|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
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>.