Identification of a protein kinase which phosphorylate alpha 4 subunit of the 26S proteasome in goldfish oocytes

摘要

To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2-phase) and mature (in M-phase) oocytes, and compared its subunits by immunoblotting. A monoclonal antibody, GC3beta (anti-goldfish 20S proteasome component 3beta) cross-reacted with two bands in the 26S proteasome from immature oocytes, however the upper band was absent in the 26S proteasome from mature oocytes. cDNAs which encode the alpha4 subunit of goldfish 20S proteasome (alpha4_ca) were isolated by an immuno-screening method using GC3beta. Phosphatase treatment of the 26S proteasome revealed that a part of alpha4_ca phosphorylated in G2-phase and dephosphorylated in M-phase. By the assay using recombinant alpha4_ca as a substrate, a kinase was purified by column chromatographs. Amino acid sequence analysis was performed for resulting partial purified fraction. A protein band, which well corresponded to the kinase activity, was identified as Casein kinase-1alpha (CK-1alpha). The result suggests that CK-1alpha phosphorylate a4 subunit of the 26S proteasome in immature oocyte of goldfish.