A novel SR-related protein is required for the second step of Pre-mRNA splicing

The SR family proteins and SR-related polypeptides are important regulators of pre-mRNA splicing. A novel SR-related protein of an apparent molecular mass of 53 kDa was isolated in a gene trap screen that identifies proteins which localize to the nuclear speckles. This novel protein possesses an arginine- and serine-rich domain and was termed SRrp53 (for SR-related protein of 53 kDa). In support for a role of this novel RS-containing protein in pre-mRNA splicing, we identified the mouse ortholog of the Saccharomyces cerevisiae U1 snRNP-specific protein Luc7p and the U2AF65-related factor HCC1 as interacting proteins. In addition, SRrp53 is able to interact with some members of the SR family of proteins and with U2AF35 in a yeast two-hybrid system and in cell extracts. We show that in HeLa nuclear extracts immunodepleted of SRrp53, the second step of pre-mRNA splicing is blocked, and recombinant SRrp53 is able to restore splicing activity. SRrp53 also regulates alternative splicing in a concentration-dependent manner. Taken together, these results suggest that SRrp53 is a novel SR-related protein that has a role both in constitutive and in alternative splicing.     

WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II

We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.     

Phage-peptide display identifies the interferon-responsive, death-activated protein kinase family as a novel modifier of MDM2 and p21WAF1

Phage-peptide display is a versatile tool for identifying novel protein-protein interfaces. Our previous work highlighted the selection of phage-peptides that bind to specific isoforms of MDM2 protein and in this work we subjected the putative MDM2-binding proteins to phage-peptide display to expand further on putative protein interaction maps. One peptide that bound MDM2 had significant homology to members of the death-activated protein kinase (DAPK) family, an enzyme family of no known direct link to the p53 pathway. We examined whether a nuclear member of the DAPK family named DAPK3 or ZIP kinase had direct links to the p53 pathway. ZIP kinase was cloned, purified, and the enzyme was able to phosphorylate MDM2 at Ser166, a site previously reported to be modified by Akt kinase, thus demonstrating that ZIP kinase is a bona fide MDM2-binding protein. Native ZIP kinase fractions were then subjected to phage-peptide display and one ZIP kinase consensus peptide motif was identified in p21(WAF1). ZIP kinase phosphorylates p21(WAF1) at Thr145 and alanine-substituted mutations in the p21(WAF1) phosphorylation site alter its ability to be phosphorylated by ZIP kinase. Thus, although ZIP kinase consensus sites were then defined as containing a minimal RKKx(T/S) consensus motif, alternate contacts in ZIP kinase binding are implicated, since amino acid residues surrounding the phospho-acceptor site can effect the specific activity of the kinase. Transfected ZIPK can promote the phosphorylation of p21(WAF1) at Thr145 in vivo and can increase the half-life of p21(WAF1), while the half-life of p21(WAF1[T145A]) is not effected by ZIP kinase. Thus, phage-peptide display identified an interferon-responsive protein kinase family as a novel modifier of two components of the p53 pathway, MDM2 and p21(WAF1), and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways.     

Cloning and characterization of IL-1HY2, a novel interleukin-1 family member

The interleukin-1 (IL-1) family members play an important role in the process of inflammation and host defense. We describe here the identification and characterization of a novel member of the IL-1 family, IL-1HY2. The human IL-1HY2 protein shares significant amino acid sequence similarity (37%) with the IL-1 receptor antagonist and has a predicted three-dimensional structure similar to that of the IL-1 receptor antagonist. The IL-1HY2 gene is located in close proximity to other IL-1 family genes on human chromosome 2, and the genomic organization of the IL-1HY2 gene is highly conserved with other IL-1 family members. IL-1HY2 protein is secreted from mammalian cells, and the purified recombinant IL-1HY2 protein binds soluble IL-1 receptor type I. IL-1HY2 is expressed in human skin, spleen, and tonsil. Immunohistochemical analysis showed that the IL-1HY2 protein is expressed in the basal epithelia of skin and in proliferating B cells of the tonsil. These data suggest that IL-1HY2 is a novel IL-1 family member and that it may participate in a network of IL-1 family members to regulate adapted and innate immune responses.     

Diva/Boo is a negative regulator of cell death in human glioma cells

Diva is a novel proapoptotic member of the Bcl-2 protein family which binds apoptosis activating factor-1 (APAF-1). Diva is identical with Boo which was identified as a novel antiapoptotic Bcl-2 family protein. Here, we report that Diva promotes the cell cycle exit of human glioma cells in response to serum deprivation and inhibits apoptosis of these cells induced by CD95 ligand or chemotherapeutic drugs. In glioma cells, Diva interferes with apoptotic signaling downstream of cytochrome c release, but upstream of caspase activation, consistent with an inhibitory effect on the mitochondrial amplification step involving the apoptosome and APAF-1.     

Purification and characterization of an active N-acetylglucosaminyltransferase enzyme complex from Streptococci

A new family of bacterial serine-rich repeat glycoproteins can function as adhesins required for biofilm formation and pathogenesis in streptococci and staphylococci. Biogenesis of these proteins depends on a gene cluster coding for glycosyltransferases and accessory secretion proteins. Previous studies show that Fap1, a member of this family from Streptococcus parasanguinis, can be glycosylated by a protein glycosylation complex in a recombinant heterogeneous host. Here we report a tandem affinity purification (TAP) approach used to isolate and study protein complexes from native streptococci. This method demonstrated that a putative glycosyltransferase (Gtf2), which is essential for Fap1 glycosylation, readily copurified with another glycosyltransferase (Gtf1) from native S. parasanguinis. This result and the similar isolation of a homologous two-protein complex from Streptococcus pneumoniae indicate the biological relevance of the complexes to the glycosylation in streptococci. Furthermore, novel N-acetylglucosaminyltransferase activity was discovered for the complexes. Optimal activity required heterodimer formation and appears to represent a novel type of glycosylation.     

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.     

Cloning and characterization of zfBLP1, a Bcl-XL homologue from the zebrafish, Danio rerio

The importance of the Bcl-2 family proteins in normal vertebrate embryogenesis is being recognized; however, their regulatory mechanism is poorly understood. We report here the cloning and characterization of a novel zebrafish Bcl-2 family protein, zfBLP1. The zfBLP1 cDNA is 1942 nucleotides long, encoding a polypeptide of 238 amino acids. The primary sequence of zfBLP1 shares 50% identity to human Bcl-XL, and contains all four conserved BH domains of the Bcl-2 family proteins. Primary sequence analysis identified a consensus ER retention signal at the C-terminal end of zfBLP1. Northern blot analysis indicated that there were two major and two minor zfBLP1 mRNA species expressed during embryonic development. Among the two major mRNA species, the short one, approx. 3 kb in size, was expressed throughout embryonic development, while the long one, approx. 7 kb long, was not detectable until the gastrula stage. These results suggest that zfBLP1 is a novel Bcl-2 family protein under complicated regulations, and is likely to play an important role in zebrafish oogenesis and embryogenesis.     

KIF1Bbeta2, capable of interacting with CHP,is localized to synaptic vesicles

Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.     

Molecular characterization of a novel RhoGAP, RRC-1 of the nematode Caenorhabditis elegans

The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPases in C. elegans.     

Glycogen synthase kinase 3alpha and 3beta mediate a glucose-sensitive antiapoptotic signaling pathway to stabilize Mcl-1

Glucose uptake and utilization are growth factor-stimulated processes that are frequently upregulated in cancer cells and that correlate with enhanced cell survival. The mechanism of metabolic protection from apoptosis, however, has been unclear. Here we identify a novel signaling pathway initiated by glucose catabolism that inhibited apoptotic death of growth factor-deprived cells. We show that increased glucose metabolism protected cells against the proapoptotic Bcl-2 family protein Bim and attenuated degradation of the antiapoptotic Bcl-2 family protein Mcl-1. Maintenance of Mcl-1 was critical for this protection, as glucose metabolism failed to protect Mcl-1-deficient cells from apoptosis. Increased glucose metabolism stabilized Mcl-1 in both cell lines and primary lymphocytes via inhibitory phosphorylation of glycogen synthase kinase 3alpha and 3beta (GSK-3alpha/beta), which otherwise promoted Mcl-1 degradation. While a number of kinases can phosphorylate and inhibit GSK-3alpha/beta, we provide evidence that protein kinase C may be stimulated by glucose-induced alterations in diacylglycerol levels or distribution to phosphorylate GSK-3alpha/beta, maintain Mcl-1 levels, and inhibit cell death. These data provide a novel nutrient-sensitive mechanism linking glucose metabolism and Bcl-2 family proteins via GSK-3 that may promote survival of cells with high rates of glucose utilization, such as growth factor-stimulated or cancerous cells.     

Caspase cleavage of Mcl-1 impairs its anti-apoptotic activity and proteasomal degradation in non-small lung cancer cells

Global cleavage of cellular proteins by activated caspases is a hallmark of apoptosis, which causes biochemical collapse of the cell. Recent studies suggest that, rather than completely destroying a protein, caspase cleavage can confer novel characteristics or functions. In this respect, the post-caspase role of Bcl-2 family proteins remains uncharacterized. Here, we showed that Mcl-1, a pro-survival member of the Bcl-2 family, was cleaved by caspase-3 in non-small cell lung cancer (NSCLC) cells undergoing chemotherapeutic agent-triggered apoptosis. Caspase cleavage partially impaired the anti-apoptotic activity of Mcl-1 by reducing its mitochondrial localization and impeding its association with the permeability transition pore-forming protein Bak. However, the stability of cleaved Mcl-1 was markedly enhanced because it was more refractory to ubiquitination-dependent proteasomal degradation, thereby improving cell viability to a greater extent than full-length Mcl-1 when transiently expressed in NSCLC cells. These findings shed new light on the role of Mcl-1 in apoptosis and suggest potential novel targets for optimizing the tumoricidal capacity of chemotherapy.     

Novel Predicted RNA-Binding Domains Associated with the Translation Machinery

Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis. A small domain consisting of 60–65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation. Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases. Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors. Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes. We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes. The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions. Received: 15 May 1998 / Accepted: 20 July 1998     

Immune-associated nucleotide-1 (IAN-1) is a thymic selection marker and defines a novel gene family conserved in plants.

Positive selection of thymocytes is a complex and crucial event in T cell development that is characterized by cell death rescue, commitment toward the helper or cytotoxic lineage, and functional maturation of thymocytes bearing an appropriate TCR. To search for novel genes involved in this process, we compared gene expression patterns in positively selected thymocytes and their immediate progenitors in mice using the differential display technique. This approach lead to the identification of a novel gene, mIAN-1 (murine immune-associated nucleotide-1), that is switched on upon positive selection and predominantly expressed in the lymphoid system. We show that mIAN-1 encodes a 42-kDa protein sharing sequence homology with the pathogen-induced plant protein aig1 and that it defines a novel family of at least three putative GTP-binding proteins. Analysis of protein expression at various stages of thymocyte development links mIAN-1 to CD3-mediated selection events, suggesting that it represents a key player of thymocyte development and that it participates to peripheral specific immune responses. The evolutionary conservation of the IAN family provides a unique example of a plant pathogen response gene conserved in animals.