Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation.
Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation.
复制标题
成纤维细胞中存在两种不同磷脂酰肌醇激酶的证据。
DOI：
10.1042/bj2470165
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Cantley,L
中科院分区：
文献类型：
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作者：
Whitman,M;Kaplan,D;Roberts,T;Cantley,L
磷脂酰肌醇（PtdIns）激酶活性从非转化和多瘤中T转化的鼠成纤维细胞进行了检查。正常和转化的3 T3成纤维细胞都有两种PtdIns激酶，它们可以通过阴离子交换色谱法分离。其中一种活性（I型）对ATP的Km为10 μ M，对腺苷、AMP或ADP的抑制有抗性，并被非离子去污剂抑制。另一种活性（II型）对ATP的Km值略高（35 μ M），并在表明细胞能荷调节该活性的浓度下被ADP、AMP和腺苷竞争性抑制。II型PtdIns激酶被非离子去污剂激活。我们先前已经报道了PtdIns激酶活性与多瘤-中-T免疫沉淀物的特异性关联[惠特曼，Kaplan，Schaffhausen，Cantley & Roberts（1985）Nature（伦敦）315，239-242; Kaplan，惠特曼，Schaffhausen，Raptis，Garcea，Pallas，Roberts & Cantley（1986）Proc. Natl. Acad. Sci. U.S.A.83，3624-3628]。免疫沉淀的PtdIns激酶与通过离子交换色谱鉴定的活性的比较表明，它是与中间T/pp 60 c-src复合物特异性结合的I型酶。该PtdIns激酶活性可与中间T和pp 60 c-src分离。I型PtdIns激酶也与来自肉瘤病毒转化细胞的pp 60 v-src免疫沉淀物相关。此外，该PtdIns激酶似乎与部分纯化的血小板衍生生长因子（PDGF）受体共沉淀。在抗磷酸酪氨酸免疫沉淀物或小麦胚芽凝集素琼脂糖沉淀物中发现的这种活性的量通过用PDGF刺激静止的Balb/C 3 T3成纤维细胞而增加50倍。这些结果表明I型PtdIns激酶受影响细胞生长和转化的试剂调节，而II型PtdIns激酶可能受局部[ATP]/[ADP]比率调节。
Phosphatidylinositol (PtdIns) kinase activities from non-transformed and polyoma-middle-T-transformed murine fibroblasts were examined. Both normal and transformed 3T3 fibroblasts have two PtdIns kinases, which can be separated by anion-exchange chromatography. One of these activities (Type I) has a Km for ATP of 10 microM, is resistant to inhibition by adenosine, AMP or ADP, and is inhibited by non-ionic detergents. The other activity (Type II) has a somewhat higher Km for ATP (35 microM) and is inhibited competitively by ADP, AMP and adenosine at concentrations suggesting regulation of this activity by the energy charge of the cell. The Type II PtdIns kinase is activated by non-ionic detergents. We have previously reported the specific association of a PtdIns kinase activity with polyoma-middle-T immunoprecipitates [Whitman, Kaplan, Schaffhausen, Cantley & Roberts (1985) Nature (London) 315, 239-242; Kaplan, Whitman, Schaffhausen, Raptis, Garcea, Pallas, Roberts & Cantley (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3624-3628]. Comparison of the immunoprecipitated PtdIns kinase with the activities identified by ion-exchange chromatography indicates that it is the Type I enzyme which specifically associates with the middle-T/pp60c-src complex. This PtdIns kinase activity is separable from both middle T and pp60c-src. Type I PtdIns kinase also associates with pp60v-src immunoprecipitates from Rous-sarcoma-virus-transformed cells. Furthermore, this PtdIns kinase appears to co-precipitate with partially purified platelet derived growth factor (PDGF) receptor. The amount of this activity found in anti-phosphotyrosine immunoprecipitates or in wheat-germ-lectin-agarose precipitates is increased 50-fold by stimulation of quiescent Balb/C 3T3 fibroblasts with PDGF. These results suggest that the Type I PtdIns kinase is regulated by agents which affect cell growth and transformation, whereas the Type II PtdIns kinase may be regulated by the local [ATP]/[ADP] ratio.