Akt-dependent T198 phosphorylation of cyclin-dependent kinase inhibitor p27kip1 in breast cancer.
The localization of the cyclin-dependent kinase inhibitor p27(kip1) is dependent on the phosphorylation of one of three key amino acid residues: S10, T157 and T198. However, it was unclear whether endogenous p27(kip1) is phosphorylated at T198 in the living cell. In the present work we describe the generation and characterization of a polyclonal antibody able to recognize recombinant, transfected as well as endogenous T198-phosphorylated p27(kip1). Using this antibody, we demonstrate that: (1) endogenous p27(kip1) is phosphorylated at T198 in 4 breast cancer cells lines (MCF7, MDA-MB231, MDA- MB436 and MDA-MB468); (2) T198 phosphorylation is increased in breast cancer cells compared with normal mammary epithelial cells (HMEC); (3) T198-phosphorylated p27(kip1) is exclusively cytoplasmic; (4) T198 phosphorylation is dependent on the activity of the PI3K-PKB/Akt pathway, being it drastically reduced by the pharmacological PI3K inhibitor LY294002 or stimulated by the constitutive activation of PKB/Akt. Finally, in primary human breast carcinomas, cytoplasmic accumulation of T198-phosphorylated p27(kip1) parallels Akt activation. We conclude that in breast cancer cells p27(kip1) is phosphorylated at T198 in a PI3K/Akt dependent manner and that this phosphorylation may contribute to p27(kip1) cytoplasmic mislocalization observed in breast cancer.
Dipartimento di Biologia e Patologia Cellulare e Molecolare L Califano Facoltà di Medicina e Chirurgia, Università Federico II, via S. Pansini 5, Napoli, Italy.
Maria Letizia
ML
Motti
Carmela
C
De Marco
Daniela
D
Califano
Alfredo
A
Fusco
Giuseppe
G
Viglietto
D000595:Amino Acid Sequence; D001940:Breast / Q000166:cytology; D001943:Breast Neoplasms / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004789:Enzyme Activation / Q000502:physiology; D004847:Epithelial Cells / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000196:embryology / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D010710:Phosphates / Q000276:immunology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D013912:Threonine / Q000276:immunology / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism* / Q000502:physiology
D018797:Cell Cycle Proteins; D010710:Phosphates; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt