Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome

In tumor cells from virtually all patients with chronic myelogenous leukemia, the Philadelphia chromosome, a fusion of chromosomes 9 and 22, directs the synthesis of the P210bcr/abl protein. The protein-tyrosine kinase activity and hybrid structure of P210bcr/abl are similar to the oncogene product of the Abelson murine leukemia virus, P160gag/v-abl, which induces acute lymphomas. To determine whether P210bcr/abl can induce chronic myelogenous leukemia, murine bone marrow was infected with a retrovirus encoding P210bcr/abl and transplanted into irradiated syngeneic recipients. Transplant recipients developed several hematologic malignancies; prominent among them was a myeloproliferative syndrome closely resembling the chronic phase of human chronic myelogenous leukemia. Tumor tissue from diseased mice harbored the provirus encoding P210bcr/abl. These results demonstrate that P210bcr/abl expression can induce chronic myelogenous leukemia. Retrovirus-mediated expression of the protein provides a murine model system for further analysis of the disease.     

Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization

Chronic myelogeneous leukemia (CML) is genetically characterized by fusion of the bcr and abl genes on chromosomes 22 and 9, respectively. In most cases, the fusion involves a reciprocal translocation t(9;22)(q34;q11), which produces the cytogenetically distinctive Philadelphia chromosome (Ph1). Fusion can be detected by Southern (DNA) analysis or by in vitro amplification of the messenger RNA from the fusion gene with polymerase chain reaction (PCR). These techniques are sensitive but cannot be applied to single cells. Two-color fluorescence in situ hybridization (FISH) was used with probes from portions of the bcr and abl genes to detect the bcr-abl fusion in individual blood and bone marrow cells from six patients. The fusion event was detected in all samples analyzed, of which three were cytogenetically Ph1-negative. One of the Ph1-negative samples was also PCR-negative. This approach is fast and sensitive, and provides potential for determining the frequency of the abnormality in different cell lineages.     

Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex

The complex containing the Mre11, Rad50, and Nbs1 proteins (MRN) is essential for the cellular response to DNA double-strand breaks, integrating DNA repair with the activation of checkpoint signaling through the protein kinase ATM (ataxia telangiectasia mutated). We demonstrate that MRN stimulates the kinase activity of ATM in vitro toward its substrates p53, Chk2, and histone H2AX. MRN makes multiple contacts with ATM and appears to stimulate ATM activity by facilitating the stable binding of substrates. Phosphorylation of Nbs1 is critical for MRN stimulation of ATM activity toward Chk2, but not p53. Kinase-deficient ATM inhibits wild-type ATM phosphorylation of Chk2, consistent with the dominant-negative effect of kinase-deficient ATM in vivo.     

Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function

As obligate intracellular parasites, viruses expertly modify cellular processes to facilitate their replication and spread, often by encoding genes that mimic the functions of cellular proteins while lacking regulatory features that modify their activity. We show that the human cytomegalovirus UL97 protein has activities similar to cellular cyclin-cyclin-dependent kinase (CDK) complexes. UL97 phosphorylated and inactivated the retinoblastoma tumor suppressor, stimulated cell cycle progression in mammalian cells, and rescued proliferation of Saccharomyces cerevisiae lacking CDK activity. UL97 is not inhibited by the CDK inhibitor p21 and lacks amino acid residues conserved in the CDKs that permit the attenuation of kinase activity. Thus, UL97 represents a functional ortholog of cellular CDKs that is immune from normal CDK control mechanisms.     

The CML-specific P210 bcr/abl protein, unlike v-abl, does not transform NIH/3T3 fibroblasts

The v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) is known to efficiently transform NIH/3T3 fibroblasts in vitro and to cause an acute lymphosarcoma in susceptible murine hosts. The role of its relative, the bcr/abl gene product, in the etiology of human chronic myelogenous leukemia (CML) remains speculative. To assess the transforming properties of the bcr/abl gene product, complementary DNA clones encoding the CML-specific P210 bcr/abl protein were expressed in NIH/3T3 fibroblasts. In contrast to the v-abl oncogene product P160, the P210 bcr/abl gene product did not transform NIH/3T3 cells. Cell lines were isolated that expressed high levels of the P210 bcr/abl protein but were morphologically normal. During the course of these experiments, a transforming recombinant of bcr/abl was isolated which fuses gag determinants derived from helper virus to the NH2-terminus of the bcr/abl protein. This suggests that a property of viral gag sequences, probably myristylation-dependent membrane localization, must be provided to bcr/abl for it to transform fibroblasts.     

Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells

Several pathways of transmembrane signaling in lymphocytes involve protein-tyrosine phosphorylation. With the exception of p56lck, a tyrosine kinase specific to T lymphoid cells that associates with the T cell transmembrane proteins CD4 and CD8, the kinases that function in these pathways are unknown. A murine lymphocyte complementary DNA that represents a new member of the src family has now been isolated and characterized. This complementary DNA, termed blk (for B lymphoid kinase), specifies a polypeptide of 55 kilodaltons that is related to, but distinct from, previously identified retroviral or cellular tyrosine kinases. The protein encoded by blk exhibits tyrosine kinase activity when expressed in bacterial cells. In the mouse and among cell lines, blk is specifically expressed in the B cell lineage. The tyrosine kinase encoded by blk may function in a signal transduction pathway that is restricted to B lymphoid cells.     

靶向bcr/abl融合基因的RNAi对慢性粒细胞白血病K562增殖活性的影响

构建含bcr/abl RNAi慢病毒重组质粒载体并包装病毒,转染K562细胞,通过Real-time PCR及Western blotting验证干扰效应。采用CCK-8法、软琼脂细胞集落形成实验检测RNAi对K562细胞的增殖影响作用。结果表明,慢病毒介导bcr/abl基因RNAi明显下调K562细胞中bcr/abl mRNA及P210bcr/abl融合蛋白,显著抑制了K562细胞的增殖,这说明靶向bcr/abl融合基因的RNAi有望成为治疗慢性粒细胞白血病的有效方法。     

NaCl胁迫对豌豆幼苗生理生化指标的动态影响

研究盐胁迫对豌豆幼苗体内丙二醛、可溶性糖、脯氨酸、可溶性蛋白质的含量及蛋白激酶活性的影响。结果表明,盐胁迫处理使豌豆幼苗叶中丙二醛、可溶性糖、脯氨酸含量和蛋白激酶活性呈现先升高、后降低的变化趋势。其峰值出现时间先后顺序为蛋白激酶、可溶性糖、丙二醛、脯氨酸。盐胁迫会阻止蛋白质的合成,从而导致其含量降低。     

The structure of a human p110alpha/p85alpha complex elucidates the effects of oncogenic PI3Kalpha mutations

PIK3CA, one of the two most frequently mutated oncogenes in human tumors, codes for p110alpha, the catalytic subunit of a phosphatidylinositol 3-kinase, isoform alpha (PI3Kalpha, p110alpha/p85). Here, we report a 3.0 angstrom resolution structure of a complex between p110alpha and a polypeptide containing the p110alpha-binding domains of p85alpha, a protein required for its enzymatic activity. The structure shows that many of the mutations occur at residues lying at the interfaces between p110alpha and p85alpha or between the kinase domain of p110alpha and other domains within the catalytic subunit. Disruptions of these interactions are likely to affect the regulation of kinase activity by p85 or the catalytic activity of the enzyme, respectively. In addition to providing new insights about the structure of PI3Kalpha, these results suggest specific mechanisms for the effect of oncogenic mutations in p110alpha and p85alpha.     

Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association

In the interleukin-2 (IL-2) system, intracellular signal transduction is triggered by the beta chain of the IL-2 receptor (IL-2R beta); however, the responsible signaling mechanism remains unidentified. Evidence for the formation of a stable complex of IL-2R beta and the lymphocyte-specific protein tyrosine kinase p56lck is presented. Specific association sites were identified in the tyrosine kinase catalytic domain of p56lck and in the cytoplasmic domain of IL-2R beta. As a result of interaction, IL-2R beta became phosphorylated in vitro by p56lck. Treatment of T lymphocytes with IL-2 promotes p56lck kinase activity. These data suggest the participation of p56lck as a critical signaling molecule downstream of IL-2R via a novel interaction.     

Regulation of a heart potassium channel by protein kinase A and C

The enzymes adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (protein kinase A) and protein kinase C regulate the activity of a diverse group of cellular proteins including membrane ion channel proteins. When protein kinase A was stimulated in cardiac ventricular myocytes with the membrane-soluble cAMP analog 8-chlorphenylthio cAMP (8-CPT cAMP), the amplitude of the delayed-rectifier potassium current (IK) doubled when recorded at 32 degrees C but was not affected at 22 degrees C. In contrast, modulation of the calcium current (ICa) by 8-CPT cAMP was independent of temperature with similar increases in ICa occurring at both temperatures. Stimulation of protein kinase C by phorbol 12,13-dibutyrate also enhanced IK in a temperature-dependent manner but failed to increase ICa at either temperature. Thus, cardiac delayed-rectifier potassium but not calcium channels are regulated by two distinct protein kinases in a similar temperature-dependent fashion.     

Integration of TGF-beta and Ras/MAPK signaling through p53 phosphorylation

During development and tissue homeostasis, cells must integrate different signals. We investigated how cell behavior is controlled by the combined activity of transforming growth factor-beta (TGF-beta) and receptor tyrosine kinase (RTK) signaling, whose integration mechanism is unknown. We find that RTK/Ras/MAPK (mitogen-activated protein kinase) activity induces p53 N-terminal phosphorylation, enabling the interaction of p53 with the TGF-beta-activated Smads. This mechanism confines mesoderm specification in Xenopus embryos and promotes TGF-beta cytostasis in human cells. These data indicate a mechanism to allow extracellular cues to specify the TGF-beta gene-expression program.     

Inhibition of myosin light chain kinase by p21-activated kinase

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation were decreased, and cell spreading was inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation.     

Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation

Glycogen synthase kinase 3beta (GSK3beta) is involved in metabolism, neurodegeneration, and cancer. Inhibition of GSK3beta activity is the primary mechanism that regulates this widely expressed active kinase. Although the protein kinase Akt inhibits GSK3beta by phosphorylation at the N terminus, preventing Akt-mediated phosphorylation does not affect the cell-survival pathway activated through the GSK3beta substrate beta-catenin. Here, we show that p38 mitogen-activated protein kinase (MAPK) also inactivates GSK3beta by direct phosphorylation at its C terminus, and this inactivation can lead to an accumulation of beta-catenin. p38 MAPK-mediated phosphorylation of GSK3beta occurs primarily in the brain and thymocytes. Activation of beta-catenin-mediated signaling through GSK3beta inhibition provides a potential mechanism for p38 MAPK-mediated survival in specific tissues.     

Activators of protein kinase C induce dissociation of CD4, but not CD8, from p56lck

The CD4 and CD8 T cell receptor accessory molecules can both be isolated from T lymphocytes in association with p56lck, a membrane-associated, cytoplasmic tyrosine protein kinase that is expressed exclusively in lymphoid cells. The enzymatic activity of p56lck may therefore be regulated by CD4 and CD8 and be important in antigen-induced T cell activation. Exposure of human T cells and some mouse T cells to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, caused the dissociation of p56lck and CD4. Activation of protein kinase C may therefore interrupt regulation of p56lck by CD4 and alter the ability of p56lck to interact with polypeptide substrates. In contrast, exposure of cells to TPA did not cause dissociation of p56lck and CD8. Regulation of p56lck by CD4 may therefore differ from regulation by CD8.     

Bypassing a kinase activity with an ATP-competitive drug

Unfolded proteins in the endoplasmic reticulum cause trans-autophosphorylation of the bifunctional transmembrane kinase Ire1, which induces its endoribonuclease activity. The endoribonuclease initiates nonconventional splicing of HAC1 messenger RNA to trigger the unfolded-protein response (UPR). We explored the role of Ire1's kinase domain by sensitizing it through site-directed mutagenesis to the ATP-competitive inhibitor 1NM-PP1. Paradoxically, rather than being inhibited by 1NM-PP1, drug-sensitized Ire1 mutants required 1NM-PP1 as a cofactor for activation. In the presence of 1NM-PP1, drug-sensitized Ire1 bypassed mutations that inactivate its kinase activity and induced a full UPR. Thus, rather than through phosphorylation per se, a conformational change in the kinase domain triggered by occupancy of the active site with a ligand leads to activation of all known downstream functions.     

冰温贮藏对羊肉中蛋白质磷酸化水平的影响

【目的】通过测定冰温贮藏及冷藏过程中肌肉肌浆蛋白与肌原纤维蛋白的磷酸化水平,分析冰温贮藏对蛋白质磷酸化水平的影响,为肉类冰温贮藏品质调控提供理论依据。【方法】选取大尾寒羊与小尾寒羊杂交公羊的背最长肌于冷藏和冰温条件下成熟,分别在0.5 h、12 h、24 h、3 d、5 d、9 d取样测定蛋白激酶活性、肌浆蛋白与肌原纤维蛋白的磷酸化水平。采用蛋白激酶活性测定试剂盒、运用酶联免疫的方法检测各处理组蛋白激酶的活性随贮藏时间的变化;通过SDS-PAGE电泳、荧光染色的方法得到全蛋白染色与磷酸化染色的肌浆蛋白与肌原纤维蛋白条带,运用Quantity One软件分析各处理组各处理时间的肌浆蛋白及肌原纤维蛋白的磷酸化水平。【结果】贮藏初期(0.5—12 h),冰温组蛋白激酶活性显著升高(P0.05),冷藏组蛋白激酶活性则显著降低(P0.05),贮藏12 h—9 d过程中,冰温组蛋白激酶活性均显著高于冷藏组(P0.05)。两处理组蛋白激酶活性均随贮藏时间的延长而逐渐降低,且冰温组蛋白激酶活性均显著高于冷藏组(P0.05)。对肌浆蛋白染色效果图中分布于15—250 k Da的17个蛋白条带逐一进行分析,结果表明贮藏温度极显著影响肌浆蛋白各蛋白条带的磷酸化水平(P0.001)。冷藏组肌浆蛋白整体磷酸化水平先升高后降低,贮藏第3天达到最大值,冰温组肌浆蛋白整体磷酸化水平先降低后升高,最小值出现在贮藏第24天,贮藏第12天至3天时,冷藏组肌浆蛋白整体磷酸化水平高于冰温组(P0.05),贮藏第9天时,冰温组肌浆蛋白整体磷酸化水平高于冷藏组(P0.05)。对肌原纤维蛋白染色效果图中分布于15—250 k Da的20个蛋白条带逐一进行分析,结果表明不同贮藏温度对所有肌原纤维蛋白条带的磷酸化水平均产生极显著影响作用(P0.001)。两处理组肌原纤维蛋白整体磷酸化水平均随贮藏时间的延长呈现先升高后降低的趋势,冷藏组与冰温组最大值分别出现在24 h和12 h,贮藏初期(0.5—12 h),两处理组间肌原纤维蛋白整体磷酸化水平无显著差异(P0.05),贮藏24 h至贮藏期结束,冷藏组肌原纤维蛋白整体磷酸化水平始终高于冰温组(P0.05)。【结论】冰温贮藏通过影响蛋白激酶的活性来调控蛋白质的磷酸化水平,进而通过影响糖酵解途径、肌肉收缩及骨架蛋白降解来间接调控肉品质。     

The phosphothreonine lyase activity of a bacterial type III effector family

Pathogenic bacteria use the type III secretion system to deliver effector proteins into host cells to modulate the host signaling pathways. In this study, the Shigella type III effector OspF was shown to inactivate mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinases 1 and 2 (Erk1/2), c-Jun N-terminal kinase, and p38]. OspF irreversibly removed phosphate groups from the phosphothreonine but not from the phosphotyrosine residue in the activation loop of MAPKs. Mass spectrometry revealed a mass loss of 98 daltons in p-Erk2, due to the abstraction of the alpha proton concomitant with cleavage of the C-OP bond in the phosphothreonine residue. This unexpected enzymatic activity, termed phosphothreonine lyase, appeared specific for MAPKs and was shared by other OspF family members.