Substitution Of Glutamic Acid Research Articles

Novel missense mutations and a 288‐bp exonic insertion in PAX9 in families with autosomal dominant hypodontia

We describe the molecular analysis of three families with hypodontia involving primarily molar teeth and report two novel mutational mechanisms. Linkage analysis of two large families revealed that the hypodontia was linked to the PAX9 locus. These two families revealed missense mutations consisting of a glutamic acid substitution for lysine and a proline substitution for leucine within the paired domain of PAX9. A pair of identical twins affected with hypodontia in a third family demonstrated a 288-bp insertion within exon 2 that resulted in a putative frameshift mutation and a premature stop codon. The insertion was associated with the loss of 7-bp from exon 2. A block of 256-bp of sequence within the insertion was completely identical to downstream sequence from the second intron of the PAX9 gene. These studies extend the spectrum of mutations in PAX9 associated with hypodontia to include heretofore undescribed categories, including missense mutations.

Characterization of an integron carrying a new class D beta-lactamase (OXA-37) in Acinetobacter baumannii.

Integrons from a clinical strain of Acinetobacter baumannii highly resistant to beta-lactams have been analyzed. The largest (2.2 kb) contained three gene cassettes: an aacA4, an open reading frame of 417 nucleotides, and an OXA-type encoding gene. The oxa gene nucleotide sequence differed from that of the oxa-20 in 2 bp, one of the mutations being silent. The nonsilent mutationgenerated a substitution of glutamic acid for aspartic acid. The new OXA has been named OXA-37.

A single amino acid substitution in DNA-PKcs explains the novel phenotype of the CHO mutant, XR-C2.

We recently described a CHO DSBR mutant belonging to the XRCC7 complementation group (XR-C2) that has the interesting phenotype of being radiosensitive, but having only a modest defect in VDJ recombination. This cell line expresses only slightly reduced levels of DNA-PKcs but has undetectable DNA-PK activity. Limited sequence analyses of DNA-PKcs transcripts from XR-C2 revealed a point mutation that results in an amino acid substitution of glutamic acid for glycine six residues from the C-terminus. To determine whether this single substitution was responsible for the phenotype in XR-C2 cells, we introduced the mutation into a DNA-PKcs expression vector. Whereas transfection of this expression vector significantly restores the VDJ recombination deficits in DNA-PKcs-deficient cells, radioresistance is not restored. Thus, expression of this mutant form of DNA-PKcs in DNA-PKcs- deficient cells substantially recapitulates the phenotype observed in XR-C2, and we conclude that this single amino acid substitution is responsible for the non-homologous end joining deficits observed in XR-C2.

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells.

Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transport-defective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, K(i)s for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.

Anionic substitutes for catalytic aspartic acids in phosphoribulokinase

Mutagenic substitution of the invariant D42 and D169 residues in phosphoribulokinase (PRK) with amino acids that contain neutral side chains (e.g., alanine or asparagine) results in large decreases in catalytic efficiency (10 5- and 10 4-fold for replacement of D42 and D169, respectively). To further evaluate the importance of anionic side chains at residues 42 and 169, substitutions of glutamic acid (D42E, D169E) and cysteine (D42C and D169C in an otherwise cysteine-free protein) have been engineered. All purified mutant enzymes bind the fluorescent alternative substrate trinitrophenyl–ATP and the allosteric effector NADH similarly to wild-type PRK. For D42E and D42C, V max exhibits substantial decreases of 135- and 220-fold, respectively. Comparable substitutions for D169 result in smaller effects; D169E and D169C exhibit decreases in V max of 39- and 26-fold, respectively. Thus, regardless of the type of substitution, changes at D42 more profoundly affect catalytic rate than do comparable changes at D169. Precedent with enzymes in which cysteine replaces an acidic residue suggests that oxidation of the thiolate to a sulfinate can convert low-activity cysteine mutants into enzymes with improved activity. Periodate oxidation of cysteine-free PRK results in a slight decrease in activity. In contrast, comparable treatment of D42C and D169C proteins increases activity by 5- and 7-fold, respectively. Thus, for reasonably efficient catalysis, PRK requires anionic character in the side chains of residues 42 and 169. The enzyme can, however, tolerate substantial structural and chemical variability at these residues.

Mitochondrial targeting of human 8-oxoguanine DNA glycosylase hOGG1 is impaired by a somatic mutation found in kidney cancer

Oxidative damage to mitochondrial DNA (mtDNA) has been implicated as a contributing factor to the origin of many pathological processes. Among the major DNA lesions generated by oxidative stress, 8-oxoguanine (8-oxoG) has a strong mutagenic potential. The main pathway for the repair of this lesion is the base excision repair (BER) initiated by the OGG1 DNA glycosylase. In human cells alternative splicing of the hOGG1 transcript yields two forms of the protein, α and β, that are localised in the nucleus and the mitochondria, respectively. We describe here a hOGG1 somatic mutation, found in a kidney cancer, that modifies the intracellular localisation of β-hOGG1. The glycine 12 to glutamic acid substitution does not affect the enzymatic activity of the enzyme but lies in what was predicted to be a mitochondrial targeting sequence (MTS). Immunocytochemical localisation experiments show that the G12E mutant protein has diffuse cytoplasmic distribution instead of the mitochondrial localisation found for the wild-type, providing an example of a single amino acid substitution capable of disrupting a MTS. These results imply that these cancer cells are deficient in their mitochondrial 8-oxoG DNA repair activity.

Characterization of an activated mutant of focal adhesion kinase: 'SuperFAK'.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in normal cellular processes such as adhesion, spreading, migration, proliferation and survival. In addition, FAK is overexpressed in a variety of cancer cells and tumours and may play a role in the development of human cancer. As a prelude to modelling the role of aberrant FAK signalling in the initiation of cancer, the goal of the present study was to engineer point mutations in FAK that would enhance enzymic activity. A number of substitutions that were reported as activating mutations in other tyrosine kinases were introduced into FAK. Glutamic acid substitutions for two lysine residues in the activation loop of FAK, based upon the K650E (Lys(650-->)Glu) mutant of fibroblast-growth-factor receptor 3, were made to create 'SuperFAK'. Two brain-specific exons were engineered into avian FAK to create FAK6.7. SuperFAK and, to a lesser extent, FAK6.7, exhibited increased catalytic activity in vitro compared with wild-type FAK. The expression of SuperFAK and FAK6.7 in fibroblasts led to hyperphosphorylation of FAK substrates. Although the catalytic activity of SuperFAK and FAK6.7 was largely independent of cell adhesion, tyrosine phosphorylation of downstream substrates was adhesion-dependent. Further, since SuperFAK exhibited the same ability as wild-type FAK to recruit Src family kinases, tyrosine phosphorylation of substrates was likely due to direct phosphorylation by FAK. In addition to enhanced biochemical signalling, SuperFAK also increased the motility of epithelial cells. SuperFAK and FAK6.7 may be valuable molecular tools to investigate the potential role of aberrant FAK signalling in human disease.

Identification of temperature-sensitive mutations in the phosphoprotein of respiratory syncytial virus that are likely involved in its interaction with the nucleoprotein.

The phosphoprotein (P) of human respiratory syncytial virus (RSV) is an essential component of the viral RNA polymerase, along with the large polymerase (L), nucleocapsid (N), and M2-1 proteins. By screening a randomly mutagenized P gene cDNA library, two independent mutations, one with a substitution of glycine at position 172 by serine (G172S) and the other with a substitution of glutamic acid at position 176 by glycine (E176G), were identified to result in the loss of N-P interaction at 37 degrees C in the yeast two-hybrid assay. Both P mutants exhibited greatly reduced activity in supporting the replication and transcription of an RSV minigenome replicon at 37 and 39 degrees C. The G172S and E176G mutations were introduced individually into the RSV A2 (rA2) antigenomic cDNA, and recombinant viruses, rA2-P172 and rA2-P176, were obtained. Both viruses replicate as well as wild-type A2 virus in both Vero and HEp-2 cells at 33 degrees C, but each mutant virus exhibited temperature-sensitive replication in both cell lines. rA2-P176 is more temperature sensitive than rA2-P172. Coimmunoprecipitation of the N protein with each P mutant from virus-infected cells demonstrates that N-P interaction is impaired at 37 degrees C. In addition, the levels of replication of rA2-P172 and rA2-P176 in the lungs of mice and cotton rats were reduced. As is the case with the in vitro assays, rA2-P176 is more restricted in replication in the lower respiratory tract of mice and cotton rats than rA2-P172. During in vitro passage at 37 degrees C, the E176G mutation in rA2-P176 was rapidly changed from glycine to predominantly aspartic acid; mutations to cysteine or serine were also detected. All of the revertants lost the temperature-sensitive phenotype. To analyze the importance of the amino acids in the region from positions 161 to 180 for the P protein function, additional mutations were introduced and their functions were analyzed in vitro. A double mutant containing both G172S and E176G changes in the P gene, substitution of the three charged residues at positions 174 to 176 by alanine, and a deletion of residues from positions 161 to 180 completely abolished the P protein function in the minigenome assay. Thus, the amino acids at positions 172 and 176 and the adjacent charged residues play critical roles in the function of the P protein.

Molecular cloning of Trypanosoma brucei CK2 catalytic subunits: the α isoform is nucleolar and phosphorylates the nucleolar protein Nopp44/46

We have demonstrated previously that Nopp44/46, an abundant nucleolar phosphoprotein of Trypanosoma brucei, is associated with a protein kinase. In many organisms multiple nucleolar proteins are phosphorylated by the protein kinase CK2, formerly known as casein kinase II. Here we report the identification of two T. brucei genes, CK2a1and CK2a2, which encode protein kinases bearing signature motifs common to CK2 catalytic subunits. The protein specified by CK2a1, designated CK2α, was capable of associating with Nopp44/46 as assessed by yeast two-hybrid analysis. An epitope-tagged version of CK2α expressed in T. brucei colocalized with Nopp44/46, with a largely nucleolar localization. This localization contrasts with the predominantly nuclear localization of mammalian CK2. When expressed in Escherichia coli, TbCK2α was catalytically active and phosphorylated Nopp44/46. Together these data demonstrate that TbCK2α is a Nopp44/46-associated kinase. Competition assays revealed that, unlike most CK2s, TbCK2α discriminates highly between ATP and GTP. This distinction may be associated with the substitution of glutamic acid and alanine for the di-asparagine motif thought to participate in purine interaction.

ATP Utilization by Yeast Replication Factor C: IV. RFC ATP-BINDING MUTANTS SHOW DEFECTS IN DNA REPLICATION, DNA REPAIR, AND CHECKPOINT REGULATION

Replication factor C is required to load proliferating cell nuclear antigen onto primer-template junctions, using the energy of ATP hydrolysis. Four of the five RFC genes have consensus ATP-binding motifs. To determine the relative importance of these sites for proper DNA metabolism in the cell, the conserved lysine in the Walker A motif of RFC1, RFC2, RFC3, or RFC4 was mutated to either arginine or glutamic acid. Arginine mutations in all RFC genes tested permitted cell growth, although poor growth was observed for rfc2-K71R. A glutamic acid substitution resulted in lethality in RFC2 and RFC3 but not in RFC1 or RFC4. Most double mutants combining mutations in two RFC genes were inviable. Except for the rfc1-K359R and rfc4-K55E mutants, which were phenotypically similar to wild type in every assay, the mutants were sensitive to DNA-damaging agents. The rfc2-K71R and rfc4-K55R mutants show checkpoint defects, most likely in the intra-S phase checkpoint. Regulation of the damage-inducible RNR3 promoter was impaired in these mutants, and phosphorylation of Rad53p in response to DNA damage was specifically defective when cells were in S phase. No dramatic defects in telomere length regulation were detected in the mutants. These data demonstrate that the ATP binding function of RFC2 is important for both DNA replication and checkpoint function and, for the first time, that RFC4 also plays a role in checkpoint regulation.

A Novel Missense Mutation in Human Lactate Dehydrogenase B-Subunit Gene

Reduced activity of serum lactate dehydrogenase (LDH; EC 1.1.1.27) was found in a male medical student during practical examinations of his own blood. Serum LDH isoenzyme pattern showed reductions in activities of the isoenzymes with lower subunit A/B ratios such as LDH1 and LDH2. These findings were indicative of a partial LDH-B subunit deficiency, which was confirmed in erythrocyte hemolysates by Western blotting. Polymerase chain reaction (PCR)-based DNA sequence analysis of the LDH-B subunit gene revealed a heterozygous nucleotide change: a guanine to adenine substitution in codon 69 (GGG → GAG) at the third exon of the LDH-B subunit gene that resulted in a glycine to glutamic acid substitution (G69E). The mutation was confirmed by PCR–restriction fragment length polymorphism (RFLP) analysis using a mismatched primer to introduce a new NcoI restriction site. The same heterozygous mutation was found in his mother but not in other family members. This mutation involves a residue belonging to αC helix in LDH-B subunit protein molecule that functions as an interface for other subunits.

Highly activated Fgfr3 with the K644M mutation causes prolonged survival in severe dwarf mice.

Several gain-of-function mutations in a receptor tyrosine kinase, fibroblast growth factor receptor 3 (FGFR3), cause dwarfism in humans. Two particularly severe dwarfisms, thanatophoric dysplasia type II (TDII) and severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), are associated with glutamic acid (E) and methionine (M) substitutions at the K650 residue in the kinase domain. TDII is lethal at birth, whereas most of the SADDAN patients survive the perinatal period. However, FGFR3 with the SADDAN mutation is more activated than FGFR3 with the TDII mutation in vitro. To find out whether the K650M mutation also causes the SADDAN phenotype, we introduced the corresponding point mutation (K644M) into the mouse Fgfr3 gene. Heterozygous mutant mice show a phenotype similar to human SADDAN, e.g. the majority of the SADDAN mice survive the perinatal period. This suggests that the survival of SADDAN patients is indeed attributed to the K650M mutation in FGFR3. The long bone abnormalities in SADDAN mice are milder than the TDII model. In addition, overgrowth of the cartilaginous tissues is observed in the rib cartilage, trachea and nasal septum. The FGF ligand at the low concentration differentially activates Map kinase in primary chondrocyte cultures from wild-type and SADDAN mice. Comparisons of the molecular bases of the phenotypic differences in SADDAN and TDII mice may increase our understanding of the factors that influence the severity in these two related skeletal dysplasias.

Refocusing of B-cell responses following a single amino acid substitution in an antigen.

Intranasal immunization of BALB/c strain mice was carried out using baculovirus-derived human chorionic gonadotrophin (hCG) beta-chain, together with Escherichia coli heat-labile enterotoxin. Gonadotrophin-reactive immunoglobulin A (IgA) was induced in a remote mucosal site, the lung, in addition to a systemic IgG response. The extensive sequence homology with luteinizing hormone (LH) results in the production of LH cross-reactive antibodies when holo-hCG is used as an immunogen. In contrast to wild-type hCGbeta, a mutated hCGbeta-chain containing an arginine to glutamic acid substitution at position 68 did not induce the production of antibodies which cross-react with LH. Furthermore, the epitopes utilized in the B-cell response to the mutated hCGbeta shifted away from the immunodominant region of the parent wild-type molecule towards epitopes within the normally weakly immunogenic C terminus. This shift in epitope usage was also seen following intramuscular immunization of rabbits. Thus, a single amino acid change, which does not disrupt the overall structure of the molecule, refocuses the immune response away from a disadvantageous cross-reactive epitope region and towards a normally weakly immunogenic but antigen-unique area. Similar mutational strategies for epitope-refocusing may be applicable to other vaccine candidate molecules.

Site-directed mutagenesis of human membrane-associated ganglioside sialidase: identification of amino-acid residues contributing to substrate specificity.

Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane-associated sialidase, which hydrolyzes only gangliosides. To cast light on the molecular basis of this narrow substrate preference, predicted active site amino-acid residues of the human membrane sialidase were altered by site-directed mutagenesis. When compared with the active site amino-acid residues proposed for Salmonella typhimurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmembrane region. Alteration of seven residues, including these five, was followed by transient expression of the mutant enzymes in COS-1 cells and characterization of their kinetic properties using various substrates. Substitution of glutamic acid (at position 51) by aspartic acid and of arginine (at position 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caused a marked reduction. The mutant enzyme E51D exhibited an increase in hydrolytic activity towards GM2 as well as sialyllactose (which are poor substrates for the wild-type) with change to a lower Km and a higher Vmax. R114Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides GD3 and GD1a that are effectively hydrolyzed by the wild-type. The inhibition experiments using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid were consistent with the results in the alteration of substrate specificity. The findings suggest that putative active-site residues of the human membrane sialidase contribute to its substrate specificity.

G76E Substitution in Type I Collagen Is the First Nonlethal Glutamic Acid Substitution in the α1(I) Chain and Alters Folding of the N-terminal End of the Helix

The majority of osteogenesis imperfecta (OI) is caused by substitutions for glycine residues in the two α chains of type I collagen. Since only 4% of possible nucleotide changes in type I collagen glycine codons would result in a glutamic acid substitution, these are predicted to be infrequent. Only one glutamic acid substitution in type I collagen has been fully reported. We describe here the clinical, biochemical, and molecular characterization of a girl with severe type III OI caused by a G76E substitution in COL1A1. This is the first delineation of a glutamic acid substitution in the α1(I) chain causing nonlethal osteogenesis imperfecta. The proband's fibroblast type I collagen chains and cyanogen bromide peptides were electrophoretically normal, while osteoblast collagen was slightly overmodified. This suggested a mutation near the N-terminal end of the collagen helix. A mismatch was detected by RNA:DNA hybrid analysis in cDNA coding for 106 amino acids at the N-terminal end of the helical region. Subclones of both alleles were sequenced and revealed a G → A (c.761G > A) mutation causing an α1(I) G76E substitution in one allele. The presence of the mutation in the proband's leukocyte gDNA, and its absence in parental gDNA, was confirmed by Tsp509I digestion. The glutamic acid substitution alters the folding of the mutant collagen helices. Pericellular processing of type I collagen by the proband's fibroblasts yielded an earlier appearance of the pC-α1(I) form and of mature α chains as compared to control cell processing. Also, the presence of the glutamic acid substitution apparently exposes the adjacent Arg75 residue in the α1 chain. Trypsin digestion of proband fibroblast collagen resulted in shortened α1 chains, as confirmed by CNBr analysis. In addition, the Tm for mutant helices from fibroblasts and osteoblasts was decreased 2–4°C versus controls, demonstrating a decrease in helix stability. These findings increase our understanding of the disruptive effect of glutamic acid substitutions in collagen.

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) activity is unaltered by phosphorylation of a periodicity/ARNT/single-minded (PAS)-region serine residue.

The aryl hydrocarbon nuclear translocator (ARNT) protein belongs to the family of basic helix-loop-helix (HLH)-periodicity/ARNT/single-minded [Per/ARNT/Sim (PAS)] transcription factors and regulates a range of cellular processes by either homodimerizing or heterodimerizing with other basic HLH-PAS proteins. To date, it has been shown that both the HLH and PAS domains are required for aryl hydrocarbon receptor (AhR) ARNT heterodimerization and that phosphorylation of ARNT is also required for this heterodimerization. Presently, regulation of ARNT with respect to phosphorylation is poorly understood. In an earlier study, murine ARNT was shown to be a phosphoprotein, to display charge heterogeneity, and to have a shift in its predominant isoforms after heterodimerization with the AhR. It was hypothesized that this shift may represent a change in ARNT phosphorylation status. Metabolic [(32)P]orthophosphate labeling of human ARNT-transfected COS-1 cells, in conjunction with phosphoamino acid analysis, Edman degradation, and phosphopeptide mapping, demonstrated that ARNT is predominantly phosphorylated on serine residues and that serine 348 (S348) in the PAS domain is phosphorylated. Alanine and glutamic acid substitutions were used to demonstrate that loss of phosphorylation at this site did not influence AhR-mediated xenobiotic response elements-driven or ARNT-mediated class B E-box-driven signaling. Additionally, the phosphorylation pattern of ARNT was unaltered after AhR heterodimerization. Although phosphorylation of S348 did not modulate AhR-ARNT or ARNT-ARNT signaling, phosphorylation of this PAS-region serine residue may be important in other ARNT-mediated gene expression systems.

Persistent Activation of NF-κB by the Tax Transforming Protein Involves Chronic Phosphorylation of IκB Kinase Subunits IKKβ and IKKγ

The Tax transforming protein encoded by human T-cell leukemia virus type 1 (HTLV1) persistently activates transcription factor NF-kappaB and deregulates the expression of downstream genes that mediate cell cycle entry. We recently found that Tax binds to and chronically stimulates the catalytic function of IkappaB kinase (IKK), a cellular enzyme complex that phosphorylates and inactivates the IkappaB inhibitory subunit of NF-kappaB. We now demonstrate that the IKKbeta catalytic subunit and IKKgamma regulatory subunit of IKK are chronically phosphorylated in HTLV1-infected and Tax-transfected cells. Alanine substitutions at Ser-177 and Ser-181 in the T loop of IKKbeta protect both of these IKK subunits from Tax-directed phosphorylation and prevent the induction of IkappaB kinase activity. Each of these inhibitory effects is recapitulated in Tax transfectants expressing the bacterial protein YopJ, a potent in vivo agonist of T loop phosphorylation. Moreover, ectopically expressed forms of IKKbeta that contain glutamic acid substitutions at Ser-177 and Ser-181 have the capacity to phosphorylate a recombinant IKKgamma substrate in vitro. We conclude that Tax-induced phosphorylation of IKKbeta is required for IKKbeta activation, phosphoryl group transfer to IKKgamma, and acquisition of the deregulated IKK phenotype.

Molecular analysis of familial androgen insensitivity syndrome due to replacement of glutamic acid 802 by lysine.

We studied a Japanese family presenting at least two cases of complete androgen insensitivity syndrome (CAIS) and negative androgen receptor binding. The index subject showed a 46, XY karyotype and a complete female phenotype. For the purpose of further diagnosis and genetic counseling, molecular analysis of the androgen receptor (AR) gene was performed. Direct sequencing of the AR gene identified a mutation at nucleotide 2935 (A-->G). This replacement was a novel missense mutation, resulting in the substitution of glutamic acid 802 by lysine which deleted a recognition site for EcoRI in exon 6 of the AR gene. We identified another affected individual, using chromosome and molecular analysis of the AR gene at exon 6. Furthermore, although heterozygote carriers could not be identified on clinical grounds, molecular identification of healthy individuals and heterozygote carriers in the family members provided definitive information for genetic counseling. We believe that the molecular analysis of familial CAIS is very informative for both the affected individuals and other family members.

The competitive interaction of actin and PIP2 with actophorin is based on overlapping target sites: design of a gain-of-function mutant.

We studied the effect of mutations in an alpha-helical region of actophorin (residues 91-108) on F-actin and PIP(2) binding. As in cofilin, residues in the NH(2)-terminal half of this region are involved in F-actin binding. We show here also that basic residues in the COOH-terminal half of the region participate in this interaction whereby we extend the previously defined actin binding interface [Lappalainen, P., et al. (1997) EMBO J. 16, 5520-5530]. In addition, we demonstrate that some of the lysines in this alpha-helical region in actophorin are implicated in PIP(2) binding. This indicates that the binding sites of F-actin and PIP(2) on actophorin overlap, explaining the mutually exclusive binding of these ligands. The Ca(2+)-dependent F-actin binding of a number of actophorin mutants (carrying a lysine to glutamic acid substitution at the COOH-terminal positions of the actin binding helical region) may mimic the behavior of members of the gelsolin family. In addition, we show that PIP(2) binding, but not actin binding, of actophorin is strongly enhanced by a point mutation that leads to a reinforcement of the positive electrostatic potential of the studied alpha-helical region.