K-562细胞胰岛素受体酪氨酸蛋白激酶活性及内源性底物的研究

本文对丁酸钠诱导分化的人白血病细胞株K-562细胞的胰岛素受体酪氨酸蛋白激酶性及细胞内源性底物进行了研究。结果表明,诱导分化后的细胞酪氨酸蛋白激活酶活性降低,胰岛素受体数量减少,酪氨酸蛋白激酶的一底物蛋白在分化后消失。该研究结果及其在该方面的进一步研究有可能为白血病发病机制的阐明以及为临床治疗白血病开辟靳途径提供一些有价值的线索。     

胰岛素受体底物家族中的两个新成员

胰岛素受体底物蛋白参与多种激素,细胞因子的信号转导,可联系下游含ＳＨ２结构域的蛋白,它是一种极为重要的信号传递中介分子,胰岛素受体底物家族有４个成员。本文从基因结构,蛋白质结构介绍新发现的两个成员（ＩＲＳ－３,ＩＲＳ－４）的一些基本特征。     

小肠平滑肌胰岛素受体的特征及其蛋白激酶活性

 本实验对狗小肠平滑肌中胰岛素受体的结构和特征进行了分析研究。通过麦胚凝集素琼脂糖和两次Sepharose-CL-6B凝胶层析从平滑肌中纯化胰岛素受体,达到电泳纯。SDS-聚丙烯酰胺凝胶电泳证明胰岛素受体是由两个亚基组成的,分子量分别为135kD和90kD。磷酸化实验证明平滑肌胰岛素受体具有胰岛素依赖性蛋白激酶活性,能催化自身的β亚基磷酸化和底物的磷酸化。Scatchard分析表明胰岛素和受体的结合呈(?)协同效应,最大结合率为13μg胰岛素/mg蛋白质。     

两种酪氨酸蛋白激酶活性在主动脉平滑肌细胞增殖周期中...

We have studied the membrane-bound tyrosine protein kinases (TPK) in cultivated aortic smooth muscle cells (ASMC) from the rabbit, and found that the cytosolic (membranous)TPK and the nuclear (membranous)TPK are two distinctive types of isozymes as judged by criteria on dynamics: (1) using synthetic poly(Glu. Ala. Tyr)n(6:3:1) as a substrate, the relative extents of stimulation by Mn2+ (450%) was more effective than that by Mg2+ (100%) on cytosolic TPK, but Mn2+ was less effective (100%) than Mg2+ (130%) on nuclear TPK: (2) the concentrations of Mn2+ and Mg2+ giving maximal stimulation on cytosolic TPK were 15 mmol/L and 50 mmol/L, whereas on nuclear TPK were 1 mmol/L and 5 mmol/L, respectively. These results indicate that the attitudes toward Mn2+ and Mg2+ can distinguish the cytosolic TPK from the nuclear one. In nucleus, with the entry of ASMC from G0 to G1 stage, TPK activity increased rapidly and reached the peak (12 times G0 activity) at the early G1 stage (3 hrs from G0), then decreased dramatically to basic line, and remained at a lower level thereafter. In cytosol, the TPK activity decreased with the entry of ASMC from G0 to G1 stage and reached its lowest point at the middle of G1 stage (6 hrs from G0), and then increased with a transient peak at the late G1 stage (9 hrs). It went down to G0 level before DNA synthesis.     

两种酪氨酸蛋白激酶活性在主动脉平滑肌细胞增殖周期中的动态变化

本文以人工合成的多肽PGAT 为底物鉴定了培养的家兔ASMC 膜性TPK。发现Mg~(2+)和Mn~(2+)对ASMC 胞浆膜性TPK 和核膜性TPK 的激活作用有两点不同:(1)它们对前者的最适激活浓度高于后者;(2)对胞浆膜性TPK,Mn~(2+)最大激活效应大于Mg~(2+)而对核膜性TPK,Mn~(2+)则低于Mg~(2+)。这两种TPK活性在其G_1期的变化特点是:胞浆膜性TPK最高活性出现在G_1晚期(9 h),核膜性TPK最高活性则出现在G_1早期(3 h)。     

LA－90细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究

ＬＡ－９０细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究夏英,高漫,颜卉君,吴国利（北京师范大学生物系生物化学及分子生物学研究室,１００８７５）关键词酪氨酸蛋白激酶;磷酸酪氨酸蛋白磷酸酶;细胞转化ｉｓ－ＲＳＶＬＡ－９０细胞是ＲＳＶ转染的小鼠３Ｔ３细胞...     

酪氨酸蛋白激酶型受体亚族的研究概况

已知许多生长因子的效应通过高亲和性受体酪氨酸激酶(Receptor Tyrosine Kinase,RTK)所介导。RTK信号通路,与已确认的第二信使系统即cAMP系统、Ca~(2 )系统和肌醇磷脂信号系统相互作用,相互协调以进行生物体正常生理活动。10年来,先后证实有9类RTK亚族(图1),各亚族成员以其独特的结构特点区别于其他亚族。 尽管RTK亚族间存在差异,但就信号发放途径而言,在很大程度上表现出共同性。配体结合胞外区城后,激活胞浆的酪氨酸激酶,导致下游大量共同发信号分子活化。经常受到激活的这类蛋白质有:磷脂酶C-γ(PLC-γ)、     

DHHC2 is a protein S-acyltransferase for Lck

Abstract

Lck is a non-receptor tyrosine kinase of the Src family that is essential for T cell activation. Dual N-terminal acylation of Lck with myristate (N-acylation) and palmitate (S-acylation) is essential for its membrane association and function. Reversible S-acylation of Lck is observed in vivo and may function as a control mechanism. Here we identify the DHHC family protein S-acyltransferase DHHC2 as an enzyme capable of palmitoylating of Lck in T cells. Reducing the DHHC2 level in Jurkat T cells using siRNA causes decreased Lck S-acylation and partial dislocation from membranes, and conversely overexpression of DHHC2 increases S-acylation of an Lck surrogate, LckN10-GFP. DHHC2 localizes primarily to the endoplasmic reticulum and Golgi apparatus suggesting that it is involved in S-acylation of newly-synthesized or recycling Lck involved in T cell signalling.     

Detection of tyrosine-specific protein kinases with gastrin as exogenous substrate

Gastrin was recently shown to be phosphorylated on its single tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine protein kinase (TPK). The TPK previously detected in the murine lymphoma (LSTRA) induced by the Moloney murine leukemia virus phosphorylates gastrin, the apparent K_m is 65 μM and the maximum rate 1900 pmol/min per mg; the kinase is more efficeint with MnCl₂ than with MgCl₂, is stimulated by NaVO₃ and inhibited by ZnCl₂. Gastrin phosphorylation is observed only when a TPK is expressed by the cell: extracts of fibroblasts infected with a temperature-sensitive mutant of the Rous sarcoma virus had no gastrin kinase activity when grown at the non-permissive temperature whereas cells grown at the permissive temperature were transformed and disclosed a clear gastrin kinase activity. Gastrin kinases were detected in various transformed cells; human lymphomas, K562 cells, cells from a patient with acute proliferative leukemia, and normal cels; human T and B lymphocytes.     

Alterations in protein kinase activity following exposure of cultured human lymphocytes to modulated microwave fields

Cultures of human tonsil lymphocytes were exposed in a Crawford cell to a 450-MHz field (peak envelope intensity 1.0 mW/cm2), sinusoidally amplitude modulated (depth 80%) at frequencies between 3 and 100 Hz for periods up to 60 min. The Crawford cell was housed in a temperature-controlled chamber (35 degrees C) and control cultures were placed in the same chamber. Activity of cAMP-dependent protein kinase relative to controls remained unaltered by fields modulated at 16 or 60 Hz with exposures of 15, 30, and 60 min. By contrast, total non-cAMP-dependent kinase activity fell to less than 50% of unexposed control levels after 15 and 30 min exposures, but, despite continuing field exposure, returned to control or preexposure levels by 45 and 60 min. A smaller reduction (20-25%) also occurred with 60-Hz modulation and was also restricted to exposure durations of 15 and 30 min. CW 450-MHz fields were without effect. Reduced enzyme activity occurred with 16-, 40-, and 60-Hz modulation frequencies, but not with 3-, 6-, 80-, or 100-Hz modulation. The specific identity of this kinase is unknown. This rapid but transient reduction in lymphocyte protein kinase activity restricted to modulation frequencies between 16 and 60 Hz and to less than 30 min exposure is consistent with "windowing" with respect to modulation frequency and exposure duration.     

Differences in the kinetic properties of thymidine kinase isoenzymes in unstimulated and phytohemagglutinin-stimulated human lymphocytes

Summary The two thymidine kinases, TK 1 and TK 2, found in phytohemagglutinin-stimulated human lymphocytes and the thymidine kinase, TK 2N, found in unstimulated human lymphocytes were purified and characterized. All three kinases had molecular weights between 70000 and 75000 which increased to 170000–200000 in the presence of 2 mM ATP.Studies on the kinetic properties of the enzymes with thymidine and ATP as the substrates and dTTP as the inhibitor showed clear differences between TK 1 and TK 2, but a close similarity between TK 2 and TK 2N. With thymidine as the variable substrate, TK 1 showed Michaelis-Menten kinetics, whereas TK 2 and TK 2N showed characteristic biphasic kinetics. With ATP as the variable substrate, all three enzymes showed positive cooperative kinetics, but TK 2 and TK 2N lost the cooperativity in the presence of dTTP. The results from inhibition studies showed, that dTTP was a cooperative inhibitor of TK 1 but a non-cooperative inhibitor of TK 2 and TK 2N.     

A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates

Regulated phosphorylation by protein tyrosine kinases (PTKs), such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the chronic myeloid leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogel-immobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semiquantitative detection of peptide phosphorylation by recombinant c-Abl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay are demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.     

Molecular Cloning of a Rat Brain cDNA,with Homology to a Tyrosine Kinase Substrate,that Induces Galactosylceramide Expression in COS-7 Cells

Abstract: A rat brain cDNA clone has been isolated, using a eukaryotic cell transient expression system in conjunction with an anti-galactosylceramide (anti-GalCer) monoclonal antibody that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). A good correlation between GalCer expression and the level of the enzyme activity of UDP-galactose:ceramide galactosyltransferase (CGT) was demonstrated. The cDNA insert encoded a polypeptide of 771 amino acids with a calculated molecular mass of 85,787 Da. The cDNA hybridized to a single mRNA of 3.1 kb in all rat organs examined, including brain, testis, and skeletal muscle. The cDNA product was determined to be a tyrosine-phosphorylated protein with a molecular mass of 110 kDa in transfected COS-7 cells and adult rat brain. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes: The transfected cells appeared to be fibroblast-like cells, whereas the parent COS-7 cells were typical epithelial-like cells. The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate but no homology to CGT. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer expression in the brain.