A mutation of keratin 18 within the coil 1A consensus motif causes widespread keratin aggregation but cell type-restricted lethality in mice

Mutations in genes encoding epidermal keratins cause skin disorders, while those in internal epithelial keratins, such as K8 and K18, are risk factors for liver diseases. The effect of dominant mutations in K8 or K18 during embryonic development and tissue homeostasis has not been examined so far. Here we demonstrate that the dominant mutation hK18 R89C, that is highly similar to hK14 R125C, causing EBS in humans, leads to cell type-specific lethality in mice, depending on the ratio of mutant to endogenous keratins. Mice expressing hK18 R89C in the absence of endogenous K19 and K18 died at mid-gestation from defects in trophoblast giant cells, accompanied by haematomas. A single, endogenous K18 allele rescued embryonic lethality but caused aggregation of keratins in all adult internal epithelia, surprisingly without spontaneous cell fragility. Closer analysis revealed that both filaments and aggregates coexisted in the same cell, depending on the ratio of mutant to endogenous keratins. Our results demonstrate that balanced overexpression of a wild-type keratin rescued the lethal consequences of a dominant-negative mutation. This has important implications for therapy approaches of keratinopathies, suggesting that suppressing the mutant allele is not necessary in vivo.     

Structural insight into mechanisms for dynamic regulation of PKM2

Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that posttranslational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a “seesaw” model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2^Y105E (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2^K305Q (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by posttranslational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.     

Dominant Negative Mutations Affect Oligomerization of Human Pyruvate Kinase M2 Isozyme and Promote Cellular Growth and Polyploidy

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM₂) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM₂ in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM₂ by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM₂ showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM₂ by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.     

Functional analysis of the Notch ligand Jagged1 missense mutant proteins underlying Alagille syndrome

Heterozygous mutations in the JAG1 gene, encoding Notch ligand Jagged1, cause Alagille syndrome (ALGS). As most of the mutations are nonsense or frameshift mutations producing inactive truncated proteins, haplo-insufficiency is considered the major pathogenic mechanism of ALGS. However, the molecular mechanisms by which the missense mutations cause ALGS remain unclear. Here we analyzed the functional properties of four ALGS missense mutant proteins, P163L, R184H, G386R and C714Y, using transfected mammalian cells. P163L and R184H showed Notch-binding activities similar to that of the wild-type when assessed by immunoprecipitation. However, their trans-activation and cis-inhibition activities were almost completely impaired. These mutant proteins localized mainly to the endoplasmic reticulum (ER), suggesting that the mutations induced improper protein folding. Furthermore, the mutant proteins bound more strongly to the ER chaperone proteins calnexin and calreticulin than the wild-type did. C714Y also localized to the ER, but possessed significant trans-activation activity and lacked enhanced binding to the chaperones, indicating a less severe phenotype. The properties of G386R were the same as those of the wild-type. Dominant-negative effects were not detected for any mutant protein. These results indicate that accumulation in the ER and binding to the chaperones correlate with the impaired signal-transduction activities of the missense mutant proteins, which may contribute to the pathogenic mechanism of ALGS. Our findings, which suggest the requirement for cell-surface localization of Jagged1 for cis-inhibition activities, also provide important information for understanding the molecular basis of Notch-signaling pathways.     

A role for histone H4K16 hypoacetylation in Saccharomyces cerevisiae kinetochore function

Hypoacetylated H4 is present at regional centromeres; however, its role in kinetochore function is poorly understood. We characterized H4 acetylation at point centromeres in Saccharomyces cerevisiae and determined the consequences of altered H4 acetylation on chromosome segregation. We observed low levels of tetra-acetylated and K16 acetylated histone H4 (H4K16Ac) at centromeres. Low levels of H4K16Ac were also observed at noncentromeric regions associated with Cse4p. Inhibition of histone deacetylases (HDAC) using nicotinamide (NAM) caused lethality in cse4 and hhf1-20 kinetochore mutants and increased centromeric H4K16Ac. Overexpression of Sas2-mediated H4K16 acetylation activity in wild-type cells led to increased rates of chromosome loss and synthetic dosage lethality in kinetochore mutants. Consistent with increased H4K16 acetylation as a cause of the phenotypes, deletion of the H4K16 deacetylase SIR2 or a sir2-H364Y catalytic mutant resulted in higher rates of chromosome loss compared to wild-type cells. Moreover, H4K16Q acetylmimic mutants displayed increased rates of chromosome loss compared to H4K16R nonacetylatable mutants and wild-type cells. Our work shows that hypoacetylated centromeric H4 is conserved across eukaryotic centromeres and hypoacetylation of H4K16 at centromeres plays an important role in accurate chromosome segregation.     

Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K(D) = 10-30 nm) and low (K(D) = 0.1-1.0 microm) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat beta2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type alpha1 and gamma2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [(3)H]Ro15-4513 binding or impaired allosteric interactions between the low affinity GABA and benzodiazepine sites. Although mutations at position 74 had little effect on [(3)H]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [(3)H]muscimol binding sites by approximately 6-fold, and the Y62S mutation led to a loss of detectable high affinity binding sites. After expression in oocytes, the EC(50) values for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude that Tyr-62 of the beta subunit is an important determinant for high affinity agonist binding to the GABA(A) receptor.     

Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli.

We investigated the functional roles of putative active site residues in Escherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for ATP. We designed these mutations to alter key positions in sequence motifs conserved in the protein histidine kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "GT block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases. Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)). Of these, three (N380D, D420N, and G422A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg(2+). The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg(2+), but reduced affinity (relative to that of wild-type CheA) in the presence of Mg(2+). The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA. Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibited severely reduced k(cat) values, but preserved K(M)(ATP) and K(d)(ATP) values close to those of wild-type CheA. Two mutants (T499S and T499A) exhibited only small effects on k(cat) and K(M)(ATP). Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT block makes little, if any, contribution. We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and Mg(2+)-bound ATP in both the ground state and the transition state for the CheA autophosphorylation reaction.     

Characterization of a novel, dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome

Andersen-Tawil syndrome is characterized by periodic paralysis, ventricular ectopy, and dysmorphic features. Approximately 60% of patients exhibit loss-of-function mutations in KCNJ2, which encodes the inwardly rectifying K(+) channel pore forming subunit Kir2.1. Here, we report the identification of a novel KCNJ2 mutation (G211T), resulting in the amino acid substitution D71Y, in a patient presenting with signs and symptoms of Andersen-Tawil syndrome. The functional properties of the mutant subunit were characterized using voltage-clamp experiments on transiently transfected HEK-293 cells and neonatal mouse ventricular myocytes. Whole-cell current recordings of transfected HEK-293 cells demonstrated that the mutant protein Kir2.1-D71Y fails to form functional ion channels when expressed alone, but co-assembles with wild-type Kir2.1 subunits and suppresses wild-type subunit function. Further analysis revealed that current suppression requires at least two mutant subunits per channel. The D71Y mutation does not measurably affect the membrane trafficking of either the mutant or the wild-type subunit or alter the kinetic properties of the currents. Additional experiments revealed that expression of the mutant subunit suppresses native I(K1) in neonatal mouse ventricular myocytes. Simulations predict that the D71Y mutation in human ventricular myocytes will result in a mild prolongation of the action potential and potentially increase cell excitability. These experiments indicate that the Kir2.1-D71Y mutant protein functions as a dominant negative subunit resulting in reduced inwardly rectifying K(+) current amplitudes and altered cellular excitability in patients with Andersen-Tawil syndrome.     

A single deletion in the membrane-proximal region of the Sindbis virus glycoprotein E2 endodomain blocks virus assembly

The envelopment of the Sindbis virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390-400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531-541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, DeltaK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, DeltaK391 produced wild-type levels of virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby virus assembly. The data suggest that these viruses have evolved properties adapted specifically for assembly in the diverse hosts in which they grow.     

Prolactin inhibits activity of pyruvate kinase M2 to stimulate cell proliferation

Mitogenic and prosurvival effects underlie the tumorigenic roles of prolactin (PRL) in the pathogenesis of breast cancer. PRL signaling is mediated through its receptor (PRLr). A proteomics screen identified the pyruvate kinase M2 (PKM2), a glycolytic enzyme known to play an important role in tumorigenesis, as a protein that constitutively interacts with PRLr. Treatment of cells with PRL inhibited pyruvate kinase activity and increased the lactate content in human cells in a manner that was dependent on the abundance of PRLr, activation of Janus kinase 2, and tyrosine phosphorylation of the intracellular domain of PRLr. Knockdown of PKM2 attenuated PRL-stimulated cell proliferation. The extent of this proliferation was rescued by the knock-in of the wild-type PKM2 but not of its mutant insensitive to PRL-mediated inhibition. We discuss a hypothesis that the inhibition of PKM2 by PRL contributes to the PRL-stimulated cell proliferation.     

Trafficking defects of a novel autosomal recessive distal renal tubular acidosis mutant (S773P) of the human kidney anion exchanger (kAE1)

Autosomal dominant and recessive distal renal tubular acidosis (dRTA) can be caused by mutations in the anion exchanger 1 (AE1 or SLC4A1) gene, which encodes the erythroid chloride/bicarbonate anion exchanger membrane glycoprotein (eAE1) and a truncated kidney isoform (kAE1). The biosynthesis and trafficking of kAE1 containing a novel recessive missense dRTA mutation (kAE1 S773P) was studied in transiently transfected HEK-293 cells, expressing the mutant alone or in combination with wild-type kAE1 or another recessive mutant, kAE1 G701D. The kAE1 S773P mutant was expressed at a three times lower level than wild-type, had a 2-fold decrease in its half-life, and was targeted for degradation by the proteasome. It could not be detected at the plasma membrane in human embryonic kidney cells and showed predominant endoplasmic reticulum immunolocalization in both human embryonic kidney and LLC-PK1 cells. The oligosaccharide on a kAE1 S773P N-glycosylation mutant (N555) was not processed to the complex form indicating impaired exit from the endoplasmic reticulum. The kAE1 S773P mutant showed decreased binding to an inhibitor affinity resin and increased sensitivity to proteases, suggesting that it was not properly folded. The other recessive dRTA mutant, kAE1 G701D, also exhibited defective trafficking to the plasma membrane. The recessive kAE1 mutants formed dimers like wild-type AE1 and could hetero-oligomerize with wild-type kAE1 or with each other. Hetero-oligomers of wild-type kAE1 with recessive kAE1 S773P or G701D, in contrast to the dominant kAE1 R589H mutant, were delivered to the plasma membrane.     

Cellular and molecular mechanisms of autosomal dominant form of progressive hearing loss, DFNA2

Despite advances in identifying deafness genes, determination of the underlying cellular and functional mechanisms for auditory diseases remains a challenge. Mutations of the human K(+) channel hKv7.4 lead to post-lingual progressive hearing loss (DFNA2), which affects world-wide population with diverse racial backgrounds. Here, we have generated the spectrum of point mutations in the hKv7.4 that have been identified as diseased mutants. We report that expression of five point mutations in the pore region, namely L274H, W276S, L281S, G285C, and G296S, as well as the C-terminal mutant G321S in the heterologous expression system, yielded non-functional channels because of endoplasmic reticulum retention of the mutant channels. We mimicked the dominant diseased conditions by co-expressing the wild-type and mutant channels. As compared with expression of wild-type channel alone, the blend of wild-type and mutant channel subunits resulted in reduced currents. Moreover, the combinatorial ratios of wild type:mutant and the ensuing current magnitude could not be explained by the predictions of a tetrameric channel and a dominant negative effect of the mutant subunits. The results can be explained by the dependence of cell surface expression of the mutant on the wild-type subunit. Surprisingly, a transmembrane mutation F182L, which has been identified in a pre-lingual progressive hearing loss patient in Taiwan, yielded cell surface expression and functional features that were similar to that of the wild type, suggesting that this mutation may represent redundant polymorphism. Collectively, these findings provide traces of the cellular mechanisms for DFNA2.     

Characterization of a novel,dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome

Andersen-Tawil syndrome is characterized by periodic paralysis, ventricular ectopy, and dysmorphic features. Approximately 60% of patients exhibit loss-of-function mutations in KCNJ2, which encodes the inwardly rectifying K+ channel pore forming subunit Kir2.1. Here, we report the identification of a novel KCNJ2 mutation (G211T), resulting in the amino acid substitution D71Y, in a patient presenting with signs and symptoms of Andersen-Tawil syndrome. The functional properties of the mutant subunit were characterized using voltage-clamp experiments on transiently transfected HEK-293 cells and neonatal mouse ventricular myocytes. Whole-cell current recordings of transfected HEK-293 cells demonstrated that the mutant protein Kir2.1-D71Y fails to form functional ion channels when expressed alone, but co-assembles with wild-type Kir2.1 subunits and suppresses wild-type subunit function. Further analysis revealed that current suppression requires at least two mutant subunits per channel. The D71Y mutation does not measurably affect the membrane trafficking of either the mutant or the wild-type subunit or alter the kinetic properties of the currents. Additional experiments revealed that expression of the mutant subunit suppresses native IK1 in neonatal mouse ventricular myocytes. Simulations predict that the D71Y mutation in human ventricular myocytes will result in a mild prolongation of the action potential and potentially increase cell excitability. These experiments indicate that the Kir2.1-D71Y mutant protein functions as a dominant negative subunit resulting in reduced inwardly rectifying K+ current amplitudes and altered cellular excitability in patients with Andersen-Tawil syndrome.     

Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import.

Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.     

Differential Behavior of Missense Mutations in the Intersubunit Contact Domain of the Human Pyruvate Kinase M2 Isozyme

In this study, we attempted to understand the mechanism of regulation of the activity and allosteric behavior of the pyruvate kinase M₂ enzyme and two of its missense mutations, H391Y and K422R, found in cells from Bloom syndrome patients, prone to develop cancer. Results show that despite the presence of mutations in the intersubunit contact domain, the K422R and H391Y mutant proteins maintained their homotetrameric structure, similar to the wild-type protein, but showed a loss of activity of 75 and 20%, respectively. Interestingly, H391Y showed a 6-fold increase in affinity for its substrate phosphoenolpyruvate and behaved like a non-allosteric protein with compromised cooperative binding. However, the affinity for phosphoenolpyruvate was lost significantly in K422R. Unlike K422R, H391Y showed enhanced thermal stability, stability over a range of pH values, a lesser effect of the allosteric inhibitor Phe, and resistance toward structural alteration upon binding of the activator (fructose 1,6-bisphosphate) and inhibitor (Phe). Both mutants showed a slight shift in the pH optimum from 7.4 to 7.0. Although this study signifies the importance of conserved amino acid residues in long-range communications between the subunits of multimeric proteins, the altered behavior of mutants is suggestive of their probable role in tumor-promoting growth and metabolism in Bloom syndrome patients with defective pyruvate kinase M₂.Pyruvate kinase (PK³; EC 2.7.1.40), a pacemaker of the glycolytic pathway, catalyzes irreversibly the transphosphorylation from P-enolpyruvate to ADP, generating pyruvate and ATP (1, 2). There are four different isozymes (L, R, M₁, and M₂) in mammalian tissues, which differ in their regulatory properties. These isozymes are allosteric in nature with the exception of the M₁ form, present in skeletal muscle and brain (3–7). PKM₂ is a ubiquitous prototype enzyme present in all tissues during the embryonic stage and is gradually replaced by other isozymic forms in specific tissues during development. The M₂, L, and R isozymes show homotropic cooperative activation with P-enolpyruvate and heterotropic cooperative activation with Fru-1,6-P₂ (8–10). The M₁ isozyme is regulated by neither P-enolpyruvate nor Fru-1,6-P₂ because of its intrinsic active conformation in the R-state (5, 6). Under unfavorable conditions such as hypoxia and lack of glucose supply, the anaerobic tissues and tumor cells rely heavily on PKM₂ for ATP production (7). Therefore, stringent control of PK activity is of great importance not only for cell metabolism but also for tumorigenic proliferation.The M₁ and M₂ isozymes are produced from a single gene locus by mutually exclusive alternative splicing (11–14). In the human M₁ and M₂ isozymes, the exon that is exchanged because of alternative splicing encodes 56 amino acids, in which a total of 22 amino acids differ within a length of 45 residues. The residues located in this region form the major intersubunit contact domain (8). The distinguishable kinetic properties of the M₁ and M₂ isozymes are attributed to these amino acid substitutions. It has been shown by x-ray crystallographic analyses and computer modeling that the corresponding regions of their polypeptides participate directly in the intersubunit contact, which is responsible for the intersubunit communication required for allosteric cooperativity (8, 15).PK has been largely conserved throughout evolution. The enzyme is usually a homotetramer composed of four identical subunits, and each subunit consists of four domains: the A-, B-, and C-domains and the N-terminal domain. The structure of human PKM₂ was recently determined in complex with inhibitors (16). In mammalian cells, PK activity is regulated by two different mechanisms: one at the level of expression and the other through allosteric regulation. The catalytic site usually composes a small part of the enzyme, but allosteric control is transmitted over a long range, thus increasing the number of possible residues involved in regulation. The allosteric transition in PK involves mutual rotations of the A- and C-domains within each subunit and the subunit within the tetramer (14). The residues at the subunit interfaces have the critical function of relaying the allosteric signal from and to the catalytic and regulatory sites. This region also transmits the allosteric signal between P-enolpyruvate- and Fru-1,6-P₂-binding sites. Despite the availability of structural details of several PK isozymes, it is difficult to identify the structural elements that play an important role in PK regulation and propagation of the allosteric signals. Although the role of some of the PK residues (positions 340, 389, 398, 401, 402, 408, 423, and 427) has been studied in allosteric regulation (10, 17–19) by in vitro site-directed mutagenesis, the absence of these mutations in any naturally occurring condition presents limitations in attributing a biological role to the introduced changes.The natural mutations H391Y and K422R (reported previously as K421R) were reported by us for the first time in the PKM₂ gene in a Bloom syndrome cell line and in the lymphocytes of an Indian Bloom syndrome patient, respectively (20). The two missense mutations, located in the region of the intersubunit contact domain (Fig. 1, A and B), presented with the biochemical phenotype of down-regulated enzyme activity to different extents (20) and were expected to influence the allosteric nature of the enzyme. The regulatory behavior of allosteric PK has been described by a two-state model that proposes an active (R) and an inactive (T) form of the macromolecule with differential affinity for ligands (15). Upon binding of the substrate or its analogs, the enzyme undergoes a transition from a low activity/low affinity conformation (T state) to a high activity/high affinity conformation (R state). The binding of phenylalanine produces a global structural change and exhibits reduced affinity for substrate P-enolpyruvate in the T state (21–23). Previous studies have demonstrated that each individual domain acts as a rigid body and that, upon transition from the T to the R state, the domain of the functional tetramer modifies its relative orientation by 29°. These movements bring conformational change to the active site, which, upon transition to the T state, undergoes a distortion of the P-enolpyruvate-binding site (24).Open in a separate windowFIGURE 1.A, ribbon diagram of the overall structure of PK showing the positions of the two mutations, H391Y and K422R, along with the active site and Fru-1,6-P₂-binding site. B, intersubunit contact domain of PK. The major amino acid residues and side chains at the tetramer interface region are shown.Because the mutations observed by us previously (20) are located at highly conserved positions not only in different isozymic forms but also across the species (supplemental Fig. S1) and are observed in the genetic background of a syndrome prone to cancer in early age, a study related to the structure-function correlations of these mutations is likely to provide insight into their possible biological importance, especially in the context of recent research highlighting the importance of PKM₂ in tumor promotion and growth. In this study, we investigated the role of the two natural missense mutations, after site-directed mutagenesis in the PKM₂ gene, in the regulation of allosteric properties as well as their effects on the secondary and tertiary structures in comparison with wild-type PKM₂ (PK-WT). An attempt has also been made to understand the effects of these mutations at the interface of the subunits on the signal transmission pathway within the protein.     

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival,Cell Invasion,Focal Adhesions,and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130^Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130^Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.     

Structural and signaling requirements for BCR-ABL-mediated transformation and inhibition of apoptosis.

BCR-ABL is a deregulated tyrosine kinase expressed in Philadelphia chromosome-positive human leukemias. Prolongation of hematopoietic cell survival by inhibition of apoptosis has been proposed to be an integral component of BCR-ABL-induced chronic myelogenous leukemia. BCR-ABL elicits transformation of both fibroblast and hematopoietic cells and blocks apoptosis following cytokine deprivation in various factor-dependent cells. To elucidate the mechanisms whereby BCR-ABL induces transformation and blocks apoptosis in hematopoietic cells, we examined the biological effects of expression of a series of BCR-ABL mutants. Single amino acid substitutions in the GRB2 binding site (Y177F), Src homology 2 domain (R552L), or an autophosphorylation site in the tyrosine kinase domain (Y793F) do not diminish the antiapoptotic and transforming properties of BCR-ABL in hematopoietic cells, although these mutations were previously shown to drastically reduce the transforming activity of BCR-ABL in fibroblasts. A BCR-ABL molecule containing all three mutations (Y177F/R552L/Y793F) exhibits a severe decrease in transforming and antiapoptotic activities compared with the wild-type BCR-ABL protein in 32D myeloid progenitor cells. Ras is activated, the SHC adapter protein is tyrosine phosphorylated and binds GRB2, and myc mRNA levels are increased following expression of all kinase active BCR-ABL proteins with the exception of the Y177F/R552L/Y793F BCR-ABL mutant in 32D cells. We propose that BCR-ABL uses multiple pathways to activate Ras in hematopoietic cells and that this activation is necessary for the transforming and antiapoptotic activities of BCR-ABL. However, Ras activation is not sufficient for BCR-ABL-mediated transformation. A BCR-ABL deletion mutant (delta 176-427) that activates Ras and blocks apoptosis but has severely impaired transforming ability in 32D cells has been identified. These data suggest that BCR-ABL requires additional signaling components to elicit tumorigenic growth which are distinct from those required to block apoptosis.     

Leukemia-associated mutations in SHIP1 inhibit its enzymatic activity, interaction with the GM-CSF receptor and Grb2, and its ability to inactivate PI3K/AKT signaling

The inositol 5-phosphatase SHIP1 is a negative regulator of the PI3K/AKT pathway, which is constitutively activated in 50-70% of acute myeloid leukemias (AML). Ten different missense mutations in SHIP1 have been described in 3% of AML patients suggesting a functional role of SHIP1 in AML. Here, we report the identification of two new SHIP1 mutations T162P and R225W that were detected in 2 and 1 out of 96 AML patients, respectively. The functional analysis of all 12 AML-associated SHIP1 mutations, one ALL-associated SHIP1 mutation (Q1076X) and a missense SNP (H1168Y) revealed that two mutations i.e. Y643H and P1039S abrogated the ability of SHIP1 to reduce constitutive PI3K/AKT signaling in Jurkat cells. The loss of function of SHIP1 mutant Y643H which is localized in the inositol phosphatase domain was due to a reduction of the specific activity by 84%. Because all other SHIP1 mutants had a normal enzymatic activity, we assumed that these SHIP1 mutants may be functionally impaired due to a loss of interaction with plasma membrane receptors or adapter proteins. In agreement with this model, we found that the SHIP1 mutant F28L located in the FLVR motif of the SH2 domain was incapable of binding tyrosine-phosphorylated proteins including the GM-CSF receptor and that the SHIP1 mutant Q1076X lost its ability to bind to the C-terminal SH3 domain of the adapter protein Grb2. In addition, SHIP1 mutant P1039S which does not reduce PI3K/AKT signaling anymore is located in a PXXP SH3 domain consensus binding motif suggesting that mutation of the conserved proline residue interferes with binding of SHIP1 to a so far unidentified SH3 domain containing protein. In summary, our data indicate that SHIP1 mutations detected in human leukemia patients impair the negative regulatory function of SHIP1 on PI3K/AKT signaling in leukemia cells either directly by reduced enzymatic activity or indirectly by disturbed protein interaction with tyrosine-phosphorylated membrane receptors or adapter proteins. These results further support a functional role of SHIP1 as tumor suppressor protein in the pathogenesis of AML.