Andrej Shevchenko

FLICE is activated by association with the CD95 death‐inducing signaling complex (DISC)

Upon activation, the apoptosis‐inducing cell membrane receptor CD95 (APO‐1/Fas) recruits a set of intracellular signaling proteins (CAP1‐4) into a death‐inducing signaling complex (DISC). In the DISC, CAP1 and CAP2 represent FADD/MORT1. CAP4 was identified recently as an ICE‐like protease, FLICE, with two death effector domains (DED). Here we show that FLICE binds to FADD through its N‐terminal DED. This is an obligatory step in CD95 signaling detected in the DISC of all CD95‐sensitive cells tested. Upon prolonged triggering of CD95 with agonistic antibodies all cytosolic FLICE gets proteolytically activated. Physiological FLICE cleavage requires association with the DISC and occurs by a two‐step mechanism. Initial cleavage generates a p43 and a p12 fragment further processed to a p10 fragment. Subsequent cleavage of the receptor‐bound p43 results in formation of the prodomain p26 and the release of the active site‐containing fragment p18. Activation of FLICE is blocked by the peptide inhibitors zVAD‐fmk, zDEVD‐fmk and zIETD‐fmk, but not by crmA or Ac‐YVAD‐CHO. Taken together, our data indicate that FLICE is the first in a cascade of ICE‐like proteases activated by CD95 and that this activation requires a functional CD95 DISC.

Exit from Mitosis Is Triggered by Tem1-Dependent Release of the Protein Phosphatase Cdc14 from Nucleolar RENT Complex

Exit from mitosis in budding yeast requires a group of essential proteins--including the GTPase Tem1 and the protein phosphatase Cdc14--that downregulate cyclin-dependent kinase activity. We identified a mutation, net1-1, that bypasses the lethality of tem1 delta. NET1 encodes a novel protein, and mass spectrometric analysis reveals that it is a key component of a multifunctional complex, denoted RENT (for regulator of nucleolar silencing and telophase), that also contains Cdc14 and the silencing regulator Sir2. From G1 through anaphase, RENT localizes to the nucleolus, and Cdc14 activity is inhibited by Net1. In late anaphase, Cdc14 dissociates from RENT, disperses throughout the cell in a Tem1-dependent manner, and ultimately triggers mitotic exit. Nucleolar sequestration may be a general mechanism for the regulation of diverse biological processes.

Gemin3 A Novel Dead Box Protein That Interacts with Smn, the Spinal Muscular Atrophy Gene Product, and Is a Component of Gems

The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.

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.

Peptide Sequencing by Mass Spectrometry for Homology Searches and Cloning of Genes

It is now possible to obtain sequence information from gel-separated proteins by mass spectrometry at levels too low for conventional approaches. Usually this tandem mass spectrometric data are used for database searches with the aim of identifying the corresponding gene. Recently it has been shown that long and accurate amino acid sequences can be obtained which are sufficient for PCR-based strategies to clone the corresponding gene [Wilm et al. (1996), Nature379, 466–469]. More than eight proteins have now been cloned based on that method. In many more cases the sequence information identified homologous proteins. Issues involved in cloning by mass spectrometric sequence information are discussed, as are two case studies. These results clearly establish mass spectrometry as a viable tool not only for the database identification of proteins, but also for the de novo sequencing of gel-separated proteins at the low-picomole to femtomole level.

Euplotes telomerase contains an La motif protein produced by apparent translational frameshifting

Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in most eukaryotes. In the ciliate Euplotes aediculatus, the protein p43 biochemically co‐purifies with active telomerase and appears to be stoichiometric with both the RNA and the catalytic protein subunit of this telomerase complex. Here we describe cloning of the gene for p43 and present evidence that it is an authentic component of the telomerase holoenzyme. Comparison of the nucleotide sequence of the cloned gene with peptide sequences of the protein suggests that production of full‐length p43 relies on a programmed ribosomal frameshift, an extremely rare translational mechanism. Anti‐p43 antibodies immunodeplete telomerase RNA and telomerase activity from E.aediculatus nuclear extracts, indicating that the vast majority of mature telomerase complexes in the cell are associated with p43. The sequence of p43 reveals similarity to the La autoantigen, an RNA‐binding protein involved in maturation of RNA polymerase III transcripts, and recombinant p43 binds telomerase RNA in vitro. By analogy to other La proteins, p43 may function in chaperoning the assembly and/or facilitating nuclear retention of telomerase.

The Bbp1p–Mps2p complex connects the SPB to the nuclear envelope and is essential for SPB duplication

In budding yeast, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope via its central plaque structure. Here, we describe the identification of BBP1 in a suppressor screen with a conditional lethal allele of SPC29. Bbp1p was detected at the central plaque periphery of the SPB and bbp1‐1 cells were found to be defective in SPB duplication. bbp1‐1 cells extend their satellite into a duplication plaque like wild‐type cells; however, this duplication plaque then fails to insert properly into the nuclear envelope and does not assemble a functional inner plaque. This function in SPB duplication is probably fulfilled by a stable complex of Bbp1p and Mps2p, a nuclear envelope protein that is also essential for duplication plaque insertion. In addition, we found that Bbp1p interacts with Spc29p and the half‐bridge component Kar1p. These interactions are likely to play a role in connecting the SPB with the nuclear envelope and the central plaque with the half‐bridge.

A major 170 kDa protein associated with bovine adrenal medulla microtubules: a member of the centrosomin family?

Microtubules isolated from bovine adrenal medulla cells contain a major 170 kDa protein (p170). p170 is heat‐labile and is associated with microtubules in an ATP‐insensitive manner. This protein was purified to near homogeneity using FPLC. A preparation containing purified p170 caused bundling of microtubules. By microsequencing of p170, two polypeptides were identified which appeared to be identical to a recently sequenced p167 centrosomin‐related protein. Polyclonal affinity‐purified anti‐p170 antibody was found to immunostain microtubules and to recognize the 170 kDa polypeptide in culture cells. We suggest that p170 is a new member of a centrosomin family and is a new structural protein associated with microtubules in some cell types.

Deciphering Functionally Important Multiprotein Complexes by Mass Spectrometry

Simple chemical reactions, such as the hydrolysis of a peptide bond or the attachment of a phosphate moiety, are catalyzed by individual enzymes. Often the enzymatic activity can be reproduced in an in vitro experiment and the chemical mechanisms of the reaction can be elucidated in detail [1]. However if the enzyme is involved in regulation of a cellular activity it seldom acts by itself. Enzymatic activity is controlled and targeted to specific substrates localized in specific cellular compartments by recruitment of other proteins acting as receptors, ligands, adaptors, etc. [2]. Thus complex multiprotein assemblies constitute important functional units of the cell regulation pathways and are involved in numerous cellular processes such as cell division, signal transduction, mRNA splicing and nuclear transport [2–5]. Study of the structure, regulation and relationship of protein complexes provides clues to understanding in molecular detail how cellular activities are carried out. Dissection of the protein complex places its individual subunits in a context of the specific cellular function carried out by the entire assembly. Taken together with specific motifs recognized in the sequence of an individual subunit, this often suggests a possible role of the protein in the concerted functioning of the whole assembly and allows the design of further suitable functional experiments.