Increased IGF-II protein affects p57kip2 expression in vivo and in vitro: Implications for Beckwith–Wiedemann syndrome

In both human and mouse, the Igf2 gene, localized on chromosomes 11 and 7, respectively, is expressed from the paternally inherited chromosome in the majority of tissues. Insulin-like growth factor-II (IGF-II) plays an important role in embryonic growth, and aberrant IGF2 expression has been documented in several human pathologies, such as Beckwith–Wiedemann syndrome (BWS), and a wide variety of tumors. Human and mouse genetic data strongly implicate another gene, CDKN1C (p57kip2), located in the same imprinted gene cluster on human chromosome II, in BWS. p57KIP2 is a cyclin-dependent kinase inhibitor and is required for normal mouse embryonic development. Mutations in CDKN1C (p57kip2) have been identified in a small proportion of patients with BWS, and removal of the gene from mice by targeted mutagenesis produces a phenotype with elements in common with this overgrowth syndrome. Patients with BWS with biallelic expression of IGF2 or with a CDKN1C (p57kip2) mutation, as well as overlapping phenotypes observed in two types of mutant mice, the p57kip2 knockout and IGF-II-overexpressing mice, strongly suggest that the genes may act in a common pathway of growth control in situations where Igf2 expression is abnormal. Herein, we show that p57kip2 expression is reduced on IGF-II treatment of primary embryo fibroblasts in a dose-dependent manner. In addition, p57kip2 expression is down-regulated in mice with high serum levels of IGF-II. These data suggest that the effects of increased IGF-II in BWS may, in part, be mediated through a decrease in p57kip2 gene expression.

c-Abl is required for development and optimal cell proliferation in the context of p53 deficiency

The c-Abl tyrosine kinase and the p53 tumor suppressor protein interact functionally and biochemically in cellular genotoxic stress response pathways and are implicated as downstream mediators of ATM (ataxia-telangiectasia mutated). This fact led us to study genetic interactions in vivo between c-Abl and p53 by examining the phenotype of mice and cells deficient in both proteins. c-Abl-null mice show high neonatal mortality and decreased B lymphocytes, whereas p53-null mice are prone to tumor development. Surprisingly, mice doubly deficient in both c-Abl and p53 are not viable, suggesting that c-Abl and p53 together contribute to an essential function required for normal development. Fibroblasts lacking both c-Abl and p53 were similar to fibroblasts deficient in p53 alone, showing loss of the G1/S cell-cycle checkpoint and similar clonogenic survival after ionizing radiation. Fibroblasts deficient in both c-Abl and p53 show reduced growth in culture, as manifested by reduction in the rate of proliferation, saturation density, and colony formation, compared with fibroblasts lacking p53 alone. This defect could be restored by reconstitution of c-Abl expression. Taken together, these results indicate that the ATM phenotype cannot be explained solely by loss of c-Abl and p53 and that c-Abl contributes to enhanced proliferation of p53-deficient cells. Inhibition of c-Abl function may be a therapeutic strategy to target p53-deficient cells selectively.

Cyclin A activates the DNA polymerase δ-dependent elongation machinery in vitro: A parvovirus DNA replication model

Replication of the single-stranded linear DNA genome of parvovirus minute virus of mice (MVM) starts with complementary strand synthesis from the 3′-terminal snap-back telomere, which serves as a primer for the formation of double-stranded replicative form (RF) DNA. This DNA elongation reaction, designated conversion, is exclusively dependent on cellular factors. In cell extracts, we found that complementary strand synthesis was inhibited by the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and rescued by the addition of proliferating cell nuclear antigen, arguing for the involvement of DNA polymerase (Pol) δ in the conversion reaction. In vivo time course analyses using synchronized MVM-infected A9 cells allowed initial detection of MVM RF DNA at the G1/S phase transition, coinciding with the onset of cyclin A expression and cyclin A-associated kinase activity. Under in vitro conditions, formation of RF DNA was efficiently supported by A9 S cell extracts, but only marginally by G1 cell extracts. Addition of recombinant cyclin A stimulated DNA conversion in G1 cell extracts, and correlated with a concomitant increase in cyclin A-associated kinase activity. Conversely, a specific antibody neutralizing cyclin A-dependent kinase activity, abolished the capacity of S cell extracts for DNA conversion. We found no evidence for the involvement of cyclin E in the regulation of the conversion reaction. We conclude that cyclin A is necessary for activation of complementary strand synthesis, which we propose as a model reaction to study the cell cycle regulation of the Pol δ-dependent elongation machinery.

Role of Syk in B-cell development and antigen-receptor signaling

Antigen receptors (BCRs) on developing B lymphocytes play two opposing roles—promoting survival of cells that may later bind a foreign antigen and inhibiting survival of cells that bind too strongly to self-antigens. It is not known how these opposing outcomes are signaled by BCRs on immature B cells. Here we analyze the effect of a null mutation in the Syk tyrosine kinase on maturing B cells displaying a transgene-encoded BCR that binds hen egg lysozyme (HEL). In the absence of HEL antigen, HEL-specific BCRs are expressed normally on the surface of Syk-deficient immature B-lineage cells, but this fails to promote maturation beyond the earliest stages of B-lineage commitment. Binding of HEL antigen, nevertheless, triggers phosphorylation of CD79α/β BCR subunits and modulation of receptors from the surface in Syk-deficient cells, but it cannot induce an intracellular calcium response. Continuous binding of low- or high-avidity forms of HEL, expressed as self-antigens, fails to restore the signal needed for maturation. Compared with the effects in the same system of null mutations in other BCR signaling elements, such as CD45 and Lyn kinase, these results indicate that Syk is essential for transmitting a signal that initiates the program of B-lymphocyte maturation.

Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate

P210 Bcr-Abl is an activated tyrosine kinase oncogene encoded by the Philadelphia chromosome associated with human chronic myelogenous leukemia (CML). The disease represents a clonal disorder arising in the pluripotent hematopoietic stem cell. During the chronic phase, patients present with a dramatic expansion of myeloid cells and a mild anemia. Retroviral gene transfer and transgenic expression in rodents have demonstrated the ability of Bcr-Abl to induce various types of leukemia. However, study of human CML or rodent models has not determined the direct and immediate effects of Bcr-Abl on hematopoietic cells from those requiring secondary genetic or epigenetic changes selected during the pathogenic process. We utilized tetracycline-regulated expression of Bcr-Abl from a promoter engineered for robust expression in primitive stem cells through multilineage blood cell development in combination with the in vitro differentiation of embryonal stem cells into hematopoietic elements. Our results demonstrate that Bcr-Abl expression alone is sufficient to increase the number of multipotent and myeloid lineage committed progenitors in a dose-dependent manner while suppressing the development of committed erythroid progenitors. These effects are reversible upon extinguishing Bcr-Abl expression. These findings are consistent with Bcr-Abl being the sole genetic change needed for the establishment of the chronic phase of CML and provide a powerful system for the analysis of any genetic change that alters cell growth and lineage choices of the hematopoietic stem cell.

Structural interactions of fibroblast growth factor receptor with its ligands

Fibroblast growth factors (FGFs) effect cellular responses by binding to FGF receptors (FGFRs). FGF bound to extracellular domains on the FGFR in the presence of heparin activates the cytoplasmic receptor tyrosine kinase through autophosphorylation. We have crystallized a complex between human FGF1 and a two-domain extracellular fragment of human FGFR2. The crystal structure, determined by multiwavelength anomalous diffraction analysis of the selenomethionyl protein, is a dimeric assemblage of 1:1 ligand:receptor complexes. FGF is bound at the junction between the two domains of one FGFR, and two such units are associated through receptor:receptor and secondary ligand:receptor interfaces. Sulfate ion positions appear to mark the course of heparin binding between FGF molecules through a basic region on receptor D2 domains. This dimeric assemblage provides a structural mechanism for FGF signal transduction.

Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (>30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock.

A human model for multigenic inheritance: Phenotypic expression in Hirschsprung disease requires both the RET gene and a new 9q31 locus

Reduced penetrance in genetic disorders may be either dependent or independent of the genetic background of gene carriers. Hirschsprung disease (HSCR) demonstrates a complex pattern of inheritance with ≈50% of familial cases being heterozygous for mutations in the receptor tyrosine kinase RET. Even when identified, the penetrance of RET mutations is only 50–70%, gender-dependent, and varies with the extent of aganglionosis. We searched for additional susceptibility genes which, in conjunction with RET, lead to phenotypic expression by studying 12 multiplex HSCR families. Haplotype analysis and extensive mutation screening demonstrated three types of families: six families harboring severe RET mutations (group I); and the six remaining families, five of which are RET-linked families with no sequence alterations and one RET-unlinked family (group II). Although the presence of RET mutations in group I families is sufficient to explain HSCR inheritance, a genome scan reveals a new susceptibility locus on 9q31 exclusively in group II families. As such, the gene at 9q31 is a modifier of HSCR penetrance. These observations imply that identification of new susceptibility factors in a complex disease may depend on classification of families by mutational type at known susceptibility genes.

In vitro hematopoietic and endothelial potential of flk-1−/− embryonic stem cells and embryos

Mice deficient in the Flk-1 receptor tyrosine kinase are known to die in utero because of defective vascular and hematopoietic development. Here, we show that flk-1−/− embryonic stem cells are nevertheless able to differentiate into hematopoietic and endothelial cells in vitro, although they give rise to a greatly reduced number of blast colonies, a measure of hemangioblast potential. Furthermore, normal numbers of hematopoietic progenitors are found in 7.5-day postcoitum flk-1−/− embryos, even though 8.5-day postcoitum flk-1−/− embryos are known to be deficient in such cells. Our results suggest that hematopoietic/endothelial progenitors arise independently of Flk-1, but that their subsequent migration and expansion require a Flk-1-mediated signal.

Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals

Various proteins with different biological activities have been observed to be translocated from the nucleus to the cytoplasm in an energy- and signal-dependent manner in eukaryotic cells. This nuclear export is directed by nuclear export signals (NESs), typically characterized by hydrophobic, primarily leucine, amino acid residues. Moreover, it has been shown that CRM1/exportin 1 is an export receptor for leucine-rich NESs. However, additional NES-interacting proteins have been described. In particular, eukaryotic initiation factor 5A (eIF-5A) has been shown to be a critical cellular cofactor for the nuclear export of the HIV type 1 (HIV-1) Rev trans-activator protein. In this study we compared the nuclear export activity of NESs of different origin. Microinjection of export substrates into the nucleus of somatic cells in combination with specific inhibitors indicated that specific nuclear export pathways exist for different NES-containing proteins. In particular, inhibition of eIF-5A blocked the nuclear export of NESs derived from the HIV-1 Rev and human T cell leukemia virus type I Rex trans-activators, whereas nucleocytoplasmic translocation of the protein kinase inhibitor-NES was unaffected. In contrast, however, inhibition of CRM1/exportin 1 blocked the nuclear export of all NES-containing proteins investigated. Our data confirm that CRM1/exportin 1 is a general export receptor for leucine-rich NESs and suggest that eIF-5A acts either upstream of CRM1/exportin 1 or forms a complex with the NES and CRM1/exportin 1 in the nucleocytoplasmic translocation of the HIV-1 Rev and human T cell leukemia virus type I Rex RNA export factors.

The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promoted cell survival and prevented DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptotic cell death. Time-course studies revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity.

Diffusible, nonfibrillar ligands derived from Aβ1–42 are potent central nervous system neurotoxins

Aβ1–42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer’s pathogenesis. Neurotoxicity of amyloid β protein (Aβ) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Aβ oligomers (referred to as ADDLs, for Aβ-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer’s disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer’s disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Aβ-derived diffusible ligands acting upon particular neural signal transduction pathways.

PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells

PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27KIP1 and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27KIP1 and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27KIP1.

Mode matches and their locations in the hydrophobic free energy sequences of peptide ligands and their receptor eigenfunctions

Patterns in sequences of amino acid hydrophobic free energies predict secondary structures in proteins. In protein folding, matches in hydrophobic free energy statistical wavelengths appear to contribute to selective aggregation of secondary structures in “hydrophobic zippers.” In a similar setting, the use of Fourier analysis to characterize the dominant statistical wavelengths of peptide ligands’ and receptor proteins’ hydrophobic modes to predict such matches has been limited by the aliasing and end effects of short peptide lengths, as well as the broad-band, mode multiplicity of many of their frequency (power) spectra. In addition, the sequence locations of the matching modes are lost in this transformation. We make new use of three techniques to address these difficulties: (i) eigenfunction construction from the linear decomposition of the lagged covariance matrices of the ligands and receptors as hydrophobic free energy sequences; (ii) maximum entropy, complex poles power spectra, which select the dominant modes of the hydrophobic free energy sequences or their eigenfunctions; and (iii) discrete, best bases, trigonometric wavelet transformations, which confirm the dominant spectral frequencies of the eigenfunctions and locate them as (absolute valued) moduli in the peptide or receptor sequence. The leading eigenfunction of the covariance matrix of a transmembrane receptor sequence locates the same transmembrane segments seen in n-block-averaged hydropathy plots while leaving the remaining hydrophobic modes unsmoothed and available for further analyses as secondary eigenfunctions. In these receptor eigenfunctions, we find a set of statistical wavelength matches between peptide ligands and their G-protein and tyrosine kinase coupled receptors, ranging across examples from 13.10 amino acids in acid fibroblast growth factor to 2.18 residues in corticotropin releasing factor. We find that the wavelet-located receptor modes in the extracellular loops are compatible with studies of receptor chimeric exchanges and point mutations. A nonbinding corticotropin-releasing factor receptor mutant is shown to have lost the signatory mode common to the normal receptor and its ligand. Hydrophobic free energy eigenfunctions and their transformations offer new quantitative physical homologies in database searches for peptide-receptor matches.

Cell locomotion and focal adhesions are regulated by substrate flexibility

Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.