Analysis of MAP kinase cascade in the immune response

丝裂原活化蛋白激酶(MAPK)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。虽然小鼠中每个MAPK的基因敲除有助于阐明其独特的功能，但很难找到其多余的功能。因此，使用MAPK功能增益突变体的替代方法可能有助于1)构建和分析具有结构性活性的MAPK融合分子。MKK6是p38的MAPKK(MAPK激酶)，它揭示了没有上游激活信号的弱激酶活性。为了创造一个具有结构性活性的MAPK突变体，我们将MKK6与p38α(MKK6-p38α)融合。P38α在MKK6-p38α中被结构性磷酸化，ATF2ATF2报告活性增强。另一方面，当测试MKK6-p38β时，p38β被结构性磷酸化，但ATF2ATF2报告活性没有增加。在某些情况下，MKK6-p38与下游底物的结合可能会受到限制。2)MAPK的建立和分析。Seven Maker(Sem)是CD区MAPK的单一氨基酸替代。我们已经证明，p38^&lt；sem&gt；和ERK^&lt；sem&gt；不能与蛋白酪氨酸磷酸酶结合，并且不能被这些磷酸酶失活。到目前为止，在p38^&lt；sem&gt；敲门小鼠中还没有观察到p38的过度磷酸化。这些小鼠是可以存活的。蛋白酪氨酸磷酸酶和丝氨酸/苏氨酸磷酸酶Wip1等冗余的磷酸酶可能参与了p38的负调控。与之形成鲜明对比的是，ERK基因敲门小鼠的ERK蛋白过度磷酸化。这些小鼠表现出较高的围产期发病率。在未来的项目中，有可能通过ERK^&lt；sem&gt；的条件表达来研究ERK^&lt；sem&gt；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>.