Conservation of function of Drosophila melanogaster abl and murine v-abl proteins in transformation of mammalian cells.

The Drosophila melanogaster abl and the murine v-abl genes encode tyrosine protein kinases (TPKs) whose amino acid sequences are highly conserved. To assess functional conservation between the two gene products, we constructed Drosophila abl/v-abl-chimeric Abelson murine leukemia viruses. In these chimeric Abelson murine leukemia viruses, the TPK and carboxy-terminal regions of v-abl were replaced with the corresponding regions of D. melanogaster abl. The chimeric Abelson murine leukemia viruses were able to mediate morphological and oncogenic transformation of NIH 3T3 cells and were able to abrogate the interleukin-3 dependence of a lymphoid cell line. We also found that a virus that contained both TPK and carboxy-terminal Drosophila abl regions had no in vitro transforming activity for primary bone marrow cells and lacked the ability to induce tumors in susceptible mice. A virus that replaced only a portion of the v-abl TPK region with that of Drosophila abl had low activity in in vitro bone marrow transformation and tumorigenesis assays. These results indicate that the transforming functions of abl TPKs are only partially conserved through evolution. These results also imply that the TPK region of v-abl is a major determinant of its efficient lymphoid cell-transforming activity.     

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.     

Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans

External signals induce the switch from a yeast to a hyphal growth form in the fungal pathogen Candida albicans. We demonstrate here that the catalytic subunit of a protein kinase A (PKA) isoform encoded by TPK2 is required for internal signalling leading to hyphal differentiation. TPK2 complements the growth defect of a Saccharomyces cerevisiae tpk1-3 mutant and Tpk2p is able to phosphorylate an established PKA-acceptor peptide (kemptide). Deletion of TPK2 blocks morphogenesis and partially reduces virulence, whereas TPK2 overexpression induces hyphal formation and stimulates agar invasion. The defective tpk2 phenotype is suppressed by overproduction of known signalling components, including Efg1p and Cek1p, whereas TPK2 overexpression reconstitutes the cek1 but not the efg1 phenotype. The results indicate that PKA activity of Tpk2p is an important contributing factor in regulating dimorphism of C. albicans.     

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.     

蛋白激酶与细胞分化—两种分化诱导剂对酪氨酸蛋白激酶的抑制

在报道视黄酸(RA)是人肝癌细胞株SMMC-7721分化诱导剂的基础上,本文继续报道8-溴-环磷酸腺苷对该细胞也有分化诱导作用,两者都抑制细胞的增殖,降低γ-谷氨酰转肽酶(γ-GT)比活力和升高(ALP)碱性磷酸酶的比活力。在10μmol/LRA和0.5mmol/L-8-Br-cAMP处理细胞1、3、5天后,胞液和膜性组分中的酪氨酸蛋白激酶(TPK)的比活力均降低,其中RA对胞液TPK的作用在早期较明显,约降低30％,而对膜性TPK的影响则随培养天数而逐渐增加,至第5天下降达50％以上。8Br-cAMP则相反,对胞浆TPK的抑制主要发生在3天以后;约抑制43—53％,而对膜性组分则抑制率逐日降低,在第一天较为明显。因TPK是一个细胞增殖恶变的标志,故RA和8-溴-cAMP对TPK的抑制进一步证明这两种分化诱导剂对SMMC-7721细胞的逆转作用。     

A membrane-anchored cytoplasmic domain of the human insulin receptor mediates a constitutively elevated insulin-independent uptake of 2-deoxyglucose

Insulin stimulates the autophosphorylation of the beta-subunit of the insulin receptor (IR) on tyrosine residues. Mutations which compromise IR autophosphorylation in vivo result in a decrease of the insulin-activated uptake of 2-deoxyglucose. These results are consistent with previous results which implicate IR autophosphorylation in the generation of the insulin response by cells. To further explore the specificity of the IR tyrosine phosphokinase (TPK) domain in IR function, we have altered the human IR (hIR) cDNA to encode truncated insulin-independent TPKs, which are expressed in chinese hamster ovary (CHO) cells as either membrane-anchored or cytosolic proteins. Both mutant hIRs exhibit TPK activity in vitro, although the cytosolic form is approximately 20 times more active. The carbohydrate moiety of the membrane-anchored form is of the high mannose type, consistent with an intracellular localization for this mutant hIR. The two mutant hIRs mediate very different physiological responses in transfected cells: the membrane-anchored, but not the cytosolic, hIR TPK mediates a constitutively elevated (135% the maximum insulin-stimulated response in CHO cells) insulin-independent uptake of 2-deoxyglucose. These results thus suggest that the hIR TPK is in fact specific for this aspect of IR function and, when membrane-associated, can mediate the insulin-independent uptake of 2-deoxyglucose. Neither of these mutant hIRs appears to transform CHO cells.     

Only site-directed antibodies reactive with the highly conserved src-homologous region of the v-abl protein neutralize kinase activity

Antisera specific for six regions of the v- abl protein were used to serologically characterize the Abelson murine leukemia virus tyrosine kinase. Chemically synthesized peptides corresponding to the predicted v- abl protein sequence and larger regions of the v- abl protein expressed as fusion proteins in bacteria were used as immunogens. The specificity of each antiserum was confirmed by immunoprecipitation analysis with defined deletion mutants of Abelson murine leukemia virus. Several of these v- abl -specific antisera display much higher titers and avidities than serum harvested from mice bearing Abelson murine leukemia virus-induced tumors, previously the only source of anti- abl -specific serum. Two antisera were found to block the in vitro autophosphorylation of the v- abl protein as well as its ability to phosphorylate a peptide substrate. Examination of the sites against which the kinase-blocking antisera were prepared revealed that both are in close proximity to the in vivo sites of tyrosine phosphorylation, which fall within the region of high homology with v-src and other tyrosine kinases. Antisera directed against other regions of v- abl did not inhibit kinase activity.     

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca~(2+)浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 + ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 + ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 + ]i 升高 ,即 PKC和 Ca2 + 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .     

High level of tyrosine protein kinase in a murine lymphoma cell line induced by Moloney leukemia virus.

Several sarcoma-inducing viruses encode protein kinases that phosphorylate tyrosine residues. Such enzymatic activities can be detected within the detergent-insoluble matrix of transformed fibroblasts. We have analysed the protein kinase activities in two murine lymphoma cell lines ( MBL2 and LSTRA) induced by Moloney murine leukemia virus (Mo-MuLV). After incubation of the detergent-insoluble matrix of these cells with [gamma-32P]ATP, several alkali-resistant phosphoproteins, including a very heavily labelled 55 000 mol. wt. protein ( p55 ), have been detected in LSTRA, reflecting the activity of a protein kinase specific to this cell line. This protein kinase activity shares some of the distinctive properties of the protein kinases of transforming viruses, i.e., specificity for tyrosine residues, association with membranous and/or cytoskeletal structures, and inhibition by a synthetic peptide derived from the phosphorylation site of pp60src. In view of the absence of a transforming gene in MoMuLV , it is likely that the high level of protein kinase detected in the LSTRA cell line arises from the expression of a cellular gene.     

Insulin and epidermal growth factor do not affect phosphoinositide metabolism in rat liver plasma membranes and hepatocytes

Recent studies with viral oncogene tyrosine kinases have suggested that these kinases may phosphorylate phosphoinositides and diacylglycerol. Since the receptors for insulin and epidermal growth factor (EGF) also possess tyrosine kinase activity, we have investigated possible effects of insulin and EGF on phosphoinositide metabolism in rat liver plasma membranes and rat hepatocytes. In plasma membranes prepared from rats injected 18 h prior with [3H]myo-inositol or incubated with [gamma-32P]ATP, phosphatidylinositol-4-P and phosphatidylinositol-4,5-P2 were formed, but there were no effects of either insulin or EGF although these agents stimulated protein tyrosine phosphorylation. In hepatocytes incubated with [3H]myo-inositol, label was incorporated into phosphatidylinositol, phosphatidylinositol-4-P, and phosphatidylinositol-4,5-P2, but there was no effect of insulin. Incubation of hepatocytes with [3H]myo-inositol plus insulin or EGF for 2 h also did not alter the formation of [3H]myo-inositol-1,4,5-P3 from [3H]phosphatidylinositol-4,5-P2 induced by vasopressin. These findings suggest that the tyrosine kinase activity of liver insulin and EGF receptors is not important in phosphoinositide formation.     

The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1

The feline c-fms proto-oncogene product is a 170 kd glycoprotein with associated tyrosine kinase activity. This glycoprotein was expressed on mature cat macrophages from peritoneal inflammatory exudates and spleen. Similarly, the receptor for the murine colony-stimulating factor, CSF-1, is restricted to cells of the mononuclear phagocytic lineage and is a 165 kd glycoprotein with an associated tyrosine kinase. Rabbit antisera to a recombinant v-fms-coded polypeptide precipitated the feline c-fms product and specifically cross-reacted with a 165 kd glycoprotein from mouse macrophages. This putative product of the murine c-fms gene exhibited an associated tyrosine kinase activity in immune complexes, specifically bound murine CSF-1, and, in the presence of the growth factor, was phosphorylated on tyrosine in membrane preparations. The murine c-fms proto-oncogene product and the CSF-1 receptor are therefore related, and possibly identical, molecules.     

T cell activation induces rapid tyrosine phosphorylation of a limited number of cellular substrates

Activation of murine T cells by antigen, antibodies binding the T cell antigen receptor, or stimulatory anti-Thy-1 antibodies results in rapid phosphorylation of the T cell receptor zeta chain on tyrosine residues. The T cell receptor is itself unlikely to be a tyrosine kinase; rather, it is probable that this receptor is coupled to a nonreceptor tyrosine kinase. To understand further this protein kinase pathway, additional targets of the tyrosine kinase have been sought by comparing anti-phosphotyrosine antibody immunoblots of cellular proteins from unactivated and activated T cell hybridomas. In addition to the T cell receptor zeta chain, two proteins of 53 and 62 kDa are phosphorylated on tyrosine residues after T cell activation. These phosphorylations require stimulatory anti-Thy-1 antibodies, antigen, or antireceptor antibody stimulation. The 53-kDa protein is preferentially phosphorylated by antigen or antireceptor antibody. Of interest is that variants of the murine T cell hybridoma lacking the T cell receptor zeta chain or lacking surface antigen receptor can nonetheless be stimulated by anti-Thy-1 antibodies to phosphorylate the 62-kDa substrate. In contrast to the tyrosine kinases of oncogenic viruses, the kinase coupled to the T cell antigen receptor appears to have a limited number of targets. These proteins are candidates for critical substrates in this protein tyrosine kinase pathway.     

Molecular cloning of human thiamin pyrophosphokinase

Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) catalyses phosphorylation of thiamin to thiamin pyrophosphate, an active enzyme cofactor. Here we describe the cloning of complete human TPK1 cDNA from an adult liver library. Human TPK1 is 89% identical to murine TPK1 at the protein level. The gene maps to chromosome 7q34-36, consists of at least eight exons, and spans a distance at least of 420 kb. The mRNA of human TPK1 is highly expressed in testis, small intestine and kidney with lesser but detectable expression in brain, liver, placenta and spleen. The availability of the human TPK1 gene will provide another useful tool for studying the role of this enzyme in human thiamin metabolism and deficiency state.     

Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase

We have isolated three genes (TPK1, TPK2, and TPK3) from the yeast S. cerevisiae that encode the catalytic subunits of the cAMP-dependent protein kinase. Gene disruption experiments demonstrated that no two of the three genes are essential by themselves but at least one TPK gene is required for a cell to grow normally. Comparison of the predicted amino acid sequences of the TPK genes indicates conserved and variable domains. The carboxy-terminal 320 amino acid residues have more than 75% homology to each other and more than 50% homology to the bovine catalytic subunit. The amino-terminal regions show no homology to each other and are heterogeneous in length. The TPK1 gene carried on a multicopy plasmid can suppress both a temperature-sensitive ras2 gene and adenylate cyclase gene.     

Various proteins modulate the kinase activity of the insulin receptor

Previous studies of the substrate specificity of the purified insulin receptor tyrosine kinase using synthetic random polymers have demonstrated that the receptor kinase phosphorylates poly (Glu, Tyr) 4:1 but not poly (Glu, Tyr) 1:1. In the present study, insulin treatment of Chinese hamster ovary cells overexpressing the human insulin receptor was found to stimulate the ability of their membrane extracts to phosphorylate poly (Glu, Tyr) 1:1. It was concluded that this activity was due to the receptor itself because: 1) it was precipitated with a monoclonal antibody to the receptor; 2) the addition of various membrane extracts to purified insulin receptor preparations stimulated the ability of these preparations to phosphorylate poly (Glu, Tyr) 1:1; and 3) certain purified proteins, including bovine serum albumin and casein, were also capable of stimulating the purified receptor to phosphorylate poly (Glu, Tyr) 1:1. The effect of albumin was dose-dependent; 0.5 and 10 mg/ml bovine serum albumin stimulated the phosphorylation of poly (Glu, Tyr) 1:1 by 2- and 230-fold, respectively. In contrast, albumin had no effect on the phosphorylation of poly (Glu, Tyr) 4:1. These results indicate that the activity of the insulin receptor kinase on certain substrates can be modulated by the presence of other proteins.     

Tyrosine protein kinase activity in renal brush-border membranes.

Tyrosine protein kinase (TPK) activity was detected in rat renal brush-border membranes (BBM) using poly(Glu80Na,Tyr20) as a substrate. Maximal TPK activity required prior detergent dispersion of the membranes with 0.05% Triton X-100 and the presence of vanadate, a potent inhibitor of phosphotyrosine protein phosphatases, in the phosphorylation medium. Optimal conditions for measurement of TPK activity were 10 mM of MgCl2 and MnCl2, at 30 degrees C and pH 7.0. TPK activity was inhibited by genistein, with a IC50 value of 15 microM, while no inhibition was observed in the presence of 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H7), an inhibitor of serine-threonine kinases. TPK activity was enriched 4-fold in the BBM fraction relative to cortex homogenate. It was co-enriched with BBM enzyme markers, but not with those of the basolateral membrane (BLM). The endogenous substrates of TPK in brush-border and basolateral membranes were determined by Western blot analysis using an antiphosphotyrosine monoclonal antibody (PY20). Various phosphotyrosine-containing proteins were found in the BBM (31, 34, 46, 50, 53, 72, 90, 118 and 170 kDa) and in the BLM (37, 48, 50, 53, 72, 90, 130 and 170 kDa). Addition of exogenous insulin receptor to BBM and BLM increased the phosphorylation of most of the substrates. Solubilization of the TPK activity from BBM with 0.5% CHAPS and subsequent gel filtration on Superdex 75 yielded two peaks of tyrosine protein kinase activity with apparent molecular masses of 49 and 66 kDa. These results provide evidence for a non-receptor TPK activity associated with the renal tubular luminal membrane.     

Use of tyrosine-containing polymers to characterize the substrate specificity of insulin and other hormone-stimulated tyrosine kinases

Synthetic copolymers containing tyrosine residues were used to characterize the substrate specificity of the insulin receptor kinase and compare it to tyrosine kinases stimulated by epidermal growth factor, insulin-like growth factor-1 and phorbol ester. In partially purified receptor preparations from eight different tissues insulin best stimulated (highest V) phosphorylation of a random copolymer composed of glutamic and tyrosine residues at a 4:1 ratio (Glu/Tyr, 4:1). The insulin-stimulated phosphorylation of this polymer was highly significant also in receptor preparations from fresh human monocytes, where insulin binding and autophosphorylation were difficult to detect. Other tyrosine-containing polymers Ala/Glu/Lys/Tyr (6:2:5:1) and Glu/Ala/Tyr (6:3:1) were also phosphorylated by the insulin-stimulated kinase but to a lower extent. A tyrosine kinase stimulated by insulin-like growth factor-1, and one stimulated by phorbol ester also best phosphorylated the polymer Glu/Tyr (4:1). The three kinases differed only in their capability to phosphorylate Glu/Ala/Tyr (6:3:1) or Ala/Glu/Lys/Tyr (6:2:5:1). Glu/Tyr (4:1) was a poor substrate for the epidermal growth factor receptor kinase which best phosphorylated the polymer Glu/Ala/Tyr (6:3:1). Three additional polymers: Glu/Tyr (1:1), Glu/Ala/Tyr (1:1:1), and Lys/Tyr (1:1) failed to serve as substrates for all four tyrosine kinases tested. Taken together these findings suggest that. Hormone-sensitive tyrosine kinases have similar yet distinct substrate specificity and are likely to phosphorylate their native substrates on tyrosines adjacent to acidic (glutamic) residues. Tyrosine-containing polymer substrates are highly sensitive and convenient tools to study (hormone-sensitive) tyrosine kinases whose native substrates are unknown or present at low concentrations.     

编码新型转录因子样蛋白的小鼠及人类同源cDNA的克隆(英文)

 通过检索GenBank的表达序列标签 (EST)数据库并结合cDNA末端快速扩增法 (RACE) ,从小鼠胸腺克隆到一个新的cDNA序列 ,并从人类肝癌组织中克隆出了其同源cDNA .根据读码框架分析 ,这两个cDNA分别编码 541和 555个氨基酸的蛋白质 两个蛋白质之间氨基酸序列一致率为77% ,和已知蛋白无显著同源性 .分子生物学软件和网上分析表明 ,两个蛋白质所含功能序列与STAT家族成员极为相似 ,均含有包括酪氨酸蛋白激酶在内的多种蛋白激酶的磷酸化位点和核定位信号 (NLS) ,可能是一种新型转录因子 .RT PCR分析显示 ,两个基因在正常组织中选择性表达 ,其分布相似 ,而且都具有一定程度的与分化或增殖相关的趋势 .     

Dual-specificity protein kinases: will any hydroxyl do?

Protein kinases are classified by the target amino acid in their substrates. Those protein kinases that phosphorylate hydroxyamino acids comprise two groups, the protein-tyrosine and protein-serine/threonine kinases, which, until recently, had been thought to be mutually exclusive. However, several new protein kinases have been discovered that, by the criterion of primary structure, would be classified as protein-serine/threonine kinases but which, surprisingly, are able to phosphorylate tyrosine residues. Even more surprising, there are reports of protein kinases that are capable of phosphorylating both tyrosine and serine/threonine residues. We review and discuss recent developments concerning these 'dal-specificity' protein kinases.     

Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1

In the yeast Saccharomyces cerevisiae, three genes TPK1, TPK2, and TPK3 encode catalytic subunits of cAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPK1 gene. In order to purify C1 completely free of C2 and C3, a strain was constructed that contained only the TPK1 gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPK1) and yeast regulatory subunit (BCY1) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-200, then bound to an anti-regulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of cAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. Kinetic analysis showed that the apparent Km for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 microM, respectively. The kcat was determined to be 640 min-1. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked.