scholarly journals Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

2002 ◽  
Vol 13 (4) ◽  
pp. 1190-1202 ◽  
Author(s):  
Hélène Defacque ◽  
Evelyne Bos ◽  
Boyan Garvalov ◽  
Cécile Barret ◽  
Christian Roy ◽  
...  
Keyword(s):  
Binding Sites ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Membrane Surface ◽  
Latex Bead ◽  
Actin Assembly ◽  
Wild Type ◽  
Membrane Surfaces ◽  
Organelle Membrane

Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P2-binding proteins ezrin and/or moesin were essential for this process ( Defacque et al., 2000b ). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P2, and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P2-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.

scholarly journals Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

2004 ◽  
Vol 48 (11) ◽  
pp. 4154-4162 ◽  
Author(s):  
Thomas Herget ◽  
Martina Freitag ◽  
Monika Morbitzer ◽  
Regina Kupfer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Human Fibroblasts ◽  
Growth Factor Receptor ◽  
Protein Kinase Activity ◽  
Hcmv Replication

ABSTRACT Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC50s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC50s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 μM, respectively) were in the range of the IC50 of ganciclovir (1.2 ± 0.2 μM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

Arabidopsis MLK3, a Plant-specific Casein Kinase 1, Negatively Regulated Flowering and Phosphorylated Histone H3 in vivo

2019 ◽  
Author(s):  
Zhen Wang ◽  
Junmei Kang ◽  
Shangang Jia ◽  
Tiejun Zhang ◽  
Zhihai Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Histone H3 ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Altered Expression ◽  
Casein Kinase 1 ◽  
Threonine Kinase

Abstract Background: Casein kinase 1 (CK1) family members are highly conserved serine/threonine kinase present in most eukaryotes with multiple biological functions. Arabidopsis MUT9-like kinases ( MLKs ) belong to a clade CK1 specific to the plant kingdom and have been implicated collectively in modulating flowering related processes. Three of the four MLKs ( MLK1/2/4 ) have been characterized, however, little is known about MLK3 , the most divergent MLKs. Results: We demonstrated that compared with wild type, mlk3 , a truncated MLK3 , flowered slightly early under long day conditions and ectopic expression of MLK3 rescued the morphological defects of mlk3 , indicating that MLK3 negatively regulates flowering. GA 3 application accelerated flowering of both wild type and mlk3 , suggesting that mlk3 had normal GA response. The recombinant MLK3-GFP was localized in the nucleus exclusively. In vitro kinase assay revealed that the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3ph). Mutation of a conserved catalytic residue (Lysine 175) abolished the kinase activity and resulted in failure to complement the early flowering phenotype of mlk3 . Interestingly, the global level of H3T3 phosphorylation in mlk3 did not differ significantly from wild type, suggesting the redundant roles of MLKs in flowering regulation. The transcriptomic analysis demonstrated that 425 genes significantly altered expression level in mlk3 relative to wild type. The mlk3 mlk4 double mutant generated by crossing mlk3 with mlk4 , a loss-of-function mutant of MLK4 showing late flowering, flowered between the two parental lines, suggesting that MLK3 played an antagonistic role to MLK4 in plant transition to flowering. Conclusions: A serine/threonine kinase encoding gene MLK3 is a casein kinase 1 specific to the plant species and represses flowering slightly. MLK3 located in nucleus catalyzes the phosphorylation of histone H3 at threonine 3 in vitro and an intact lysine residue (K175) is indispensible for the kinase activity. This study sheds new light on the delicate control of flowering by the plant-specific CK1 in Arabidopsis.

The characterization of ligand-specific maize (Zea mays) profilin mutants

2001 ◽  
Vol 358 (1) ◽  
pp. 49-57 ◽  
Author(s):  
David R. KOVAR ◽  
Bj⊘rn K. DRØBAK ◽  
David A. COLLINGS ◽  
Christopher J. STAIGER
Keyword(s):  
Zea Mays ◽  
Hair Cells ◽  
Plant Cells ◽  
Single Amino Acid ◽  
Actin Assembly ◽  
Wild Type ◽  
Ligand Interaction ◽  
Binding Partners

Profilins are low-molecular-mass (12–15kDa) cytosolic proteins that are major regulators of actin assembly in all eukaryotic cells. In general, profilins from evolutionarily diverse organisms share the ability to bind to G-actin, poly-(l-proline) (PLP) and proline-rich proteins, and polyphosphoinositides. However, the functional importance of each of these interactions remains unclear and might differ between organisms. We investigated the importance of profilin's interaction with its various ligands in plant cells by characterizing four maize (Zea mays) profilin 5 (ZmPRO5) mutants that had single amino acid substitutions in the presumed sites of ligand interaction. Comparisons in vitro with wild-type ZmPRO5 showed that these mutations altered ligand association specifically. ZmPRO5-Y6F had a 3-fold increased affinity for PLP, ZmPRO5-Y6Q had a 5-fold decreased affinity for PLP, ZmPRO5-D8A had a 2-fold increased affinity for PtdIns(4,5)P2 and ZmPRO5-K86A had a 35-fold decreased affinity for G-actin. When the profilins were microinjected into Tradescantia stamen hair cells, ZmPRO5-Y6F increased the rate of nuclear displacement in stamen hairs, whereas ZmPRO5-K86A decreased the rate. Mutants with a decreased affinity for PLP (ZmPRO5-Y6Q) or an enhanced affinity for PtdIns(4,5)P2 (ZmPRO5-D8A) were not significantly different from wild-type ZmPRO5 in affecting nuclear position. These results indicate that plant profilin's association with G-actin is extremely important and further substantiate the simple model that profilin acts primarily as a G-actin-sequestering protein in plant cells. Furthermore, interaction with proline-rich binding partners might also contribute to regulating profilin's effect on actin assembly in plant cells.

scholarly journals Drosophila DBT Lacking Protein Kinase Activity Produces Long-Period and Arrhythmic Circadian Behavioral and Molecular Rhythms

2007 ◽  
Vol 27 (23) ◽  
pp. 8049-8064 ◽  
Author(s):  
Michael J. Muskus ◽  
Fabian Preuss ◽  
Jin-Yuan Fan ◽  
Edward S. Bjes ◽  
Jeffrey L. Price
Keyword(s):  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Wild Type ◽  
Endogenous Gene ◽  
Long Period ◽  
Short Period ◽  
Lower Expression ◽  
General Reduction

ABSTRACT A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBTK/R) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBTK/R antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered “set point” for period length that is not altered by overexpression. Overexpression of the DBTK/R produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.

The Rho Family GTPase Cdc42 Regulates Multiple Hematopoietic Stem/Progenitor Cell Functions and Erythropoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 32-32
Author(s):  
Lei Wang ◽  
Linda Yang ◽  
Marie–Dominique Filippi ◽  
David A. Williams ◽  
Yi Zheng
Keyword(s):  
Bone Marrow ◽  
Fetal Liver ◽  
Brdu Incorporation ◽  
Low Density ◽  
Actin Assembly ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Rho Family

Abstract The Rho family GTPase Cdc42 has emerged as a key signal transducer in cell regulation. To investigate its physiologic function in hematopoiesis, we have generated mice carrying a gene targeted null allele of cdc42gap, a major negative regulatory gene of Cdc42 and mice with conditional targeted cdc42 allele (cdc42flox/flox). Deletion of the respective gene products in mice was confirmed by PCR genotyping and Western blotting. Low-density fetal liver or bone marrow cells from Cdc42GAP−/− mice displayed ~3 fold elevated Cdc42 activity and normal RhoA, Rac1 or Rac2 activity, indicating that cdc42gap deletion has a specific effect on Cdc42 activity. The Cdc42GAP-deficient hematopoietic stem/progenitor cells (HSC/Ps, Lin−c-Kit+) generated from Cdc42GAP−/− E14.5 fetal liver and the Cdc42−/− HSC/Ps derived by in vitro expression of Cre via a retrovirus vector from Cdc42flox/flox low density bone marrow showed a growth defect in liquid culture that was associated with increased apoptosis but normal cell cycle progression. Cdc42GAP-deficient HSC/Ps displayed impaired cortical F-actin assembly with extended actin protrusions upon exposure to SDF–1 in vitro and a punctuated actin structure after SCF stimulation while Cdc42−/− but not wild type HSC/Ps responded to SDF-1 in inducing membrane protrusions. Both Cdc42−/− and Cdc42GAP−/− HSC/Ps were markedly decreased in adhesion to fibronectin. Moreover, both Cdc42−/− and Cdc42GAP−/− HSC/Ps showed impaired migration in response to SDF-1. These results demonstrate that Cdc42 regulation is essential for multiple HSC/P functions. To understand the in vivo hematopoietic function of Cdc42, we have characterized the Cdc42GAP−/− mice further. The embryos and newborns of homozygous showed a ~30% reduction in hematopoietic organ (i.e. liver, bone marrow, thymus and spleen) cellularity, consistent with the reduced sizes of the animals. This was attributed to the increased spontaneous apoptosis associated with elevated Cdc42/JNK/Bid activities but not to a proliferative defect as revealed by in vivo TUNEL and BrdU incorporation assays. ~80% of Cdc42GAP−/− mice died one week after birth, and the surviving pups attained adulthood but were anemic. Whereas Cdc42GAP−/− mice contained small reduction in the frequency of HSC markers and normal CFU-G, CFU-M, and CFU-GM activities, the frequency of BFU-E and CFU-E were significantly reduced. These results suggest an important role of Cdc42 in erythropoiesis in vivo. Taken together, we propose that Cdc42 is essential for multiple HSC/P functions including survival, actin cytoskeleton regulation, adhesion and migration, and that deregulation of its activity can have a significant impact on erythropoiesis. Cdc42 regulates HSC/P functions and erythropoiesis Genotype/phenotype Apoptosis increase Adhesion decrease Migration decrease F-actin assembly HSC frequency decrease BFU-E, CFU-E decrease The numbers were indicated as fold difference compared with wild type. ND:not determined yet. Cdc42GAP−/− 2.43, p<0.005 0.97, p<0.01 1.01, p<0.01 protrusion (SDF-1); punctruated (SCF) 0.34, p<0.05 0.92, p<0.01; 0.38, p<0 Cdc42−/− 3.68, p<0.005 0.98, p<0.001 3.85, p<0.005 protrusion (SDF-1) ND ND

A Conserved Mechanism of Transcription Factor Competition Controls Hematopoietic Progenitor Self-Renewal.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 91-91
Author(s):  
Shane R. Horman ◽  
Chinamenveni S. Velu ◽  
Tristan Bourdeau ◽  
Avinash Baktula ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Conditional Deletion

Abstract An intrinsic mechanism of self-renewal is critical for the maintenance of hematopoietic stem cells (HSC), but this HSC function is extinguished during differentiation of progenitors. Here we show that the self-renewal capacity of hematopoietic progenitor cells is regulated through physical competition for occupancy of select DNA binding sites. Initially, we found that conditional deletion of the Growth factor independent-1 (Gfi1) gene results in the accumulation of abnormally persistent myeloid progenitors in vivo. Specifically, while germline Gfi1 deletion induces defective HSC self renewal and a block to granulopoiesis, we find that conditional deletion of Gfi1 induces a severe but transient block to neutrophil development with repopulation of the bone marrow by the remaining wild type HSC within 8 weeks post deletion. However, even though normal levels of granulocyte colony forming units (G-CFU) returned by 8 weeks post deletion, an abnormal Gfi1−/− myeloid progenitor remained in the bone marrow in vivo. Subsequently, we find in vitro that both wild-type bone marrow cells expressing Gfi1-dominant-negative mutants, and Gfi1−/− Lin- bone marrow contain cells that replate indefinitely. We hypothesized that Gfi1 is critical to extinguish self renewal in hematopoietic progenitors. In seemingly unrelated work, we discovered antagonism between the drosophila orthologs of Gfi1 and the Hoxa9/Pbx1/Meis1 transcription factor complex during drosophila embryo segmentation. We extended our drosophila findings to discover that a subset of mammalian DNA regulatory sequences encode DNA binding sites for both Gfi1 and Hoxa9/Pbx1/Meis1. These DNA sequences are able to bind either factor, and function as a molecular switch. Interestingly, composite Gfi1/ Hoxa9/Pbx1/Meis1 binding sites are present in the regulatory regions of the gene encoding Hoxa9. We note that Gfi1 expression is normally induced, while Hoxa9 expression is down-regulated, during the transition from common myeloid progenitor (CMP) to the granulocyte-monocyte progenitor (GMP). CMP have greater self renewal potential than GMP. Conditional deletion of Gfi1 in sorted CMP or GMP both increases Hoxa9 expression and generates progenitors capable of replating indefinitely in vitro. Thus, Gfi1 is critical to limit self renewal in these progenitors. Deregulated Hoxa9 expression or activity appears pivotal to this new Gfi1-null phenotype, because Gfi1 dominant-negative mutants immortalize wild-type (or Hoxa7−/−) but not Hoxa9−/− bone marrow cells in vitro. An abnormal gain of self-renewal can unleash the leukemic potential of progenitor cells. We find that both limiting Gfi1 gene dosage and expression of Gfi1 dominant-negative mutants significantly increases Nup98-Hoxa9-mediated colony formation. In contrast, forced expression of Gfi1 prevents Nup98-Hoxa9 immortalization. Notably, the expression of Hoxa9 (independent of cases with Nup98-Hoxa9 fusions) has been reported to be of significant prognostic value in human acute myeloid leukemia. In conclusion, Gfi1 and the Hoxa9/Pbx1/Meis1 complex compete to control the expression of genes (such as Hoxa9) which are critical to extinguish self renewal and limit the leukemogenic potential of hematopoietic progenitors. The antagonism between these transcription factor complexes is conserved from drosophila segment formation to mammalian hematopoietic progenitor biology.

A Novel ENU-Induced Intronic Mutation of Ank1 Causing Quantitative Defect of Ankyrin-1 Models Hemolytic Hereditary Spherocytosis in Mouse

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 989-989
Author(s):  
Xin Du ◽  
Hua Huang ◽  
Lara Krieg
Keyword(s):  
Cell Biology ◽  
Hereditary Spherocytosis ◽  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Regulatory Region ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Intronic Mutation ◽  
Vertical Linkages

Abstract Abstract 989 The ability of red blood cells (RBCs) to maintain the surface area and deformability is vital for their survival. The maintenance of membrane surface is dependent upon the strong cohesion between the lipid bilayer and the skeletal network, achieved by vertical linkages between transmembrane proteins and spectrin tetramers. Mutations causing functional deficiencies in these proteins have been identified in various hemolytic anemias. Here we reported a mild hereditary spherocytosis (HS) and hemolytic anemia phenotype in mouse, named hema6, induced by N-ethyl-N-nitrosourea (ENU) mutagenesis. Hema6 phenotype is transmitted as a semidominant trait as heterozygous mice are less severely affected than homozygotes. The causal mutation was traced to a single nucleotide transition in the deep intronic region of intron 13 of gene Ank1, encoding the anchorage protein ankyrin-1 in RBC. In vitro minigene assay revealed two abnormally spliced transcripts in addition to wild-type mRNA. The wild-type Ank1 transcript was detected in the homozygous mutant mouse with 30% reduction in expression level compared to that in wild type mouse. The aberrant transcripts presumably encoded a 509 amino acids protein, which lacks beta-spectrin binding domain and C-terminal regulatory region. The truncated protein was not detected by western blotting using currently available antibodies against full-length ankyrin-1 in homozygous hema6 erythrocyte ghosts, whereas the wild-type Ankyrin-1 are present with reduced quantity. Employing biochemical and cell biology assays, we characterized the mechanism by which Ank1hema6 mutation causes hemolytic hereditary spherocytosis in mouse. Hema6 strain provides a novel tool to study ankyrin-1 and its pathogenesis role in HS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mechanisms of p34cdc2 regulation.

1993 ◽  
Vol 13 (3) ◽  
pp. 1675-1685 ◽  
Author(s):  
S Atherton-Fessler ◽  
L L Parker ◽  
R L Geahlen ◽  
H Piwnica-Worms
Keyword(s):  
Active Site ◽  
Kinase Activity ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Cyclin B ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
Maximal Inhibition

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.

scholarly journals Identification of Inhibitors for a Virally Encoded Protein Kinase by 2 Different Screening Systems: In Vitro Kinase Assay and In-Cell Activity Assay

2005 ◽  
Vol 10 (1) ◽  
pp. 36-45 ◽  
Author(s):  
Helmut Mett ◽  
Kerstin Hölscher ◽  
Heidrun Degen ◽  
Christina Esdar ◽  
Birgit Felden De Neumann ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Cell Activity ◽  
Kinase Assay ◽  
Activity Assay ◽  
Drug Candidates ◽  
Cellular Context ◽  
In Vitro Kinase Assay

The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 μM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates. ( Journal of Biomolecular Screening 2005:36-45)

scholarly journals Cell surface expression of v-fms-coded glycoproteins is required for transformation.

1984 ◽  
Vol 4 (10) ◽  
pp. 1999-2009 ◽  
Author(s):  
M F Roussel ◽  
C W Rettenmier ◽  
A T Look ◽  
C J Sherr
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Cell Surface ◽  
Gene Product ◽  
Kinase Activity ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Wild Type ◽  
Specific Protein Kinase

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.

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