Analysis of MAP kinase cascade in the immune response

丝裂原活化蛋白激酶（MAPKs）如p38和ERK是调节免疫系统的关键信号分子。每个MAPK都有独特的功能和冗余的功能。虽然敲除小鼠中每个MAPK的基因有助于阐明其独特功能，但很难发现其冗余功能。因此，使用MAPK的功能获得突变体的替代方法可能会有所帮助。1)本构活性MAPK融合分子的生成与分析。MKK6是p38的一个MAPKK （MAPK激酶），显示出弱的激酶活性，没有上游激活信号。为了创建一个具有组成活性的MAPK突变体，我们将MKK6与p38α融合（MKK6-p38α）。MKK6中的p38α - p38α组成性磷酸化，ATF2报告基因活性增强。因此，可以通过将p38与MKK6融合来制造本构活性p38。另一方面，当检测MKK6-p38β时，p38β被组成性磷酸化，但ATF2报告基因活性没有增加。在某些情况下，MKK6-p38与下游底物的结合可能受到限制。2) MAPK^<sem>-敲入小鼠的建立与分析。7 - maker （sem）是MAPK在CD区的单氨基酸取代。其他人和我们已经证明p38^<sem>和ERK^<sem>不能与蛋白酪氨酸磷酸酶结合，并且抵抗这些磷酸酶的失活。到目前为止，在p38^<sem>敲入小鼠中未观察到p38的过度磷酸化。这些老鼠是可以存活的。蛋白质-酪氨酸-磷酸酶和一种冗余磷酸酶如Wip1（一种丝氨酸/苏氨酸磷酸酶）可能在p38的负调控中起作用。与此形成鲜明对比的是，在ERK^<sem>敲入小鼠中观察到ERK的过度磷酸化。这些小鼠显示出高围产期发病率。在未来的项目中，有可能通过条件表达ERK^<sem>来研究ERK^<sem>在免疫系统中的作用。

Mitogen-activated protein kinases (MAPKs) such as p38 and ERK are key signaling molecules in the regulation of the immune system. Each MAPK has unique functions and redundant functions. Though the gene-knockout of each MAPK in mice is useful to elucidate the unique functions, it is difficult to find out its redundant functions. Therefore alternative approach using gain-of-function mutants of MAPK might be helpful.1) Generation and analysis of constitutively active MAPK fusion molecules.MKK6, a MAPKK (MAPK kinase) of p38, reveals a weak kinase activity without upstream activation signals. To create a constitutively active MAPK mutant, we fused MKK6 with p38α (MKK6-p38α). p38α in MKK6- p38α was constitutively phosphorylated and ATF2 reporter activity was augmented. Thus, it is possible to make a constitutively active p38 by fusing it to MKK6.On the other hand, when MKK6-p38β was tested, p38β was constitutively phosphorylated but ATF2 reporter activity was not increased. Accession of MKK6-p38 with downstream substrates may be limited in some cases.2) Establishment and analysis of MAPK^<sem>-knockin mice.Sevenmaker (sem) is a single amino acid substitution of MAPK in the CD region. Others and we have demonstrated that p38^<sem> and ERK^<sem> fail to associate with protein-tyrosine-phosphatases and are resistant to inactivation by these phosphatases.No hyperphosphorylation of p38 has been observed in the p38^<sem> knockin mice, so far. These mice are viable. It is possible that protein-tyrosine-phosphatases and a redundant phosphatase such as Wip1, a serine/threonine phosphatase, might be working in the negative regulation of p38.In a sharp contrast, hyperphosphorylation of ERK was observed in the ERK^<sem> knockin mice. These mice revealed high perinatal morbidity. In the future project, it is possible to study the effect of ERK^<sem> in the immune system by conditional expression of ERK^<sem>.