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.     

De Novo Mutations in Ataxin-2 Gene and ALS Risk

Pathogenic CAG repeat expansion in the ataxin-2 gene (ATXN2) is the genetic cause of spinocerebellar ataxia type 2 (SCA2). Recently, it has been associated with Parkinsonism and increased genetic risk for amyotrophic lateral sclerosis (ALS). Here we report the association of de novo mutations in ATXN2 with autosomal dominant ALS. These findings support our previous conjectures based on population studies on the role of large normal ATXN2 alleles as the source for new mutations being involved in neurodegenerative pathologies associated with CAG expansions. The de novo mutations expanded from ALS/SCA2 non-risk alleles as proven by meta-analysis method. The ALS risk was associated with SCA2 alleles as well as with intermediate CAG lengths in the ATXN2. Higher risk for ALS was associated with pathogenic CAG repeat as revealed by meta-analysis.     

PDCD10 Gene Mutations in Multiple Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.     

FOXC2 Mutations in Familial and Sporadic Spinal Extradural Arachnoid Cyst

Spinal extradural arachnoid cyst (SEDAC) is a cyst in the spinal canal that protrudes into the epidural space from a defect in the dura mater. Most cases are sporadic; however, three familial SEDAC cases have been reported, suggesting genetic etiological factors. All familial cases are associated with lymphedema-distichiasis syndrome (LDS), whose causal gene is FOXC2. However, FOXC2 mutation analysis has been performed in only 1 family, and no mutation analysis has been performed on sporadic (non-familial) SEDACs. We recruited 17 SEDAC subjects consisting of 2 familial and 7 sporadic cases and examined FOXC2 mutations by Sanger sequencing and structural abnormalities by TaqMan copy number assay. We identified 2 novel FOXC2 mutations in 2 familial cases. Incomplete LDS penetrance was noted in both families. Four subjects presented with SEDACs only. Thus, SEDAC caused by the heterozygous FOXC2 loss-of-function mutation should be considered a feature of LDS, although it often manifests as the sole symptom. Seven sporadic SEDAC subjects had no FOXC2 mutations, no symptoms of LDS, and showed differing clinical characteristics from those who had FOXC2 mutations, suggesting that other gene(s) besides FOXC2 are likely to be involved in SEDAC.     

Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly

The Caenorhabditis elegans unc-45 locus has been proposed to encode a protein machine for myosin assembly. The UNC-45 protein is predicted to contain an NH₂-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p. CRO1 and She4p are fungal proteins required for the segregation of other molecules in budding, endocytosis, and septation. Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residues within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent translation of the COOH-terminal domain. Electron microscopy shows that thick filament accumulation in vivo is decreased by ∼50% in the CB286 ts mutant grown at the restrictive temperature. The thick filaments that assemble have abnormal structure. Immunofluorescence and immunoelectron microscopy show that myosins A and B are scrambled, in contrast to their assembly into distinct regions at the permissive temperature and in wild type. This abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortened lengths upon isolation. These results implicate the UNC-45 CRO1/She4p-like region in the assembly of myosin isoforms in C. elegans and suggest a possible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) protein family.     

De Novo Mutations in Moderate or Severe Intellectual Disability

Genetics is believed to have an important role in intellectual disability (ID). Recent studies have emphasized the involvement of de novo mutations (DNMs) in ID but the extent to which they contribute to its pathogenesis and the identity of the corresponding genes remain largely unknown. Here, we report a screen for DNMs in subjects with moderate or severe ID. We sequenced the exomes of 41 probands and their parents, and confirmed 81 DNMs affecting the coding sequence or consensus splice sites (1.98 DNMs/proband). We observed a significant excess of de novo single nucleotide substitutions and loss-of-function mutations in these cases compared to control subjects, suggesting that at least a subset of these variations are pathogenic. A total of 12 likely pathogenic DNMs were identified in genes previously associated with ID (ARID1B, CHD2, FOXG1, GABRB3, GATAD2B, GRIN2B, MBD5, MED13L, SETBP1, TBR1, TCF4, WDR45), resulting in a diagnostic yield of ∼29%. We also identified 12 possibly pathogenic DNMs in genes (HNRNPU, WAC, RYR2, SET, EGR1, MYH10, EIF2C1, COL4A3BP, CHMP2A, PPP1CB, VPS4A, PPP2R2B) that have not previously been causally linked to ID. Interestingly, no case was explained by inherited mutations. Protein network analysis indicated that the products of many of these known and candidate genes interact with each other or with products of other ID-associated genes further supporting their involvement in ID. We conclude that DNMs represent a major cause of moderate or severe ID.     

OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency

Background

Studies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.

Results

We identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level).Obscurin levels in DCM controls, donor heart and myectomy samples were the same.

Conclusions

OBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations.     

Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences

The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776–782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The β-aggregation predictor TANGO found highly β-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were β-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High β-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.Protein amyloids are characteristic of pathological conditions, including neurodegenerative diseases (4, 11, 17, 38). These protein aggregates can also occur naturally in adhesive bacterial curli (3), melanosomes (14), condensed peptide hormone arrays (24), as regulatory prions in yeast (2, 5), and fungal hydrophobins, which are nonantigenic coats to some fungi (1, 33, 39). Nevertheless, such natural occurrences are relatively few, considering the negative free energy for amyloid formation (28).We have recently discovered that there are amyloid-forming sequences in the cell surface Als adhesins of Candida albicans. Cells that express these adhesins aggregate readily, and the aggregation has amyloid-like properties, including protein conformational shifting, surface birefringence, and ability to bind the amyloid-active dyes Congo red and amino-naphthalene sulfonic acid (ANS) (29). A five- to seven-residue sequence in Als1p, Als3p, and Als5p has extremely high potential for formation of β-aggregates, according to the protein state prediction program TANGO (13, 27, 31). Such β-aggregates include amyloids, which are ordered structures with paracrystalline regions of stacked parallel β-strands that are perpendicular to the long axis of micrometer-long fibrils. The strands are stabilized by interaction of identical sequences from many protein molecules (31, 32). Where TANGO analyses have shown that specific sequences have β-aggregate potentials greater than 20%, an insoluble β-aggregate state is likely to form. These β-aggregates nucleate formation of amyloids if the proteins can associate to form fibers (13, 27, 31). Sequences in the conserved 127-residue T region of Als1p, Als3p, and Als5p have β-aggregation potentials of >90% (27). An oligopeptide with this sequence, as well as 412- and 645-residue fragments of Als5p formed authentic amyloids, as determined by characteristic dye binding and fiber morphology. The amyloid-forming sequences were rich in the β-branched amino acids Thr, Val, and Ile. This amino acid composition is unusual among proteins in general, but is common in the Thr-rich mid-piece domains of yeast adhesins.Yeasts display many cell-wall-bound adhesins that mediate colonial and biofilm interactions as well as host-pathogen binding (9, 21, 41). Such adhesins have a common mosaic structure. In general, the adhesins have N-terminal globular binding domains (often immunoglobulin-like or lectin-like), Thr-rich mid-piece sequences including tandem repeats, and 300- to 800-residue heavily glycosylated Ser and Thr-rich “stalk” domains near the C-terminal domain that extend the active regions from the surface of the wall. The adhesins are covalently cross-linked to wall polysaccharides through modified glycosylphosphatidylinositol (GPI) anchors and/or glycosyl esters of glutamic acid (9, 18).Because the yeast adhesins share this common modular domain structure, we searched among known and putative yeast adhesins for sequences with high β-aggregation potential. We have found that many of these proteins share amyloid-forming sequences and amyloid-like behavior on activation.     

WD Repeat Domain of Dictyostelium Myosin Heavy Chain Kinase C Functions in both Substrate Targeting and Cellular Localization

Myosin II disassembly in Dictyostelium discoideum is regulated by three structurally related myosin heavy chain kinases (myosin II heavy chain kinase A [MHCK-A], -B, and -C). We show that the WD repeat domain of MHCK-C is unique in that it mediates both substrate targeting and subcellular localization, revealing a target for regulation that is distinct from those of the other MHCKs.The ability of a cell to undergo highly specific modifications in shape during processes such as cytokinesis, cell migration, cell adhesion, and receptor capping is dependent, in large part, on the proper control of where and when myosin II contracts actin filaments in the cell (3, 4). In Dictyostelium discoideum, myosin II filament disassembly is regulated by at least three myosin II heavy chain kinases (myosin II heavy chain kinase A [MHCK-A], MHCK-B, and MHCK-C). The Dictyostelium MHCKs possess alpha kinase domains and carboxyl-terminal WD repeat domains (11, 13, 17). The WD repeat domains of MHCK-A and MHCK-B facilitate myosin II heavy chain phosphorylation by these kinases by binding directly to myosin II filaments (14, 15). However, the WD repeat domains play no detectable role in determining the subcellular localization of these kinases. Similar functions for the WD repeat domain of MHCK-C have not been explored, and there is nothing known about the signaling events regulating MHCK-C localization and activity, thus limiting comparisons among the MHCKs that could ultimately reveal distinct functions and mechanisms of regulation for these seemingly redundant enzymes.     

Schistosoma japonicum Eggs Induce a Proinflammatory,Anti-Fibrogenic Phenotype in Hepatic Stellate Cells

Hepatic fibrosis induced by egg deposition is the most serious pathology associated with chronic schistosomiasis, in which the hepatic stellate cell (HSC) plays a central role. While the effect of Schistosoma mansoni eggs on the fibrogenic phenotype of HSCs has been investigated, studies determining the effect of eggs of S . japonicum on HSCs are lacking. Disease caused by S . japonicum is much more severe than that resulting from S. mansoni infection so it is important to compare the pathologies caused by these two parasites, to determine whether this phenotype is due to the species interacting differently with the mammalian host. Accordingly, we investigated the effect of S . japonicum eggs on the human HSC cell line, LX-2, with and without TGF-β (Transforming Growth Factor beta) co-treatment, so as to determine the impact on genes associated with fibrogenesis, inflammation and matrix re-organisation. Activation status of HSCs was assessed by αSMA (Alpha Smooth Muscle Actin) immunofluorescence, accumulation of Oil Red O-stained lipid droplets and the relative expression of selected genes associated with activation. The fibrogenic phenotype of HSCs was inhibited by the presence of eggs both with or without TGF-β treatment, as evidenced by a lack of αSMA staining and reduced gene expression of αSMA and Col1A1 (Collagen 1A1). Unlike S. mansoni-treated cells, however, expression of the quiescent HSC marker PPAR-γ (Peroxisome Proliferator-Activated Receptor gamma) was not increased, nor was there accumulation of lipid droplets. In contrast, S . japonicum eggs induced the mRNA expression of MMP-9 (Matrix Metalloproteinase 9), CCL2 (Chemokine (C-C motif) Ligand 2) and IL-6 (Interleukin 6) in HSCs indicating that rather than inducing complete HSC quiescence, the eggs induced a proinflammatory phenotype. These results suggest HSCs in close proximity to S . japonicum eggs in the liver may play a role in the proinflammatory regulation of hepatic granuloma formation.     

Effect of Starvation on the Endocytic Pathway in Dictyostelium Cells

Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.Dictyostelium discoideum is a widely used model organism for studying the organization and function of the endocytic pathway. In Dictyostelium, the organization of the endocytic pathway is similar to that in higher eukaryotes. The pathway in Dictyostelium can be divided into four steps (see Fig. S1 in the supplemental material): uptake at the plasma membrane of particles and medium, transfer through early acidic endocytic compartments (lysosomes), passage into less acidic postlysosomes (PLs), and finally, exocytosis of undigested materials (17, 20). Thus, Dictyostelium recapitulates many of the functions of the endocytic pathway in mammalian cells, including some features observed in most cell types (lysosome biogenesis) and some observed only in specialized cells (phagocytosis, macropinocytosis, and lysosome secretion).Dictyostelium amoebae live in the soil, where they feed by ingesting and digesting other microorganisms. In addition, axenic laboratory strains can macropinocytose medium to ensure their growth. Accordingly, both in natural situations and in laboratory settings, the endocytic pathway plays a key role in the acquisition of nutrients by Dictyostelium cells. In agreement with this notion, several observations suggest that the physiology of the endocytic pathway is sensitive to nutrient availability. In particular, starvation induces secretion of lysosomal enzymes by an unknown mechanism (11). The morphology of the endocytic pathway is also sensitive to nutritional cues, as shown for example by the observation that formation of multilamellar endosomes is enhanced in cells fed with bacteria (18).Here, we analyzed the effect of starvation on the organization as well as the dynamics of the endocytic pathway. We found that, while the overall organization was not extensively modified in starved cells, the dynamics of endocytic compartments were altered. Moreover, analysis of two specific knockout mutants, the apm3 (6) and lvsB (8) strains, revealed that their phenotype was profoundly altered upon starvation, providing further insight about the role of Apm3 and LvsB in the endocytic pathway.     

U2AF1 Mutations in Chinese Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome

Somatic mutations of U2AF1 gene have recently been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we analyzed the frequency and clinical impact of U2AF1 mutations in a cohort of 452 Chinese patients with myeloid neoplasms. Mutations in U2AF1 were found in 2.5% (7/275) of AML and 6.3% (6/96) of MDS patients, but in none of 81 CML. All mutations were heterozygous missense mutations affecting codon S34 or Q157. There was no significant association of U2AF1 mutation with blood parameters, FAB subtypes, karyotypes and other gene mutations in AML. The overall survival (OS) of AML patients with U2AF1 mutation (median 3 months) was shorter than those without mutation (median 7 months) (P = 0.035). No difference in the OS was observed between MDS patients with and without U2AF1 mutations. Our data show that U2AF1 mutation is a recurrent event at a low frequency in AML and MDS.     

Noonan Syndrome/Leukemia-associated Gain-of-function Mutations in SHP-2 Phosphatase (PTPN11) Enhance Cell Migration and Angiogenesis

Mutations in SHP-2 phosphatase (PTPN11) that cause hyperactivation of its catalytic activity have been identified in Noonan syndrome and various childhood leukemias. Recent studies suggest that the gain-of-function (GOF) mutations of SHP-2 play a causal role in the pathogenesis of these diseases. However, the molecular mechanisms by which GOF mutations of SHP-2 induce these phenotypes are not fully understood. Here, we show that GOF mutations in SHP-2, such as E76K and D61G, drastically increase spreading and migration of various cell types, including hematopoietic cells, endothelial cells, and fibroblasts. More importantly, in vivo angiogenesis in SHP-2 D61G knock-in mice is also enhanced. Mechanistic studies suggest that the increased cell migration is attributed to the enhanced β1 integrin outside-in signaling. In response to β1 integrin cross-linking or fibronectin stimulation, activation of ERK and Akt kinases is greatly increased by SHP-2 GOF mutations. Also, integrin-induced activation of RhoA and Rac1 GTPases is elevated. Interestingly, mutant cells with the SHP-2 GOF mutation (D61G) are more sensitive than wild-type cells to the suppression of cell motility by inhibition of these pathways. Collectively, these studies reaffirm the positive role of SHP-2 phosphatase in cell motility and suggest a new mechanism by which SHP-2 GOF mutations contribute to diseases.SHP-2, a multifunctional SH2 domain-containing protein-tyrosine phosphatase implicated in diverse cell signaling processes (1–3), plays a critical role in cellular function. Homozygous deletion of Exon 2 (4) or Exon 3 (5) of the SHP-2 gene (PTPN11) in mice leads to early embryonic lethality prior to and at midgestation, respectively. SHP-2 null mutant mice die much earlier, at peri-implantation (4). Exon 3 deletion mutation of SHP-2 blocks hematopoietic potential of embryonic stem cells both in vitro and in vivo (6–8), whereas SHP-2 null mutation causes inner cell mass death and diminished trophoblast stem cell survival (4). Recent studies on SHP-2 conditional knock-out or tissue-specific knock-out mice have further revealed an array of important functions of this phosphatase in various physiological processes (9–12). The phenotypes demonstrated by loss of SHP-2 function are apparently attributed to the role of SHP-2 in the cell signaling pathways induced by growth factors/cytokines. SHP-2 generally promotes signal transmission in growth factor/cytokine signaling in both catalytic-dependent and -independent fashion (1–3). The positive role of SHP-2 in the intracellular signaling processes, in particular, the ERK³ and PI3K/Akt kinase pathways, has been well established, although the underlying mechanism remains elusive, in particular, the signaling function of the catalytic activity of SHP-2 in these pathways is poorly understood.In addition to the role of SHP-2 in cell proliferation and differentiation, the phenotypes induced by loss of SHP-2 function may be associated with its role in cell migration. Indeed, dominant negative SHP-2 disrupts Xenopus gastrulation, causing tail truncations (13, 14). Targeted Exon 3 deletion mutation in SHP-2 results in decreased cell spreading, migration (15, 16), and impaired limb development in the chimeric mice (7). The role of SHP-2 in cell adhesion and migration has also been demonstrated by catalytically inactive mutant SHP-2-overexpressing cells (17–20). The molecular mechanisms by which SHP-2 regulates these cellular processes, however, have not been well defined. For example, the role of SHP-2 in the activation of the Rho family small GTPases that is critical for cell motility is still controversial. Both positive (19, 21, 22) and negative roles (18, 23) for SHP-2 in this context have been reported. Part of the reason for this discrepancy might be due to the difference in the cell models used. Catalytically inactive mutant SHP-2 was often used to determine the role of SHP-2 in cell signaling. In the catalytically inactive mutant SHP-2-overexpressing cells, the catalytic activity of endogenous SHP-2 is inhibited. However, as SHP-2 also functions independent of its catalytic activity, overexpression of catalytically deficient SHP-2 may also increase its scaffolding function, generating complex effects.The critical role of SHP-2 in cellular function is further underscored by the identification of SHP-2 mutations in human diseases. Genetic lesions in PTPN11 that cause hyperactivation of SHP-2 catalytic activity have been identified in the developmental disorder Noonan syndrome (24) and various childhood leukemias, including juvenile myelomonocytic leukemia (JMML), B cell acute lymphoblastic leukemia, and acute myeloid leukemia (25, 26). In addition, activating mutations in SHP-2 have been identified in sporadic solid tumors (27). The SHP-2 mutations appear to play a causal role in the development of these diseases as SHP-2 mutations and other JMML-associated Ras or Neurofibromatosis 1 mutations are mutually exclusive in the patients (24–27). Moreover, single SHP-2 gain-of-function (GOF) mutations are sufficient to induce Noonan syndrome, cytokine hypersensitivity in hematopoietic progenitor cells, and JMML-like myeloproliferative disease in mice (28–32). Gain-of-function cell models derived from the newly available SHP-2 GOF mutation (D61G) knock-in mice (28) now provide us with a good opportunity to clarify the role of SHP-2 in cell motility. Unlike the dominant negative approach in which overexpression of mutant forms of SHP-2 generates complex effects, the SHP-2 D61G knock-in model eliminates this possibility as the mutant SHP-2 is expressed at the physiological level (28). Additionally, defining signaling functions of GOF mutant SHP-2 in cell movement can also help elucidate the molecular mechanisms by which SHP-2 mutations contribute to the relevant diseases.