Identification of the nuclear localization signal of p21(cip1) and consequences of its mutation on cell proliferation.
Overexpression of p21(cip1) induces cell cycle arrest. Although this ability has been correlated with its nuclear localization, the evidence is not conclusive. The mutants that were used to inhibit its nuclear translocation could no longer bind to several proteins known to interact with the last 25 amino acids of p21(cip1). Here we used point mutation analysis and fusion of the proteins to DsRed to identify which amino acids are essential for the nuclear localization of p21(cip1). We conclude that amino acids RKR(140-142) are essential for nuclear translocation of p21(cip1). While wild-type DsRed-p21 induces cell cycle arrest in 95% of transfected cells, overexpression of cytoplasmatic p21AAA(140-142) arrested only 20% of transfected cells. We conclude that cytoplasmatic p21, with no deletion in the C-terminal region, had a much lower capacity to arrest the cell cycle.
Rodríguez-Vilarrupla
Aina
A
Departament de Biologia Cellular i Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, C/Casanova, Barcelona, Spain.
Díaz
Carmen
C
Canela
Núria
N
Rahn
Hans Peter
HP
Bachs
Oriol
O
Agell
Neus
N
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002147:Calmodulin / Q000378:metabolism; D002453:Cell Cycle; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D004252:DNA Mutational Analysis; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D017354:Point Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors
D002147:Calmodulin; C493957:Cdkn1a protein, mouse; D002868:Chromosomal Proteins, Non-Histone; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D019913:Nuclear Localization Signals; D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors