Lenormand P, Sardet C, Pagès G, L'Allemain G, Brunet A, Pouysségur J
Centre de Biochimie-CNRS, Université de Nice, France.
J Cell Biol. 1993 Sep;122(5):1079-88. doi: 10.1083/jcb.122.5.1079.
Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.
丝裂原活化蛋白激酶（p42mapk和p44mapk）是丝氨酸/苏氨酸激酶，在受到各种细胞外信号刺激的细胞中会迅速被激活。这种激活是通过丝裂原活化蛋白激酶激酶（p45mapkk）介导的，p45mapkk是一种双特异性激酶，它可磷酸化丝裂原活化蛋白激酶的两个关键调节性苏氨酸和酪氨酸残基。我们先前报道过，丝裂原活化蛋白激酶激活的持续阶段对于有丝分裂原刺激的细胞通过细胞周期的“限制点”至关重要。在此，我们使用特异性多克隆抗体以及表位标记的重组丝裂原活化蛋白激酶进行转染，证明这些信号蛋白激酶在生长因子刺激的细胞中经历了独特的时空定位。在G0期停滞的仓鼠成纤维细胞中，激活剂p45mapkk和丝裂原活化蛋白激酶（p42mapk、p44mapk）主要位于细胞质中。在血清或α-凝血酶进行有丝分裂原刺激后，两种丝裂原活化蛋白激酶同工型均转位至细胞核。这种转位迅速（在15分钟内可见）、持续（至少在整个G1期直至6小时）、可逆（通过去除有丝分裂原刺激），并且显然与有丝分裂原潜能“耦合”；对诸如α-凝血酶受体合成肽和佛波酯等不能持续激活丝裂原活化蛋白激酶的非有丝分裂原剂无反应。当p42mapk和p44mapk以高水平稳定表达时，它们存在于静息细胞的细胞核中；激酶缺陷型突变体（p44mapk T192A或Y194F）也有明显的核定位。与之形成鲜明对比的是，即使在长时间的生长因子刺激期间以及转染达到高表达水平后，p45mapkk激活剂仍保留在细胞质中。我们提出，在整个G0-G1期p42mapk和p44mapk的快速且持续的核转运对于这些激酶介导生长反应的功能至关重要。
