|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 2000 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1999 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1998 : ¥1,200,000 (Direct Cost : ¥1,200,000)
The main purpose of the project was to investigate in vitro and in vivo effects of IDA MAPK peptide, corresponding to the activation segment of MAPK, and elucidate the molecular mechanisms of its action, as well as to investigate the involvement of MAPK pathway in the cell cycle control and signal transduction.
Therefore, we synthesized phosphorylated and unphosphorylated forms of the peptide and investigated their in vitro effect on the activity of various protein kinases. We found that unphosphorylated IDA MAPK peptide could inhibit in vitro both MAPK and MAPK kinase, but not some other protein kinases, including MAPK homologue p38 kinase, suggesting high specificity of inhibition.
In vivo, we used IDA MAPK peptide to inhibit the activation of MAPK pathway in Xenopus oocytes upon maturation and found that unphosphorylated peptide suppressed markedly both MAPK activation and oocyte maturation in response to progesterone. IDA MAPK peptide effectively inhibited MAPK pathway in cytostat
ic factor-arrested and cycling Xenopus egg extracts with little effect on the activity of Cdc2 kinase. The peptide shortened duration of mitosis, abolishing the spindle assembly checkpoint in cycling extracts.
Using anti-IDA MAPK antibody we demonstrated that a rearrangement of MAPK activation segment takes place at low pH.It promotes a stable low-activity conformation of the enzyme which is favorable for intramolecular autophosphorylation.
Monitoring MAPK activity we found that the fertilization-induced tyrosine dephosphorylation of MAPK, as well as the calcium response, are inhibited by the Src kinase inhibitor PP1 and the PLC inhibitor U-73122 and further demonstrated that tyrosine kinase-dependent activation of phospholipase Cγ is required for calcium transient in Xenopus egg fertilization.
Using MAPK reactivation as a marker of cell cycle progression, we demonstrated that calcium oscillations in cell-free Xenopus egg cycling extracts were coordinated with cell cycle events and described three enzymatically and morphologically distinctive blocks of cell cycle that are characterized by different activities of MAPK and Cdc2 kinase. Less