COOH-terminal Deletion Mutants Research Articles

Nitric Oxide Inhibits the Proliferation and Invasion of Pancreatic Cancer Cells through Degradation of Insulin Receptor Substrate-1 Protein

Nitric oxide (NO), which plays a role in the posttranslational modification of proteins, exhibits tumoricidal activity. However, the mechanism remains largely unclear. We investigated whether the regulation of insulin receptor substrate (IRS)-1 protein expression and insulin/insulin-like growth factor (IGF) signaling by NO is involved in the proliferation and invasion of pancreatic cancer cells. NO donor inhibited insulin/IGF-I-stimulated phosphorylation of insulin receptor/IGF-I receptor, IRS-1, Akt/PKB, and glycogen synthase kinase-3beta along with decreased expression of IRS-1 protein in MIAPaCa-2 cells, whereas NO donor enhanced the phosphorylation of extracellular signal-regulated kinase-1/2. In contrast, a selective inducible nitric oxide synthase inhibitor, 1400W, upregulated the expression of IRS-1 protein and the phosphorylation of IRS-1, Akt/PKB, and glycogen synthase kinase-3beta, along with enhanced proliferation and invasion of Panc-1 cells expressing inducible nitric oxide synthase protein. NO donor induced IRS-1 protein reduction through increased ubiquitination and degradation. For the detection of the site responsible for NO-induced ubiquitination, IRS-1 deletion mutant genes were transfected and overexpressed in MIAPaCa-2 cells. The results indicate that the COOH terminus of the IRS-1 protein is required for NO donor-induced ubiquitination and protein degradation. Cells stably transfected with COOH-terminal deletion mutants of IRS-1 exhibited reduced IGF signaling and cell proliferation compared with vector alone-transfected cells, with no influence of NO on IGF signaling and invasion, although stable transfectants with full-length IRS-1 protein exhibited remarkable NO-induced reduction in IGF signaling, cell proliferation, and invasion. These findings indicate that NO inhibits the proliferation and invasion of pancreatic cancer cells, at least in part, through upregulation of IRS-1 protein degradation and resultant downregulation of the insulin/IGF-I-Akt pathway.

Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method

The transfection of cardiac myocytes is difficult, and so most of the data regarding the regulation of trafficking and targeting of cardiac ion channels have been obtained using heterologous expression systems. Here we apply the fast biolistic transfection procedure to adult cardiomyocytes to show that biolistically introduced exogenous voltage-gated potassium channel, Kv1.5, is functional and, like endogenous Kv1.5, localizes to the intercalated disc, where it is expressed at the surface of that structure. Transfection efficiency averages 28.2 +/- 5.7% of surviving myocytes at 24 h postbombardment. Ventricular myocytes transfected with a tagged Kv1.5 exhibit an increased sustained current component that is approximately 40% sensitive to 100 microM 4-aminopyridine and which is absent in myocytes transfected with a fluorescent protein-encoding construct alone. Kv1.5 deletion mutations known to reduce the surface expression of the channel in heterologous cells similarly reduce the surface expression in transfected ventricular myocytes, although targeting to the intercalated disc per se is generally unaffected by both NH(2)- and COOH-terminal deletion mutants. Expressed current levels in wild-type Kv1.5, Kv1.5DeltaSH3(1), Kv1.5DeltaN209, and Kv1.5DeltaN135 mutants were well correlated with apparent surface expression of the channel at the intercalated disc. Our results conclusively demonstrate functionality of channels present at the intercalated disc in native myocytes and identify determinants of trafficking and surface targeting in intact cells. Clearly, biolistic transfection of adult cardiac myocytes will be a valuable method to study the regulation of surface expression of channels in their native environment.

TrkBT1 Induces Liver Metastasis of Pancreatic Cancer Cells by Sequestering Rho GDP Dissociation Inhibitor and Promoting RhoA Activation

Many genetic and molecular alterations, such as K-ras mutation and NF-kappaB activation, have been identified in pancreatic cancer. However, the mechanisms by which pancreatic cancer metastasizes still remain to be determined. Although we previously showed that the tropomyosin-related kinase B (TrkB) was significantly correlated with the development of liver metastasis, its function in pancreatic cancer metastasis remained unresolved. In the present study, we showed that overexpressed TrkB is an alternatively spliced transcript variant of TrkB (TrkBT1) with a unique COOH-terminal 12-amino acid sequence and is mainly localized in the cytoplasm. Our results showed that overexpression of Flag-tagged TrkBT1 but not a Flag-tagged TrkBT1 COOH-terminal deletion mutant (Flag-TrkBT1DeltaC) in nonmetastatic pancreatic cancer cells enhanced cell proliferation, promoted formation of colonies in soft agar, stimulated tumor cell invasion, and induced liver metastasis in an orthotopic xenograft mouse model of pancreatic cancer. TrkBT1 interacted with Rho GDP dissociation inhibitor (GDI) in vivo, but Flag-TrkBT1DeltaC did not. Furthermore, overexpression of Flag-TrkBT1 and knockdown of RhoGDI expression by RhoGDI short hairpin RNAs promoted RhoA activation, but Flag-TrkBT1DeltaC overexpression did not. Therefore, our results showed that TrkBT1 overexpression induces liver metastasis of pancreatic cancer and uncovered a unique signaling mechanism by which TrkBT1 sequesters GDI and activates RhoA signaling.

Molecular Basis of Nuclear Factor-κB Activation by Astrocyte Elevated Gene-1

Malignant glioma is a consistently fatal brain cancer. The tumor invades the surrounding tissue, limiting complete surgical removal and thereby initiating recurrence. Identifying molecules critical for glioma invasion is essential to develop targeted, effective therapies. The expression of astrocyte elevated gene-1 (AEG-1) increases in malignant glioma and AEG-1 regulates in vitro invasion and migration of malignant glioma cells by activating the nuclear factor-kappaB (NF-kappaB) signaling pathway. The present studies elucidate the domains of AEG-1 important for mediating its function. Serial NH(2)-terminal and COOH-terminal deletion mutants were constructed and functional analysis revealed that the NH(2)-terminal 71 amino acids were essential for invasion, migration, and NF-kappaB-activating properties of AEG-1. The p65-interaction domain was identified between amino acids 101 to 205, indicating that p65 interaction alone is not sufficient to mediate AEG-1 function. Coimmunoprecipitation assays revealed that AEG-1 interacts with cyclic AMP-responsive element binding protein-binding protein (CBP), indicating that it might act as a bridging factor between NF-kappaB, CBP, and the basal transcription machinery. Chromatin immunoprecipitation assays showed that AEG-1 is associated with the NF-kappaB binding element in the interleukin-8 promoter. Thus, AEG-1 might function as a coactivator for NF-kappaB, consequently augmenting expression of genes necessary for invasion of glioma cells. In these contexts, AEG-1 represents a viable potential target for the therapy of malignant glioma.

Proapoptotic effects of NARC 1 (= PCSK9), the gene encoding a novel serine proteinase

NARC 1/PCSK9 encodes a novel serine proteinase known to play a role in cholesterol homeostasis. NARC 1 mRNA expression in cerebellar granule neurons (CGNs) was discovered to be induced following an apoptotic injury. Coregulation of known apoptotic mediators (caspase-3 and death receptor 6) raises the possibility that NARC 1 might be involved in the propagation of apoptotic signaling in neurons. CGNs were transfected with EGFP-fusion constructs of wild-type and mutant NARC 1, and a laser scanning cytometry-based method of scoring cell death in transfectants was applied. Use of the poly-caspase inhibitor BAF allowed assessment of the caspase-dependence of the NARC 1 proapoptotic effect. Wild-type NARC 1 was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the catalytic domain resulted in a reduced level of cell death, consistent with loss of the BAF-sensitive component of cell death. NH(2)-terminal deletion constructs of NARC 1 had effects similar to wild-type, both in the absence and presence of BAF, whereas expression of COOH-terminal deletion mutants produced a rate of cell death similar to wild-type in the absence of BAF treatment, but which lacked the capacity to be reduced by treatment with BAF. The mechanism by which NARC 1-EGFP over-expression induces cell death in cultured CGNs remains unclear. Mutation analysis established a positive correlation between the presence of the Narc 1 active site serine in the transiently expressed protein and induction of the BAF-sensitive component of the cell death phenotype. A caspase-independent component proved sufficiently complex to map discretely within the Narc 1 protein.

NF-κB regulates the stability and activity of p73 by inducing its proteolytic degradation through a ubiquitin-dependent proteasome pathway

Nuclear factor kappa B (NF-kappaB), which exists as heterodimeric complexes composed of p50 and p65, has been shown to play an important role in cell survival processes. In the present study, we found for the first time that NF-kappaB has an ability to induce the ubiquitin-dependent proteasomal degradation of proapoptotic p73alpha. The activation of NF-kappaB in tumor necrosis factor alpha (TNF-alpha)-stimulated H1299 cells resulted in a significant reduction in the amounts of the endogenous p73alpha. Consistent with these results, TNF-alpha-mediated downregulation of p73alpha was observed in wild-type (WT) mouse embryonic fibroblasts (MEFs) but not in p65-deficient MEFs. Ectopic expression of NF-kappaB decreased a half-life of p73alpha by increasing its ubiquitination levels, and thereby inhibiting the transcriptional activity as well as proapoptotic function of p73alpha, whereas NF-kappaB had undetectable effects on p53. Immunoprecipitation experiments demonstrated that, under our experimental conditions, NF-kappaB does not bind to p73alpha in mammalian cultured cells. In contrast to WT p65, the COOH-terminal deletion mutant of p65 (p65DeltaC) failed to reduce the expression levels of p73alpha, suggesting that NF-kappaB-mediated proteolytic degradation of p73alpha requires the transcriptional activity of NF-kappaB. Taken together, our present results imply that NF-kappaB-mediated degradation of proapoptotic p73 is a novel inhibitory mechanism of p73 that regulates cell survival and death.

Identification of basolateral membrane targeting signal of human sodium‐dependent dicarboxylate transporter 3

Sodium-dependent dicarboxylate transporters (NaDC) include low-affinity NaDC1 and high-affinity NaDC3. Despite high similarities structurally and functionally, both are localized to opposite surfaces of renal tubular cells. The molecular mechanisms and localization signals leading to this polarized distribution remain unknown. In this study, distribution of NaDC3 in human kidney tissue was firstly observed by immunohistochemistry and immunofluorescence. Then, EGFP-fused wild-type, NH2- and COOH-terminal deletion and point mutants of NaDC3, and chimera between NaDC3 and NaDC1, were generated and transfected into polarized renal cells lines, LLC-PK1 and MDCK. Their subcellular localizations were analyzed by laser confocal microscopy. Immunolocalization results revealed that NaDC3 was expressed at basolateral membrane of human renal proximal tubular epithelia. Confocal examinations showed that wild-type NaDC3 was targeted to the basolateral membrane of MDCK and LLC-PK1. Deletion mutations indicated that the basolateral targeting signal of NaDC3 located within a short sequence AKKVWSARR of its amino-terminal cytoplasmic domain. Addition of this sequence could redirect apical NaDC1 to the basolateral membrane of LLC-PK1. Point mutagenesis revealed that mutation of either of two hydrophobic amino acids V and W in this short sequence largely redirected NaDC3 to both apical and basolateral surfaces of LLC-PK, indicating that the two hydrophobic amino acids are critical for the basolateral targeting of NaDC3. Our studies provide direct evidence of the localization of NaDC3 at the basolateral membrane of human renal proximal tubule cells and identify a di-hydrophobic amino acid motif VW as basolateral localization signal in the N-terminal cytoplasmic domain of NaDC3.

Analysis of mGluR1a constitutive internalization using a pulse–chase enzyme-linked immuno-sorbant assay (ELISA)

The surface expression of G protein-coupled receptors is regulated by internalization. For many receptors, a constitutive level of internalization in the absence of agonist has been reported. The constitutive internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse–chase ELISA method that allows the investigation of mGluR1a constitutive internalization. When investigated by pulse–chase ELISA, the constitutive internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse–chase labelling procedure. The application of the pulse–chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive internalization. Interestingly, only DMII underwent significant constitutive internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse–chase ELISA appears to be an efficient tool to analyze the constitutive internalization of different mGluR1 splice variants.

Serine 68 of phospholemman is critical in modulation of contractility, [Ca2+]itransients, and Na+/Ca2+exchange in adult rat cardiac myocytes

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)-K(+)-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.

The Core Histone N-Terminal Tail Domains Negatively Regulate Binding of Transcription Factor IIIA to a Nucleosome Containing a 5S RNA Gene via a Novel Mechanism

Reconstitution of a DNA fragment containing a 5S RNA gene from Xenopus borealis into a nucleosome greatly restricts binding of the primary 5S transcription factor, TFIIIA. Consistent with transcription experiments using reconstituted templates, removal of the histone tail domains stimulates TFIIIA binding to the 5S nucleosome greater than 100-fold. However, we show that tail removal increases the probability of 5S DNA unwrapping from the core histone surface by only approximately fivefold. Moreover, using site-specific histone-to-DNA cross-linking, we show that TFIIIA binding neither induces nor requires nucleosome movement. Binding studies with COOH-terminal deletion mutants of TFIIIA and 5S nucleosomes reconstituted with native and tailless core histones indicate that the core histone tail domains play a direct role in restricting the binding of TFIIIA. Deletion of only the COOH-terminal transcription activation domain dramatically stimulates TFIIIA binding to the native nucleosome, while further C-terminal deletions or removal of the tail domains does not lead to further increases in TFIIIA binding. We conclude that the unmodified core histone tail domains directly negatively influence TFIIIA binding to the nucleosome in a manner that requires the C-terminal transcription activation domain of TFIIIA. Our data suggest an additional mechanism by which the core histone tail domains regulate the binding of trans-acting factors in chromatin.

Localization of the 12.6-kDa FK506-binding Protein (FKBP12.6) Binding Site to the NH2-terminal Domain of the Cardiac Ca2+ Release Channel (Ryanodine Receptor)

The 12.6-kDa FK506-binding protein (FKBP12.6) interacts with the cardiac ryanodine receptor (RyR2) and modulates its channel function. However, the molecular basis of FKBP12.6-RyR2 interaction is poorly understood. To investigate the significance of the isoleucine-proline (residues 2427-2428) dipeptide epitope, which is thought to form an essential part of the FKBP12.6 binding site in RyR2, we generated single and double mutants, P2428Q, I2427E/P2428A, and P2428A/L2429E, expressed them in HEK293 cells, and assessed their ability to bind GST-FKBP12.6. None of these mutations abolished GST-FKBP12.6 binding, indicating that this isoleucine-proline motif is unlikely to form the core of the FKBP12.6 binding site in RyR2. To systematically define the molecular determinants of FKBP12.6 binding, we constructed a series of internal and NH(2)- and COOH-terminal deletion mutants of RyR2 and examined the effect of these deletions on GST-FKBP12.6 binding. These deletion analyses revealed that the first 305 NH(2)-terminal residues and COOH-terminal residues 1937-4967 are not essential for GST-FKBP12.6 binding, whereas multiple sequences within a large region between residues 305 and 1937 are required for GST-FKBP12.6 interaction. Furthermore, an NH(2)-terminal fragment containing the first 1937 residues is sufficient for GST-FKBP12.6 binding. Co-expression of overlapping NH(2) and COOH-terminal fragments covering the entire sequence of RyR2 produced functional channels but did not restore GST-FKBP12.6 binding. These data suggest that FKBP12.6 binding is likely to be conformationdependent. Binding of FKBP12.6 to the NH(2)-terminal domain may play a role in stabilizing the conformation of this region.

A Role for KH Domain Proteins (Sam68-like Mammalian Proteins and Quaking Proteins) in the Post-transcriptional Regulation of HIV Replication

Overexpression of Sam68 functionally substitutes for, as well as synergizes with, human immunodeficiency virus type 1 (HIV-1) Rev in RRE (Rev response element)-mediated gene expression and virus replication. In addition, COOH-terminal deletion and/or point mutants of Sam68 exhibit a transdominant negative phenotype for HIV replication. Sam68 is a member of KH domain family that includes SLM-1, SLM-2 (Sam68 like mammalian); and QKI-5, QKI-6, and QKI-7 (mouse quaking) proteins. The objective of this study was to examine the effects of these KH family proteins on RRE- and CTE (constitutive transport element of type-D retrovirus)-mediated transactivation. We now report that SLM-1 and SLM-2 proteins, which are the closest relatives of Sam68, marginally enhanced RRE-mediated transactivation, while QK isoforms that are distant relatives of Sam68 had no effect. Interestingly, these proteins still enhanced the effect of Rev in RRE-mediated gene expression. The increase in chloramphenicol acetyltransferase activity was also reflected at the levels of cytoplasmic RRE-chloramphenicol acetyltransferase mRNAs, indicating that Sam68 and KH proteins may have been involved in the stability or export of unspliced RNA. The increase in Rev activity was sensitive to leptomycin B, but not to olomoucine, indicating that the effect of SLM-1, SLM-2, QKI-5, QKI-6, and QKI-7 is exerted through a CRM-1-dependent mRNA export pathway. Thus, KH family proteins play an important role in the post-transcriptional regulation of HIV.

Human survivin is a kinetochore-associated passenger protein.

Survivin, a dimeric baculovirus inhibitor of apoptosis repeat (BIR) motif protein that is principally expressed in G2 and mitosis, has been associated with protection against apoptosis of cells that exit mitosis aberrantly. Mammalian survivin has been reported to associate with centrosomes and with the mitotic spindle. We have expressed a human hemagglutinin-tagged survivin plasmid to determine its localization, and find instead that it clearly acts as a passenger protein. In HeLa cells, survivin first associates with the kinetochores, and then translocates to the spindle midzone during anaphase and, finally, to the midbody during cell cleavage. Its localization is similar to that of TD-60, a known passenger protein. Both a point mutation in the baculovirus IAP repeat motif (C84A) and a COOH-terminal deletion mutant (Delta106) of survivin fail to localize to either kinetochores or midbodies, but neither interferes with cell cleavage. The interphase localization of survivin is cell cycle regulated since in permanently transfected NIH3T3 cells it is excluded from the nuclei until G2, where it localizes with centromeres. Survivin remains associated with mitotic kinetochores when microtubule assembly is disrupted and its localization is thus independent of microtubules. We conclude that human survivin is positioned to have an important function in the mechanism of cell cleavage.

Biochemical Characterization of the Catalytic Domain of Human Matrix Metalloproteinase 19: EVIDENCE FOR A ROLE AS A POTENT BASEMENT MEMBRANE DEGRADING ENZYME

We have recently cloned MMP-19, a novel matrix metalloproteinase, which, due to unique structural features, was proposed to represent the first member of a new MMP subfamily (Pendás, A. M., Knäuper, V. , Puente, X. S., Llano, E., Mattei, M. G., Apte, S., Murphy, G., and López-Otin, C. (1997) J. Biol. Chem. 272, 4281-4286). A recombinant COOH-terminal deletion mutant of MMP-19 (proDelta(260-508)MMP-19), comprising the propeptide and the catalytic domain, was expressed in Escherichia coli, refolded, and purified. Interestingly, we found that proDelta(260-508)MMP-19 has the tendency to autoactivate, whereby the Lys(97)-Tyr(98) peptide bond is hydrolyzed, resulting in free catalytic domain. Mutation of two residues (Glu(88) --> Pro and Pro(90) --> Val) within the propeptide latency motif did not prevent autoactivation but the autolysis rate was somewhat reduced. Analysis of the substrate specificity revealed that the catalytic domain of MMP-19 was able to hydrolyze the general MMP substrate Mca-Pro-Leu-Gly-Dpa-Ala-Arg-NH(2) and, with higher efficiency, the stromelysin substrate Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH(2). Kinetic analysis of the interactions of the catalytic domain of MMP-19 with the natural MMP inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), showed strong inhibition using TIMP-2, TIMP-3, and TIMP-4, while TIMP-1 was less efficient. We also demonstrated that synthetic hydroxamic acid-based compounds efficiently inhibited the enzyme. The catalytic domain of MMP-19 was able to hydrolyze the basement membrane components type IV collagen, laminin, and nidogen, as well as the large tenascin-C isoform, fibronectin, and type I gelatin in vitro, suggesting that MMP-19 is a potent proteinase capable of hydrolyzing a broad range of extracellular matrix components. Neither the catalytic domain nor the full-length MMP-19 was able to degrade triple-helical collagen. Finally, and in contrast to studies with other MMPs, MMP-19 catalytic domain was not able to activate any of the latent MMPs tested in vitro.

MAGI-2 Works Its Magic on PTEN

PTEN is a tumor suppressor protein that inhibits phosphatidylinositol-3' kinase (PI-3K) activity. Wu et al . used the yeast two-hybrid method to identify a protein, membrane-associated guanylate kinase inverted-2 (MAGI-2), which associates with PTEN. Protein interactions involved the second PDZ domain of MAGI-2 and the PDZ domain-binding motif located at the COOH-terminus of PTEN. Cellular localization studies indicated that some MAGI-2 and PTEN are situated at the plasma membrane near tight junctions. Much of the MAGI-2 protein localizes to the nucleus, but the significance of this is unknown. Coexpression of MAGI-2 and PTEN in 293T cells revealed that MAGI-2 enhanced the efficiency of PTEN in repressing phosphoinositide-dependent Akt activity. Adding the PTEN COOH-terminal PDZ domain-binding motif to PTEN COOH-terminal deletion mutants increased the half-life of PTEN, perhaps through sequestration by MAGI-2 at the plasma membrane. Chromosomal deletions that include the MAGI-2 locus have been observed in certain cancers. Thus, MAGI-2 may stabilize PTEN and enhance its catalytic activity, MAGI-2 might thus act as a tumor suppressor. Wu, X., Hepner, K., Castelino-Prabhu, S., Do, D., Kaye, M.B., Yuan, X.-J., Wood, J., Ross, C., Sawyers, C.L., and Whang, Y.E. (2000) Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proc. Natl. Acad. Sci. U.S.A. 97 : 4233-4238. [Abstract] [Full Text]

DAP-kinase Regions That Regulate Cell Death

The death-associated protein kinase (DAP-kinase) promotes apoptotic signaling by Interferon γ, Fas, and Tumor Necrosis Factor α (TNF-α). Raveh et al . overexpressed random DAP-kinase fragments in cells to determine whether specific regions of the protein could prevent apoptosis. Four fragments that blocked apoptosis included: (i) an ankyrin repeat region, (ii) the linker region, (iii) the death domain central core, and (iv) the COOH-terminal 17 residues. Each fragment inhibited TNF-α-directed apoptosis in cells possessing DAP-kinase, but not in DAP-kinase deficient cells, indicating that fragment-dependent inhibition occurs through a dominant negative effect on DAP-kinase. To determine whether the COOH-terminal fragment regulated DAP-kinase catalytic activity, a COOH-terminal DAP-kinase deletion mutant, Δtail, was overexpressed in cells. Although Δtail possessed kinase activity similar to wild-type DAP-kinase, the ability of Δtail to effect apoptosis was greatly increased, equaling apoptosis mediated by a constitutively active DAP-kinase mutant. Thus, the COOH-terminus could be involved in negative regulation of DAP-kinase through an intramolecular mechanism that tightly regulates DAP-kinase interaction with downstream effectors. Raveh, T., Berissi, H., Eisenstein, M., Spivak, T., and Kimchi, A. (2000) A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity. Proc. Natl. Acad. Sci. U.S.A. 97 : 1572-1577. [Abstract] [Full Text]

Structural Requirements for α-Latrotoxin Binding and α-Latrotoxin-stimulated Secretion: A STUDY WITH CALCIUM-INDEPENDENT RECEPTOR OF α-LATROTOXIN (CIRL) DELETION MUTANTS

Stimulation of neurotransmitter release by alpha-latrotoxin requires its binding to the calcium-independent receptor of alpha-latrotoxin (CIRL), an orphan neuronal G protein-coupled receptor. CIRL consists of two noncovalently bound subunits, p85, a heptahelical integral membrane protein, and p120, a large extracellular polypeptide with domains homologous to lectin, olfactomedin, mucin, the secretin receptor family, and a novel structural motif common for large orphan G protein-coupled receptors. The analysis of CIRL deletion mutants indicates that the high affinity alpha-latrotoxin-binding site is located within residues 467-891, which comprise the first transmembrane segment of p85 and the C-terminal half of p120. The N-terminal lectin, olfactomedin, and mucin domains of p120 are not required for the interaction with alpha-latrotoxin. Soluble p120 and all its fragments, which include the 467-770 residues, bind alpha-latrotoxin with low affinity suggesting the importance of membrane-embedded p85 for the stabilization of the complex of the toxin with p120. Two COOH-terminal deletion mutants of CIRL, one with the truncated cytoplasmic domain and the other with only one transmembrane segment left of seven, supported both alpha-latrotoxin-induced calcium uptake in HEK293 cells and alpha-latrotoxin-stimulated secretion when expressed in chromaffin cells, although with a different dose dependence than wild-type CIRL and its N-terminal deletion mutant. Thus the signaling domains of CIRL are not critically important for the stimulation of exocytosis in intact chromaffin cells by alpha-latrotoxin.

Glycosylation and NH 2-terminal domain mutants of the tissue inhibitor of metalloproteinases-1 (TIMP-1)

Mutants in the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein have been created by site-directed mutagenesis and expressed in HeLa cells, using a recombinant vaccinia virus system. Removal of either or both glycosylation sites yielded proteins which retained wild-type inhibitory activity against both human fibroblast-type collagenase (FIB-CL) and M r 72 000 gelatinase (GL). However, the double glycosylation mutant protein was expressed at a level that was 2–4-fold lower than that of the wild-type or the single site glycosylation mutants. The ‘tiny-TIMP’ COOH-terminal deletion mutant that lacks the last 57 residues was also inhibitory, but the dose–response curve suggested that the interaction with the M r 72 000 gelatinase had been altered. A number of replacement mutants in the highly conserved NH 2-terminal domain, including replacement of P5A and P8A or a double mutation in the VIRAK sequence which is absolutely conserved in all TIMPs in all species (VIRAK to VIAAA), also yielded functional proteins capable of inhibiting FIB-CL and M r 72 000 GL and of forming SDS-resistant complexes with FIB-CL. None of the above manipulations abolished inhibitory function suggesting that binding of the inhibitor by the enzyme involves multiple interactions.

Fas-induced Arachidonic Acid Release Is Mediated by Ca2+-independent Phospholipase A2 but Not Cytosolic Phospholipase A2, Which Undergoes Proteolytic Inactivation

Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release from membrane glycerophospholipids, indicating phospholipase A2 (PLA2) activation. During apoptosis, type IV cytosolic PLA2 (cPLA2), a PLA2 isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA2 correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor. A mutant cPLA2 protein in which Asp522 was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXD downward arrowX), was resistant to apo-ptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA2 truncated at Asp522 comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage eventually leads to destruction of a catalytic triad essential for cPLA2 activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA2 (iPLA2), a PLA2 isozyme implicated in phospholipid remodeling, remained intact during apoptosis. Inhibitors of iPLA2, but neither cPLA2 nor secretory PLA2 inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas. Therefore, we conclude that iPLA2-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the apoptotic cell death process.

Characterization of the p125 Subunit of Human DNA Polymerase δ and Its Deletion Mutants: INTERACTION WITH CYCLIN-DEPENDENT KINASE-CYCLINS

The catalytic subunit of human DNA polymerase (pol) delta was overexpressed in an active, soluble form by the use of a baculovirus system in insect cells. The recombinant enzyme was separated from endogenous DNA polymerases by phosphocellulose, Mono Q-Sepharose, and single-stranded DNA-cellulose chromatography. Recombinant DNA pol delta was also purified by immunoaffinity chromatography. The enzymatic properties of the purified catalytic subunit were characterized. The enzyme was active and possessed both DNA polymerase and associated 3' to 5' exonuclease activities. NH2-terminal deletion mutants retained polymerase activity, whereas the core and COOH-terminal deletion mutants were devoid of any measurable activities. Coinfection of Sf9 cells with recombinant baculovirus vectors for pol delta and cyclin-dependent kinase (cdk)-cyclins followed by metabolic labeling with 32Pi showed that the recombinant catalytic subunit of pol delta could be hyperphosphorylated by G1 phase-specific cdk-cyclins. When cdk2 was coexpressed with pol delta in Sf9 cells, pol delta was found to coimmunoprecipitate with antibodies against cdk2. Experiments with deletion mutants of pol delta showed that the NH2-terminal region was essential for this interaction. Coimmunoprecipitation and Western blot experiments in Molt 4 cells confirmed the interaction in vivo. Preliminary experiments showed that phosphorylation of the catalytic subunit of pol delta by cdk2-cyclins had little or no effect on the specific activity of the enzyme.