Mutation of Cys105 inhibits dimerization of p12CDK2-AP1 and its growth suppressor effect.
p12(CDK2-AP1) (p12) is a CDK2-associated protein that negatively regulates its kinase activity. Growth arrest of normal diploid cells by contact inhibition resulted in an induction of p27(kip1) and reduction of CDK2 levels. Interestingly, we observed concomitantly in growth-arrested cells, there was a reduction of nuclear p12 and the appearance of a nuclear 25-kDa molecule (p25) recognized by anti-p12 polyclonal antibody. Biochemical analysis showed that bacterial His-tagged p12 could be converted into a dimeric p25 in a reducing agent-dependent manner, and mutating the only cysteine residue of p12 (Cys(105) --> Ala(105)) abolished the dimerization. Transient transfection of wild type p12 into U2OS cells showed a reducing agent-sensitive dimerization that was also abolished by the C105A mutation. Furthermore, reduction of p12 expression by a short interfering RNA resulted in a parallel reduction of p25. These data supports the possibility that p25 is a homodimeric form of p12 through the cysteine residue. More interestingly, transient transfection of p12 (C105A) into the normal diploid lung fibroblast CCD18LU cells resulted in a reduction of the growth-inhibitory effect of p12 and abolished the inhibitory effect of p12 on CDK2 kinase activity. In addition, we found that the C105A mutation did not alter nuclear localization of p12, but it prevented association with CDK2. Taken together, our data suggest that p12 forms a nuclear homodimers in contact inhibited normal diploid cells and dimerization of p12 is a necessary process for the growth inhibition effect by p12.
Kim
Yong
Y
School of Dentistry and Dental Research Institute, UCLA, Los Angeles, California 90095, USA.
Ohyama
Hiroe
H
Patel
Vipel
V
Figueiredo
Marxa
M
Wong
David T
DT
D000409:Alanine / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004171:Diploidy; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008907:Mink; D009154:Mutation; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000378:metabolism
D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D006639:Histidine; D005982:Glutathione Transferase; D011494:Protein Kinases; C496849:Cdk2ap1 protein, mouse; D003545:Cysteine; D000409:Alanine