Cell cycle-dependent interaction of Mad2 with conserved Box1/2 region of human granulocyte-macrophage colony-stimulating factor receptor common betac.
Box1 and 2 (box1/2) are conserved cytoplasmic motifs located in the membrane proximal region of cytokine receptors, including the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor common betac. Deletion of box1/2 abrogated all the examined activities of GM-CSF, and this phenomenon is explained by the loss of binding by Jak2. To test if a molecule other than Jak2 interacting with the box1/2 region plays a role in GM-CSF receptor signal transduction, we screened for molecules interacting with the box1/2 region by a pull-down assay using recombinant purified protein of GST fused with the betac box1/2 region and a Ba/F3 cell lysate. The mouse homologue of Mad2 protein, which plays an important role in the M phase of the cell cycle, was revealed to associate with the box1/2 region specifically. Peptides corresponding to the box1 sequence also bound to Mad2, and mutation of the box1 decreased the Mad2 interaction. Deletion analysis indicated that interaction with box1/2 occurred through the C-terminal portion of Mad2. Mad2 is known to change affinity for binding partners cell cycle dependently. Binding affinity of Mad2 to box1/2 increased in the late M phase, suggesting the possibility that GM-CSF participates in regulation of the M phase check point through interaction with Mad2.
Takeda
M
M
Department of Molecular and Developmental Biology, Institute of Medical Science, Core Research for Evolutional Science and Technology, Japan.
Dohmae
N
N
Takio
K
K
Arai
K
K
Watanabe
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D006801:Humans; D064246:Mad2 Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012097:Repressor Proteins
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins