G-CSFR Ubiquitination Critically Regulates Myeloid Cell Survival and Proliferation

The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis. Mutations in the G-CSFR in patients with severe congenital neutropenia (SCN) transforming to acute myelogenous leukemia (AML) have been shown to induce hypersensitivity and enhanced growth responses to G-CSF. Recent studies have demonstrated the importance of the ubiquitin/proteasome system in the initiation of negative signaling by the G-CSFR. To further investigate the role of ubiquitination in regulating G-CSFR signaling, we generated a mutant form of the G-CSFR (K762R/G-CSFR) which abrogates the attachment of ubiquitin to the lysine residue at position 762 of the G-CSFR that is deleted in the Δ716 G-CSFR form isolated from patients with SCN/AML. In response to G-CSF, mono-/polyubiquitination of the G-CSFR was impaired in cells expressing the mutant K762R/G-CSFR compared to cells transfected with the WT G-CSFR. Cells stably transfected with the K762R/G-CSFR displayed a higher proliferation rate, increased sensitivity to G-CSF, and enhanced survival following cytokine depletion, similar to previously published data with the Δ716 G-CSFR mutant. Activation of the signaling molecules Stat5 and Akt were also increased in K762R/G-CSFR transfected cells in response to G-CSF, and their activation remained prolonged after G-CSF withdrawal. These results indicate that ubiquitination is required for regulation of G-CSFR-mediated proliferation and cell survival. Mutations that disrupt G-CSFR ubiquitination at lysine 762 induce aberrant receptor signaling and hyperproliferative responses to G-CSF, which may contribute to leukemic transformation

Genome-Wide Screen of Three Herpesviruses for Protein Subcellular Localization and Alteration of PML Nuclear Bodies

Herpesviruses are large, ubiquitous DNA viruses with complex host interactions, yet many of the proteins encoded by these viruses have not been functionally characterized. As a first step in functional characterization, we determined the subcellular localization of 234 epitope-tagged proteins from herpes simplex virus, cytomegalovirus, and Epstein–Barr virus. Twenty-four of the 93 proteins with nuclear localization formed subnuclear structures. Twelve of these localized to the nucleolus, and five at least partially localized with promyelocytic leukemia (PML) bodies, which are known to suppress viral lytic infection. In addition, two proteins disrupted Cajal bodies, and 19 of the nuclear proteins significantly decreased the number of PML bodies per cell, including six that were shown to be SUMO-modified. These results have provided the first functional insights into over 120 previously unstudied proteins and suggest that herpesviruses employ multiple strategies for manipulating nuclear bodies that control key cellular processes

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Analyses of pig genomes provide insight into porcine demography and evolution

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model

Prolonged Akt activation in response to G-CSF in BaF3 cells transfected with the K762R/G-CSFR.

<p>BaF3 cells stably transfected with either the WT G-CSFR or K762R/G-CSFR were washed and incubated in cytokine-free media (RPMI/0.1%BSA) at 37°C for 4 hrs. Cells were then treated with G-CSF (100 ng/ml) at 37°C for 10 min, washed, incubated in cytokine-free media again for the indicated times, and lysed. Whole cell lysates were immunoprecipitated with anti-Akt antibody, and blotted with anti-phospho-Ser-Akt antibody (upper panel). The blot was stripped and re-blotted with anti-Akt antibody to confirm equal protein loading (lower panel). Cells stimulated with activated orthovanadate at room temperature for 20 min are shown as a positive control.</p

Impaired mono-/polyubiquitination of the K762R/G-CSFR in response to ligand binding.

<p>CHO ts20 cells were transiently transfected with HA-tagged ubiquitin alone (HA only), HA-Ubiquitin plus WT G-CSFR (HA+WT), or HA-Ubiquitin plus K762R/G-CSFR (HA+K762R). At 48 hrs after transfection, cells were washed and incubated in serum-free media at 4°C for 4 hrs before being transferred to 37°C and incubated with G-CSF (100 ng/mL) for the indicated times. Cells were then lysed, immunoprecipitated with anti-V5 antibody, and blotted with (A) the FK2 antibody which recognizes both poly- and monoubiquitinated proteins. (B) The blot in (A) was stripped and re-blotted with anti-V5 antibody as a control for receptor protein loading.</p

Enhanced proliferation and viability of 32D cells expressing the K762R/G-CSFR mutant.

<p>32Dcl3 cells were stably transfected with the WT G-CSFR or the K762R/G-CSFR. (A) Pooled transfectants were grown in 2 ng/ml of G-CSF and cell numbers determined over a period of 72 h. (B) Pooled transfectants were washed out of IL-3, grown in 10 ng/ml of G-CSF overnight, washed, and the viability of the cells measured using the CellTiter-Glo Luminescent Cell Viability Assay over a period of 12 h. Error bars indicating the SEM from three independent experiments are shown.</p

Prolonged G-CSF-induced Stat5 activation in K762R/G-CSFR transfectants.

<p>BaF3 cells stably transfected with either the WT G-CSFR or K762R/G-CSFR were washed and incubated in cytokine-free media (RPMI/0.1%BSA) at 37°C for 4 hrs. Cells were then treated with G-CSF (100 ng/ml) at 37°C for 10 min, washed, incubated in media depleted of growth factors for the indicated times, and lysed. Proteins from whole cell lysates were separated on SDS-PAGE and transferred onto nitrocellulose membranes, which were blotted with anti-phospho-Stat5 antibody (upper panel). The blot was stripped and re-blotted with anti-Stat5 antibody to confirm equal protein loading (lower panel). Cells stimulated with activated orthovanadate at room temperature for 20 min are shown as a positive control.</p

G-CSF-induced Stat3 activation is similar in WT and K762R transfectants.

<p>BaF3 cells stably transfected with either the WT G-CSFR or K762R/G-CSFR were washed and incubated in cytokine-free media (RPMI/0.1%BSA) at 37°C for 4 hrs. Cells were then treated with G-CSF (100 ng/ml) at 37°C for 10 min, washed, and incubated again in cytokine-free media for the indicated times, then lysed. Proteins from whole cell lysates were separated on SDS-PAGE and transferred onto nitrocellulose membranes, which were blotted with anti-phospho-Stat3 antibody (upper panel). The blot was stripped and re-blotted with anti-Stat3 antibody to confirm equal protein loading (lower panel). Cells stimulated with activated orthovanadate at room temperature for 20 min are shown as a positive control.</p