Fairhurst R M, Daeipour M, Amaral M C, Nel A E
Department of Medicine, UCLA School of Medicine 90024-1680.
Immunology. 1993 May;79(1):112-8.
We investigated activation of mitogen-activated protein (MAP) kinase, also known as microtubule associated protein-2 kinase (MAP-2K), by recombinant interleukin-2 (rIL-2) in phytohaemagglutinin (PHA)-induced peripheral blood lymphoblasts (PBL). MAP-kinase activation has been implicated in growth of lymphocytes and other cell types. Enzyme activity was purified from cell lysates by ion-exchange chromatography and activity measured by the ability to phosphorylate the substrates MAP-2 and myelin basic protein peptide (APRTPGGRR) in vitro. Recombinant IL-2 stimulated a variable (two-to 10-fold) and evanescent MAP-2K response which was dose dependent over the range 0-50 U/ml. In contrast to MAP-kinase activation by the CD3 receptor, activation by the IL-2 receptor (IL-2R) proceeded independently from protein kinase C (PKC) and extracellular-free Ca2+. MAP-kinase activation by CD3 involves an activation cascade which depends on Ca2+ influx and PKC activation. These events culminate in tyrosine phosphorylation and activation of MAP kinase. Recombinant IL-2 induced tyrosine phosphorylation of several intracellular proteins, including a 40,000 MW substrate which co-electrophoresed with ERK-2 on SDS-PAGE. The ERK-2 gene encodes a 41,000 MW MAP-2K and is subject to regulation by a variety of mitogens and growth factors in lymphocytes and non-lymphoid cells. MAP-kinase activation by rIL-2 was abrogated when PHA blasts were pretreated with the tyrosine protein kinase (TPK) inhibitor, methyl-2,5-dihydroxy-cinnamate. Although the TPK, p56lck, has been implicated in the activation of MAP kinase and the function of IL-2R, we found no mobility shift from a 56,000 to a 60,000 MW position as seen during PKC activation. Together these data suggest that tyrosine phosphorylation is critical to IL-2-mediated signal transduction and that MAP kinase is one of the cellular intermediates involved in this pathway.
我们研究了重组白细胞介素-2（rIL-2）在植物血凝素（PHA）诱导的外周血淋巴细胞（PBL）中对丝裂原活化蛋白（MAP）激酶（也称为微管相关蛋白-2激酶（MAP-2K））的激活作用。MAP激酶的激活与淋巴细胞及其他细胞类型的生长有关。通过离子交换色谱法从细胞裂解物中纯化酶活性，并通过体外磷酸化底物MAP-2和髓鞘碱性蛋白肽（APRTPGGRR）的能力来测量活性。重组IL-2刺激了可变的（2至10倍）且短暂的MAP-2K反应，该反应在0 - 50 U/ml范围内呈剂量依赖性。与CD3受体激活MAP激酶不同，IL-2受体（IL-2R）激活MAP激酶独立于蛋白激酶C（PKC）和细胞外游离Ca2+。CD3激活MAP激酶涉及一个依赖于Ca2+内流和PKC激活的激活级联反应。这些事件最终导致酪氨酸磷酸化和MAP激酶的激活。重组IL-2诱导了几种细胞内蛋白的酪氨酸磷酸化，包括一种分子量为40,000的底物，其在SDS-PAGE上与ERK-2共电泳。ERK-2基因编码一种分子量为41,000的MAP-2K，并且在淋巴细胞和非淋巴细胞中受多种有丝分裂原和生长因子的调节。当PHA刺激的淋巴细胞用酪氨酸蛋白激酶（TPK）抑制剂甲基-2,5-二羟基肉桂酸预处理时，rIL-2对MAP激酶的激活作用被消除。尽管TPK p56lck与MAP激酶的激活和IL-2R的功能有关，但我们未发现如PKC激活过程中所见的从56,000到60,000分子量位置的迁移。这些数据共同表明酪氨酸磷酸化对于IL-2介导的信号转导至关重要，并且MAP激酶是该途径中涉及的细胞中间产物之一。
