Binding of calmodulin to the carboxy-terminal region of p21 induces nuclear accumulation via inhibition of protein kinase C-mediated phosphorylation of Ser153.
Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.
Rodríguez-Vilarrupla
Aina
A
Department de Biologia Cel . lular, Fac. Medicina, Barcelona, Spain.
Jaumot
Montserrat
M
Abella
Neus
N
Canela
Núria
N
Brun
Sonia
S
Díaz
Carmen
C
Estanyol
Josep M
JM
Bachs
Oriol
O
Agell
Neus
N
D021581:Active Transport, Cell Nucleus; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002147:Calmodulin / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D003593:Cytoplasm / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012694:Serine / Q000737:chemistry*; D013347:Subcellular Fractions
D002147:Calmodulin; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018809:Proliferating Cell Nuclear Antigen; D049452:Green Fluorescent Proteins; D012694:Serine; D005982:Glutathione Transferase; D011493:Protein Kinase C