthesized mRNA.

Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.

The 25S [U4/U6·U5] tri‐snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre‐mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri‐snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6·U5] tri‐snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri–snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm‐like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA‐binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre‐mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri‐snRNPs and the U2 snRNP are co‐precipitated with protein A‐tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2·U4/U6·U5] tetra‐snRNP complex.

Gemin5, a Novel WD Repeat Protein Component of the SMN Complex That Binds Sm Proteins

The survival of motor neurons (SMN) protein is the product of the disease gene of spinal muscular atrophy and is found both in the cytoplasm and the nucleus, where it is concentrated in gems. SMN is part of a multi-protein complex that includes Gemin2, Gemin3, and Gemin4. The SMN complex plays an important role in the cytoplasmic assembly of small nuclear ribonucleoproteins (snRNPs) and likely other RNPs in pre-mRNA splicing and in the assembly of transcriptosomes. Here, we report the identification of an additional component of the SMN complex, a novel WD repeat protein termed Gemin5. Gemin5 binds SMN directly and is a component of the SMN complex. Furthermore, Gemin5 interacts with several of the snRNP core proteins including SmB, SmD1, SmD2, SmD3, and SmE, suggesting that it participates in the activities of the SMN complex in snRNP assembly. Immunolocalization studies demonstrate that Gemin5 is found in the cytoplasm and in the nucleus, where it colocalizes with SMN in gems. The presence of 13 WD repeat domains in the amino-terminal half of Gemin5 and a coiled-coil motif near its carboxyl terminus indicate that it may form a large heteromeric complex and engage in multiple interactions.