Livin, a novel inhibitor of apoptosis protein family member

A novel human inhibitor of apoptosis protein (IAP) family member termed Livin was identified, containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. The mRNA for livin was not detectable by Northern blot in most normal adult tissues with the exception of the placenta, but was present in developmental tissues and in several cancer cell lines. Highest levels were observed in two melanoma-derived cell lines, G361 and SK-Mel29. Transfection of livin in HeLa cells resulted in protection from apoptosis induced by expression of FADD, Bax, RIP, RIP3, and DR6. Similar to other IAP family members, the anti-apoptotic activity of Livin was dependent on the BIR domain. Livin was also capable of inhibiting DEVD-like caspase activity triggered by tumor necrosis factor-alpha. In vitro binding studies demonstrated a direct interaction between Livin and the active form of the downstream caspases, caspase-3 and -7, that was dependent on the BIR domain of Livin. In addition, the unprocessed and cleaved forms of caspase-9 co-immunoprecipitated with Livin in vivo, and recombinant Livin could inhibit the activation of caspase-9 induced by Apaf-1, cytochrome c, and dATP. The subcellular distribution of the transfected Livin was analyzed by immunofluorescence. Both Livin and Survivin were expressed in the nucleus and in a filamentous pattern throughout the cytoplasm. In contrast to the apoptotic activity, the COOH-terminal RING domain mediated its subcellular localization patterning. Further studies found that transfection of an antisense construct against livin could trigger apoptosis specifically in cell lines expressing livin mRNA. This was associated with an increase in DNA fragmentation and in DEVD-like caspase activity. Thus, disruption of Livin may provide a strategy to induce apoptosis in certain cancer cells.     

KIAP, a novel member of the inhibitor of apoptosis protein family

We have identified a novel human gene, kiap (kidney inhibitor of apoptosis protein) that encodes a single BIR domain and a RING zinc finger domain. kiap has been assigned to the q13.3 region of human chromosome 20 by fluorescent in situ hybridization analysis. Northern blot analysis indicates that KIAP is expressed mainly in placenta, lymph node and fetal kidney. In this report, we show that overexpression of KIAP blocks apoptosis induced by menadione or by overexpression of BAX. In addition, we show that overexpression of KIAP enhances apoptosis induced by etoposide, and, that KIAP fails to block apoptosis induced by overexpression of Fas. Thus, KIAP, a new member of the inhibitor of apoptosis protein (IAP) family, has pleiotropic effects on apoptosis induced by various stimuli.     

Role of XIAP protein, a human member of the inhibitor of apoptosis (IAP) protein family, in phytohemagglutinin-induced apoptosis of human T cell lines

A new family of human genes xiap, hiap-1 and hiap-2, which are homologous to the baculovirus iap (inhibitor of apoptosis) genes cp-iap and op-iap, has been recently cloned and shown to suppress apoptosis after serum withdrawal or exposure to a free radical inducer. In order to examine the role of one of these human genes, namely xiap, in lymphoid cells, we studied XIAP expression, after PHA stimulation in three different human T cell lines. We report here that stimulation with PHA resulted in the human T cell lines undergoing apoptosis, as assessed by DNA fragmentation and by propidiumiodide (PI) staining and flow cytometry. When XIAP protein expression was evaluated by Western blot, we observed that the induction of apoptosis by PHA was associated with a parallel decrease of XIAP expression. We also provide evidence that stably transfected Jurkat cells containing the xiap open reading frame became resistant to PHA-induced apoptosis. These data suggest a role for XIAP protein in the regulation of apoptosis in lymphoid cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family

Inhibitor of apoptosis protein (IAP)-like protein-1 (ILP-1) (also known as X-linked IAP [XIAP] and mammalian IAP homolog A [MIHA]) is a potent inhibitor of apoptosis and exerts its effects, at least in part, by the direct association with and inhibition of specific caspases. Here, we describe the molecular cloning and characterization of a human gene related to ILP-1, termed ILP-2. Despite high homology to ILP-1, ILP-2 is encoded by a distinct gene, which in normal tissues is expressed solely in testis. In contrast to ILP-1, overexpression of ILP-2 had no protective effect on apoptosis mediated by Fas (also known as CD95) or tumor necrosis factor. However, ILP-2 potently inhibited apoptosis induced by overexpression of Bax or by coexpression of caspase 9 with Apaf-1, and preincubation of cytosolic extracts with ILP-2 abrogated caspase activation in vitro. A processed form of caspase 9 could be coprecipitated with ILP-2 from cells, suggesting a physical interaction between ILP-2 and caspase 9. Thus, ILP-2 is a novel IAP family member with restricted specificity for caspase 9.     

Role of TAK1 and TAB1 in BMP signaling in early Xenopus development.

Transforming growth factor-beta (TGF-beta) superfamily members elicit signals through stimulation of serine/threonine kinase receptors. Recent studies of this signaling pathway have identified two types of novel mediating molecules, the Smads and TGF-beta activated kinase 1 (TAK1). Smads were shown to mimic the effects of bone morphogenetic protein (BMP), activin and TGF-beta. TAK1 and TAB1 were identified as a MAPKKK and its activator, respectively, which might be involved in the up-regulation of TGF-beta superfamily-induced gene expression, but their biological role is poorly understood. Here, we have examined the role of TAK1 and TAB1 in the dorsoventral patterning of early Xenopus embryos. Ectopic expression of Xenopus TAK1 (xTAK1) in early embryos induced cell death. Interestingly, however, concomitant overexpression of bcl-2 with the activated form of xTAK1 or both xTAK1 and xTAB1 in dorsal blastomeres not only rescued the cells but also caused the ventralization of the embryos. In addition, a kinase-negative form of xTAK1 (xTAK1KN) which is known to inhibit endogenous signaling could partially rescue phenotypes generated by the expression of a constitutively active BMP-2/4 type IA receptor (BMPR-IA). Moreover, xTAK1KN could block the expression of ventral mesoderm marker genes induced by Smad1 or 5. These results thus suggest that xTAK1 and xTAB1 function in the BMP signal transduction pathway in Xenopus embryos in a cooperative manner.     

Cell division regulation by BIR1, a member of the inhibitor of apoptosis family in yeast

The inhibitor of apoptosis (IAP) gene family comprises molecules that block the activity of pro-apoptotic caspase proteases. Paradoxically, yeasts contain IAP proteins but no caspases and no apoptotic program. To determine the function of these proteins in vivo, we disrupted the BIR1 gene, encoding the only known IAP in yeast Saccharomyces cerevisiae. Sporulation of heterozygous diploids yielded no viable mutant haploids, indicating that BIR1 is an essential gene. By flow cytometry, some heterozygous mutants were polyploid accumulating >4 N DNA content. These cells exhibited a 20-40% reduction in growth rate, which was rescued by plasmid-borne over-expression of BIR1 but not by its human counterpart, survivin. Deletion analysis revealed that the N-terminal domain of Bir1, containing the conserved baculovirus IAP repeat, was able to partially complement the cell growth defect caused by BIR1 deletion. Moreover, the full-length and truncated forms of Bir1 accelerated cell division in wild-type cells. Finally, BIR1 heterozygous mutants exhibited grossly altered cell morphology with misshapen or abnormally long buds connected to an unusually large mother cell. These findings identify a novel function of IAP proteins in the pleiotropic control of cell division, in addition to their role in the suppression of apoptosis.     

MAB21L2, a vertebrate member of the Male-abnormal 21 family,modulates BMP signaling and interacts with SMAD1

Background  

Through in vivo loss-of-function studies, vertebrate members of the Male abnormal 21 (mab-21) gene family have been implicated in gastrulation, neural tube formation and eye morphogenesis. Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs.     

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling

The cytokines IL-1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF-kappaB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1- and TNF-induced activation of the NF-kappaB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-kappaB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-kappaB is not impaired in TAB2-deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-kappaB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. Further more, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1- and TNF-induced activation of TAK1 and NF-kappaB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction.     

Nephrocan, a novel member of the small leucine-rich repeat protein family, is an inhibitor of transforming growth factor-beta signaling

In a search of new, small leucine-rich repeat proteoglycan/protein (SLRP) family members, a novel gene, nephrocan (NPN), has been identified. The gene consists of three exons, and based on the deduced amino acid sequence, NPN has 17 leucine-rich repeat motifs and unique cysteine-rich clusters both in the N and C termini, indicating that this gene belongs to a new class of SLRP family. NPN mRNA was predominantly expressed in kidney in adult mice, and during mouse embryogenesis, the expression was markedly increased in 11-day-old embryos at a time when early kidney development takes place. In the adult mouse kidney, NPN protein was located in distal tubules and collecting ducts. When NPN was overexpressed in cell culture, the protein was detected in the cultured medium, and upon treatment with N-glycosidase F, the molecular mass was lowered by approximately 14 kDa, indicating that NPN is a secreted N-glycosylated protein. Furthermore, transforming growth factor-beta (TGF-beta)-responsive 3TP promoter luciferase activity was down-regulated, and TGF-beta-induced Smad3 phosphorylation was also inhibited by NPN, suggesting that NPN suppresses TGF-beta/Smad signaling. Taken together, NPN is a novel member of the SLRP family that may play important roles in kidney development and pathophysiology by functioning as an endogenous inhibitor of TGF-beta signaling.     

cGMP-dependent protein kinase type I inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes

Cardiac myocyte apoptosis during ischemia and reperfusion (I/R) is tightly controlled by a complex network of stress-responsive signaling pathways. One pro-apoptotic pathway involves the interaction of the scaffold protein TAB1 with p38 mitogen-activated protein kinase (p38 MAPK) leading to the autophosphorylation and activation of p38 MAPK. Conversely, NO and its second messenger cGMP protect cardiac myocytes from apoptosis during I/R. We provide evidence that the cGMP target cGMP-dependent protein kinase type I (PKG I) interferes with TAB1-p38 MAPK signaling to protect cardiac myocytes from I/R injury. In isolated neonatal cardiac myocytes, activation of PKG I inhibited the interaction of TAB1 with p38 MAPK, p38 MAPK phosphorylation, and apoptosis induced by simulated I/R. During I/R in vivo, mice with a cardiac myocyte-restricted deletion of PKG I displayed a more pronounced interaction of TAB1 with p38 MAPK and a stronger phosphorylation of p38 MAPK in the myocardial area at risk during reperfusion and more apoptotic cardiac myocytes in the infarct border zone as compared with wild-type littermates. Notably, adenoviral expression of a constitutively active PKG I mutant truncated at the N terminus(PKGI-DeltaN1-92) did not inhibit p38 MAPK phosphorylation and apoptosis induced by simulated I/R in vitro, indicating that the N terminus of PKG I is required. As shown by co-immunoprecipitation experiments in HEK293 cells, cGMP-activated PKG I, but not constitutively active PKG I-DeltaN1-92 or PKG I mutants carrying point mutations in the N-terminal leucine-isoleucine zipper, interacted with p38 MAPK, and prevented the binding of TAB1 to p38 MAPK. Together, our data identify a novel interaction between the cGMP target PKG I and the TAB1-p38 MAPK signaling pathway that serves as a defense mechanism against myocardial I/R injury.     

The motor protein kinesin-1 links neurofibromin and merlin in a common cellular pathway of neurofibromatosis

Mutations in either of the two tumor suppressor genes NF1 (neurofibromin) and NF2 (merlin) result in Neurofibromatosis, a condition predisposing individuals to developing a variety of benign and malignant tumors of the central and peripheral nervous systems. Here we report the identification of two distinct NF1-containing complexes, one in the soluble and the other in the particulate fraction of HeLa extract. We show that the soluble NF1 complex delineates a large holo-NF1 complex (2 MDa) encompassing the components of a smaller particulate core-NF1 complex (400 kDa). Purification of the core-NF1 complex followed by mass spectrometric analysis revealed the motor protein, kinesin-1 heavy chain (HsuKHC/KIF5B), as a catalytic subunit of both NF-1-containing complexes. Importantly, although NF1 and NF2 are not in a stable association, NF2 is also a component of a distinct kinesin-1-containing complex. These results point to kinesin-1 as a common denominator between NF1 and NF2.     

Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family

Several of the inhibitor of apoptosis protein (IAP) family members regulate apoptosis in response to various cellular assaults. Some members are also involved in cell signalling, mitosis and targeting proteins to the ubiquitin-proteasome degradation machinery. The most intensively studied family member, X-linked IAP (XIAP), is a potent inhibitor of caspase activity; hence, it is generally assumed that direct caspase inhibition is an important conserved function of most members of the family. Biochemical and structural studies have precisely mapped the elements of XIAP required for caspase inhibition. Intriguingly, these elements are not conserved among IAPs. Here, we review current knowledge of the caspase-inhibitory potential of the human IAPs and show that XIAP is probably the only bona fide caspase inhibitor, suggesting that the other family members never gained the ability to directly inhibit caspase activity.     

Microtubule-associated protein 1S, a short and ubiquitously expressed member of the microtubule-associated protein 1 family

The related high molecular mass microtubule-associated proteins (MAPs) MAP1A and MAP1B are predominantly expressed in the nervous system and are involved in axon guidance and synaptic function. MAP1B is implicated in fragile X mental retardation, giant axonal neuropathy, and ataxia type 1. We report the functional characterization of a novel member of the microtubule-associated protein 1 family, which we termed MAP1S (corresponding to sequence data bank entries for VCY2IP1 and C19ORF5). MAP1S contains the three hallmark domains of the microtubule-associated protein 1 family but hardly any additional sequences. It decorates neuronal microtubules and copurifies with tubulin from brain. MAP1S is synthesized as a precursor protein that is partially cleaved into heavy and light chains in a tissue-specific manner. Heavy and light chains interact to form the MAP1S complex. The light chain binds, bundles, and stabilizes microtubules and binds to actin. The heavy chain appears to regulate light chain activity. In contrast to MAP1A and MAP1B, MAP1S is expressed in a wide range of tissues in addition to neurons and represents the non-neuronal counterpart of this cytolinker family.