Carboxyl-terminal Catalytic Domain Research Articles

Identification and characterization of caspase-7 in pufferfish (Takifugu obscurus) in response to bacterial infection and cell apoptosis

Caspase-7 is a key effector caspase that takes part in apoptosis, inflammation, and in immune responses in mammals. To date, studies on the expression and functional characterization of caspase-7 upon apoptosis in teleosts are limited. Here, we identify a caspase-7 homolog (pfcasp-7) from pufferfish (Takifugu obscurus) and characterize its expression pattern during bacterial challenge. The open reading frame of pfcasp-7 is 942 base pairs in length, encoding a polypeptide of 313 amino acids. The deduced amino acid sequence is highly homologous to other known caspase-7 s, which contain a carboxyl-terminal catalytic domain and a pentapeptide active-site motif (192QACRG196). Quantitative real-time qPCR analysis indicated that pfcasp-7 is expressed in all examined tissues (kidney, blood, gills, gut, heart, spleen, liver, brain, and muscle), with the highest expression in the kidney (>117-fold higher than the lowest expression in muscle), followed by expression in the blood and gills. Challenging with Aeromonas hydrophila induced apoptosis in the blood and kidney and resulted in the significant upregulation of pfcasp-7 expression, which was consistent with the detection of apoptosis by flow cytometry. Pfcasp-7 expression was also upregulated in the kidney leukocytes following in vitro stimulation with lipopolysaccharide and inactivated A. hydrophila. Taken together, these results suggest that pfcasp-7 is involved in apoptosis in pufferfish after bacterial infection.

Molecular and functional characterization of caspase-8 from the big-belly seahorse (Hippocampus abdominalis)

Apoptosis is a physiological process that can also participate in host immune defense mechanisms, including tumor growth suppression along with homeostasis and maturation of immune cells. Caspases are known to be involved in cellular apoptotic signaling; among them, caspase-8 plays an important role in the initiation phase of the apoptotic death cascade. In the current study, we molecularly characterized a caspase-8 homolog (designated as HaCasp-8) from Hippocampus abdominalis. The HaCasp-8 gene harbors a 1476 bp open reading frame (ORF) that codes for a protein of 492 amino acids (aa) with a predicted molecular mass of 55 kDa. HaCasp-8 houses the typical domain architecture of known initiator caspases, including the death effector domain and the carboxyl-terminal catalytic domain. As expected, phylogenetic analysis reflected a closer evolutionary relationship of HaCasp-8 with its teleostean similitudes. The results of our qPCR assays confirmed the ubiquitous expression of HaCasp-8 in physiologically important tissues examined, with pronounced expression levels in ovary tissues, followed by blood cells. HaCasp-8 expression at the mRNA level was found to be significantly modulated by lipopolysaccharide, polyinosinic:polycytidylic acid, Streptococcus iniae, and Edwardsiella tarda injection. Overexpression of HaCasp-8 could trigger a significant level of cell death in HEK293T cells, suggesting its putative role in cell death. Taken together, our findings suggest that HaCasp-8 is an important component in the caspase cascade, and its expression can be significantly modulated under pathogen stress conditions in the big-belly seahorse.

PARP inhibitors.

Poly (ADP-ribose) polymerases, abbreviated as PARPs, are a group of familiar proteins that play a central role in DNA repair employing the base excision repair (BER) pathway. There about 17 proteins in this family out of which the primary nuclear PARPs are PARP-1, PARP-2, PARP-3, and tankyrases 1 and 2 (PARP-5a and -5b) .The PARP family members are known to engage in a wide range of cellular activities, for example, DNA repair, transcription, cellular signaling, cell cycle regulation and mitosis amongst others. The chief functional units of PARP-1 are an amino terminal DNA binding domain (DBD), a central auto modification domain (AMD), and a carboxyl-terminal catalytic domain (CD). PARP inhibitors are currently undergoing clinical trials as targeted treatment modalities of breast, uterine, colorectal and ovarian cancer. This review summarizes current insights into the mechanism of action of PARP inhibitors, its recent clinical trials, and potential next steps in the evaluation of this promising class of anti-cancer drugs.

Crystal structure of UDP‐galactose 4‐epimerase‐like l‐threonine dehydrogenase belonging to the intermediate short‐chain dehydrogenase‐reductase superfamily

The crystal structure of a L-threonine dehydrogenase (L-ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1, which shows no sequence similarity to conventional L-ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two-fold rotation axis. Each monomer consisted of a Rossmann-fold domain and a carboxyl-terminal catalytic domain. The overall fold of F. frigidimaris L-ThrDH showed significant similarity to that of UDP-galactose 4-epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD-binding loop) around the substrate and NAD binding sites. In F. frigidimaris L-ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP-glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimaris L-ThrDH. Consequently, the cofactor binds to F. frigidimaris L-ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the β-hydroxyl hydrogen of L-threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE-like L-ThrDHs from GalEs.

A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase

Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific.

Determinants of sequence-specificity within human AID and APOBEC3G

Human APOBEC3G (A3G) and activation-induced deaminase (AID) belong to a family of DNA-cytosine deaminases. While A3G targets the last C in a run of C's, AID targets C in the consensus sequence WRC (W is A or T and R is a purine). Guided by the structures of the A3G carboxyl-terminal catalytic domain (A3G-CTD), we identified two potential regions (region 1 and region 2) that may interact with DNA and swapped the corresponding regions between a variant of A3G-CTD and AID. The resulting hybrids were expressed in Escherichia coli and two different genetic assays and a biochemical assay were used to determine the sequence selectivity of the hybrids in promoting C to T mutations. The results show that while the 10 amino acid region 2 of A3G was its principal sequence-specificity determinant, region 1 of A3G enhanced the target cytosine preference conferred by region 2. In contrast, neither of the two regions in AID individually or in combination were sufficient to confer the DNA sequence preference of this protein upon A3G. Instead, introduction of AID sequences in A3G relaxed the sequence-specificity of the latter protein. Our results show that the sequence selectivity of APOBEC family of enzymes is determined by at least two separate sequence segments and there may be additional regions of the protein involved in DNA sequence recognition.

N-Terminal Domain of Phosphodiesterase-11A4 (PDE11A4) Decreases Affinity of the Catalytic Site for Substrates and Tadalafil, and is Involved in Oligomerization

The phosphodiesterase-11A (PDE11) family consists of four splice variants (PDE11A1-PDE11A4) that contain a conserved carboxyl-terminal (C-terminal) catalytic domain that hydrolyzes cAMP and cGMP; the amino-termini (N-termini) vary in length and amino acid sequence. PDE11A2, PDE11A3, and PDE11A4 contain one or more GAF (cGMP-binding phosphodiesterase, Anabaena adenylyl cyclase, and Escherichia coli FhlA) subdomains. In the present study, PDE11A1 and PDE11A2 demonstrated higher affinity for cAMP and cGMP when directly compared to that of the longest isoform, PDE11A4. Moreover, PDE11A3, PDE11A2, and PDE11A1, which contain progressively shorter N-termini, were more sensitive than PDE11A4 to inhibition by two structurally unrelated inhibitors, tadalafil (Cialis) and vardenafil (Levitra). The substrate and inhibitor affinity differences among the PDE11 isozymes could not be ascribed to differences in their quaternary structure since PDE11A4, PDE11A3, and PDE11A2 were determined to be dimers, and PDE11A1 was a tetramer. These data also demonstrate that PDE11 isozymes containing at least 123 C-terminal amino acids of the GAF-B domain are stable oligomers and that GAF-A is not required for oligomerization. The isolated PDE11 catalytic domain (Met-563-Asn-934) displayed both monomeric and dimeric forms, and upon dilution, this domain was primarily monomeric, indicating that the main oligomerization contacts are within the N-termini of PDE isozymes. This report is the first to describe an inhibitory effect of the N-terminal region of PDE11A4 on the affinity of the catalytic domain for both substrates and inhibitors and the first to define the quaternary structure and the regions that contribute to this structure within the human PDE11A family.

Phosphodiesterase 2A Forms a Complex with the Co-chaperone XAP2 and Regulates Nuclear Translocation of the Aryl Hydrocarbon Receptor

Phosphodiesterase type 2A (PDE2A) hydrolyzes cyclic nucleotides cAMP and cGMP, thus efficiently controlling cNMP-dependent signaling pathways. PDE2A is composed of an amino-terminal region, two regulatory GAF domains, and a catalytic domain. Cyclic nucleotide hydrolysis is known to be activated by cGMP binding to GAF-B; however, other mechanisms may operate to fine-tune local cyclic nucleotide levels. In a yeast two-hybrid screening we identified XAP2, a crucial component of the aryl hydrocarbon receptor (AhR) complex, as a major PDE2A-interacting protein. We mapped the XAP2 binding site to the GAF-B domain of PDE2A. PDE assays with purified proteins showed that XAP2 binding does not change the enzymatic activity of PDE2A. To analyze whether PDE2A could affect the function of XAP2, we studied nuclear translocation of AhR, i.e. the master transcription factor controlling the expression of multiple detoxification genes. Notably, regulation of AhR target gene expression is initiated by tetrachlorodibenzodioxin (TCDD) binding to AhR and by a poorly understood cAMP-dependent pathway followed by the translocation of AhR from the cytosol into the nucleus. Binding of PDE2A to XAP2 inhibited TCDD- and cAMP-induced nuclear translocation of AhR in Hepa1c1c7 hepatocytes. Furthermore, PDE2A attenuated TCDD-induced transcription in reporter gene assays. We conclude that XAP2 targets PDE2A to the AhR complex, thereby restricting AhR mobility, possibly by a local reduction of cAMP levels. Our results provide first insights into the elusive cAMP-dependent regulation of AhR.

β-1,3-Glucan inducible expression of prophenoloxidase-activating proteinase from eri-silkworm, Samia cynthia ricini

A cDNA clone encoding possible prophenoloxidase-activating serine protease (PAP) was isolated by screening the cDNA library from immunized larval fat body of the wild silkmoth, Samia cynthia ricini. The cDNA encodes a 438 amino acid open reading frame with a predicted 20 residue signal peptide. Samia PAP has high sequence similarity to Bombyx mori and Manduca sexta PAPs, which contain two amino terminal clip domains followed by a carboxyl-terminal catalytic domain. The expression of the gene was barely detectable in the fat body of naive larvae, but induced after injection of the larvae with β-1,3-glucans or bacterial cells.

Amino-terminal Dimerization, NRDP1-Rhodanese Interaction, and Inhibited Catalytic Domain Conformation of the Ubiquitin-specific Protease 8 (USP8)

Ubiquitin-specific protease 8 (USP8) hydrolyzes mono and polyubiquitylated targets such as epidermal growth factor receptors and is involved in clathrin-mediated internalization. In 1182 residues, USP8 contains multiple domains, including coiled-coil, rhodanese, and catalytic domains. We report the first high-resolution crystal structures of these domains and discuss their implications for USP8 function. The amino-terminal domain is a homodimer with a novel fold. It is composed of two five-helix bundles, where the first helices are swapped, and carboxyl-terminal helices are extended in an antiparallel fashion. The structure of the rhodanese domain, determined in complex with the E3 ligase NRDP1, reveals the canonical rhodanese fold but with a distorted primordial active site. The USP8 recognition domain of NRDP1 has a novel protein fold that interacts with a conserved peptide loop of the rhodanese domain. A consensus sequence of this loop is found in other NRDP1 targets, suggesting a common mode of interaction. The structure of the carboxyl-terminal catalytic domain of USP8 exhibits the conserved tripartite architecture but shows unique traits. Notably, the active site, including the ubiquitin binding pocket, is in a closed conformation, incompatible with substrate binding. The presence of a zinc ribbon subdomain near the ubiquitin binding site further suggests a polyubiquitin-specific binding site and a mechanism for substrate induced conformational changes.

Functional Characterization in Vitro of All Two-component Signal Transduction Systems from Escherichia coli

Bacteria possess a signal transduction system, referred to as a two-component system, for adaptation to external stimuli. Each two-component system consists of a sensor protein-histidine kinase (HK) and a response regulator (RR), together forming a signal transduction pathway via histidyl-aspartyl phospho-relay. A total of 30 sensor HKs, including as yet uncharacterized putative HKs (BaeS, BasS, CreC, CusS, HydH, RstB, YedV, and YfhK), and a total of 34 RRs, including putative RRs (BaeR, BasR, CreB, CusR, HydG, RstA, YedW, YfhA, YgeK, and YhjB), have been suggested to exist in Escherichia coli. We have purified the carboxyl-terminal catalytic domain of 27 sensor HKs and the full-length protein of all 34 RRs to apparent homogeneity. Self-phosphorylation in vitro was detected for 25 HKs. The rate of self-phosphorylation differed among HKs, whereas the level of phosphorylation was generally co-related with the phosphorylation rate. However, the phosphorylation level was low for ArcB, HydH, NarQ, and NtrB even though the reaction rate was fast, whereas the level was high for the slow phosphorylation species BasS, CheA, and CreC. By using the phosphorylated HKs, we examined trans-phosphorylation in vitro of RRs for all possible combinations. Trans-phosphorylation of presumed cognate RRs by HKs was detected, for the first time, for eight pairs, BaeS-BaeR, BasS-BasR, CreC-CreB, CusS-CusR, HydH-HydG, RstB-RstA, YedV-YedW, and YfhK-YfhA. All trans-phosphorylation took place within less than 1/2 min, but the stability of phosphorylated RRs differed, indicating the involvement of de-phosphorylation control. In addition to the trans-phosphorylation between the cognate pairs, we detected trans-phosphorylation between about 3% of non-cognate HK-RR pairs, raising the possibility that the cross-talk in signal transduction takes place between two-component systems.

Functional Characterization of the Yeast Ppz1 Phosphatase Inhibitory Subunit Hal3: A MUTAGENESIS STUDY

Saccharomyces cerevisiae Hal3 is a conserved protein that binds the carboxyl-terminal catalytic domain of the PP1c (protein phosphatase 1)-related phosphatase Ppz1 and potently inhibits its activity, thus modulating all of the characterized functions so far of the phosphatase. It is unknown how Hal3 binds to Ppz1 and inhibits its activity. Although it contains a putative protein phosphatase 1c binding-like sequence (263KLHVLF268), mutagenesis analysis suggests that this motif is not required for Ppz1 binding and inhibition. The mutation of the conserved His378 (possibly involved in dehydrogenase catalytic activity) did not impair Hal3 functions or Ppz1 binding. Random mutagenesis of the 228 residue-conserved central region of Hal3 followed by a loss-of-function screen allowed the identification of nine residues important for Ppz1-related Hal3 functions. Seven of these residues cluster in a relatively small region spanning from amino acid 446 to 480. Several mutations affected Ppz1 binding and inhibition in vitro, whereas changes in Glu460 and Val462 did not alter binding but resulted in Hal3 versions unable to inhibit the phosphatase. Therefore, there are independent Hal3 structural elements required for Ppz1 binding and inhibition. S. cerevisiae encodes a protein (Vhs3) structurally related to Hal3. Recent evidence suggests that both mutations are synthetically lethal. Surprisingly, versions of Hal3 carrying mutations that strongly affected Ppz1 binding or inhibitory capacity were able to complement lethality. In contrast, the mutation of His378 did not. This finding suggests that Hal3 may have both Ppz1-dependent and independent functions involving different structural elements.

Phosphorylation of the Histone Deacetylase 7 Modulates Its Stability and Association with 14-3-3 Proteins

Class II histone deacetylases (HDACs) play a role in myogenesis and inhibit transcriptional activation by myocyte enhancer factors 2. A distinct feature of class II HDACs is their ability to shuttle between the nucleus and the cytoplasm in a cell type- and signal-dependent manner. We demonstrate here that treatment with the 26 S proteosome inhibitors, MG132 and ALLN, leads to detection of ubiquitinated HDAC7 and causes accumulation of cytoplasmic HDAC7. We also show that treatment with calyculin A, a protein phosphatase inhibitor, leads to a marked increase of HDAC7 but not HDAC5. The increase in HDAC7 is accompanied by enhanced interaction between 14-3-3 proteins and HDAC7. HDAC7 mutations that prevent the interaction with 14-3-3 proteins also block calyculin A-mediated stabilization. Expression of constitutively active calcium/calmodulin-dependent kinase I stabilizes HDAC7 and causes an increased association between HDAC7 and 14-3-3. Together, our results suggest that calcium/calmodulin-dependent kinase I-mediated phosphorylation of HDAC7 acts, in part, to promote association of HDAC7 with 14-3-3 and stabilizes HDAC7.

Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs

Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg 51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta serine proteinase homolog-1 and -2 were present. These proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS-stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes.

Association of COOH-terminal-binding Protein (CtBP) and MEF2-interacting Transcription Repressor (MITR) Contributes to Transcriptional Repression of the MEF2 Transcription Factor

The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors. Association of these HDACs with MEF2 factors represses transcription of MEF2 target genes. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal extensions of class II HDACs and also acts as a transcriptional repressor, but lacks a histone deacetylase catalytic domain. This suggests that MITR represses transcription by recruiting other corepressors. We show that the amino-terminal regions of MITR and class II HDACs interact with the transcriptional corepressor, COOH-terminal-binding protein (CtBP), through a CtBP-binding motif (P-X-D-L-R) conserved in MITR and HDACs 4, 5, and 7. Mutation of this sequence in MITR abolishes interaction with CtBP and impairs, but does not eliminate, the ability of MITR to inhibit MEF2-dependent transcription. The residual repressive activity of MITR mutants that fail to bind CtBP can be attributed to association with other HDAC family members. These findings reveal CtBP-dependent and -independent mechanisms for transcriptional repression by MITR and show that MITR represses MEF2 activity through recruitment of multicomponent corepressor complexes that include CtBP and HDACs.

The Mechanism of Action of Inhibitors of Bacterial Two-component Signal Transduction Systems

Two-component signal transduction systems allow bacteria to sense and respond rapidly to changes in their environment leading to specific gene activation or repression. These two-component systems are integral in the ability of pathogenic bacteria to mount and establish a successful infection within the host and, consequently, have been recognized as targets for new anti-microbial agents. In this paper, we define the site and mechanism of action of several previously identified inhibitors of bacterial two-component systems. We show that the most potent inhibitors target the carboxyl-terminal catalytic domain of the sensor kinase and exert their affect by causing structural alterations of the kinase leading to aggregation. Recognition of this phenomenon has important implications for the development of novel inhibitors of two-component systems and should facilitate the rapid identification and elimination of compounds with nonspecific affects from medicinal chemistry drug discovery programs.

Inhibition of Signaling Through the B Cell Antigen Receptor by the Protooncogene Product, c-Cbl, Requires Syk Tyrosine 317 and the c-Cbl Phosphotyrosine-Binding Domain

The Syk protein-tyrosine kinase couples the B cell Ag receptor (BCR) to intracellular biochemical pathways. Syk becomes phosphorylated on multiple tyrosine residues upon receptor cross-linking. Tyrosine 317 is a site of phosphorylation located within the linker region of Syk that separates the amino-terminal, tandem pair of SH2 domains from the carboxyl-terminal catalytic domain. The amino acid sequence surrounding phosphotyrosine 317 matches the consensus sequence for recognition by the phosphotyrosine-binding (PTB) domain of the protooncogene product, c-Cbl. The overexpression of c-Cbl in DT40 B cells inhibits Ag receptor-mediated activation of the NF-AT transcription factor. The ability of overexpressed c-Cbl to inhibit signaling requires both Syk tyrosine 317 and a functional c-Cbl PTB domain. Mutant forms of Syk lacking tyrosine 317 exhibit an enhanced ability to couple the BCR to pathways leading to the activation of both NF-AT and Elk-1. Coimmunoprecipitation experiments indicate that Syk phosphotyrosine 317 and the c-Cbl PTB domain enhance, but are not required for, all interactions between these two proteins. In unstimulated cells, c-Cbl and Syk can be isolated in a complex that also contains tubulin. A mutant form of Syk lacking tyrosine at position 317 exhibits an enhanced ability to interact with a diphosphopeptide modeled on the immunoreceptor tyrosine-based activation motif of the CD79a component of the Ag receptor. These studies indicate that c-Cbl may contribute to the regulation of BCR signaling by modulating the ability of Syk to associate with the BCR and couple the receptor to intracellular signaling pathways.

Identification and Characterization of PKNβ, a Novel Isoform of Protein Kinase PKN: Expression and Arachidonic Acid Dependency Are Different from Those of PKNα

The cDNA clone encoding a novel isoform of protein kinase PKN, termed PKNβ, was isolated from a HeLa cDNA library. PKNβ had high sequence homology with PKNα, originally isolated PKN, especially in the repeats of charged amino acid-rich region with leucine-zipper like sequences (CZ region/HR1), in the carboxyl-terminal catalytic domain, and in ∼130 amino acid stretch (D region/HR2), located between CZ region/HR1 and the catalytic domain. However, the amino acid sequence of PKNβ differed from that of PKNα in the region immediately amino-terminal to the catalytic domain, which contained two distinct proline-rich sequences consistent with the class II consensus sequence, PXXPXR, for binding to SH3 domain. Distribution of PKNβ differed from that of PKNα in the following two respects: (1) Northern blotting indicated that PKNβ mRNA could not be detected in human adult tissues, but was expressed abundantly in human cancer cell lines; (2) immunochemical analysis indicated that PKNβ localized in nucleus and perinuclear Golgi apparatus, and was almost absent in cytoplasmic region in NIH3T3 cells. Recombinant PKNβ expressed in COS7 cells displayed autophosphorylation and peptide kinase activity, but was found to be significantly less responsive to arachidonic acid than PKNα. The identification of this novel isoform underscores the diversity of PKN signaling pathway.

Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation.

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21-41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (Ndelta15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion Ndelta15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (Ndelta30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (Ndelta41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.

Proteolytic activation of PKN by caspase-3 or related protease during apoptosis.

PKN, a fatty acid- and Rho-activated serine/threonine kinase having a catalytic domain highly homologous to protein kinase C (PKC), was cleaved at specific sites in apoptotic Jurkat and U937 cells on Fas ligation and treatment with staurosporin or etoposide, respectively. The cleavage of PKN occurred with a time course similar to that of PKCdelta, a known caspase substrate. This proteolysis was inhibited by a caspase inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde. The cleavage fragments were generated in vitro from PKN by treatment with recombinant caspase-3. Site-directed mutagenesis of specific aspartate residues prevented the appearance of these fragments. These results indicate that PKN is cleaved by caspase-3 or related protease during apoptosis. The major proteolysis took place between the amino-terminal regulatory domain and the carboxyl-terminal catalytic domain, and it generated a constitutively active kinase fragment. The cleavage of PKN may contribute to signal transduction, eventually leading to apoptosis.