Transforming growth factor-beta induces Cdk2 relocalization to the cytoplasm coincident with dephosphorylation of retinoblastoma tumor suppressor protein.
BACKGROUND
The transforming growth factor-beta (TGF-beta) signaling pathway functions to prevent tumorigenesis, and loss of sensitivity to TGF-beta-mediated cell cycle arrest is nearly ubiquitous among human cancers. Our previous studies demonstrated that rapamycin potentiates TGF-beta-induced cell cycle arrest in nontransformed epithelial cells and partially restores TGF-beta-induced growth arrest of some human cancer cell lines. Growth arrest correlated with increased binding of p21 and p27 to cyclin-dependent kinase-2 (Cdk2), and inhibition of Cdk2 kinase activity. However, it was unclear how TGF-beta caused increased binding of p21 and p27 to Cdk2.


METHODS
Cell fractionation and immunofluorescence microscopy experiments were performed to examine the effect of TGF-beta on the intracellular localization of Cdk2, p21, and p27. Kinase assays were performed on cytoplasmic and nuclear extracts to determine how TGF-beta altered Cdk2 activity in both subcellular compartments.


RESULTS
In breast epithelial cells treatment with TGF-beta induced a decrease in nuclear Cdk2 concentrations and relocalization of Cdk2 to the cytoplasm. Cdk2 relocalization to the cytoplasm correlated with dephosphorylation of nuclear retinoblastoma tumor suppressor protein and decreased nuclear Cdk2 activity. In these epithelial cell lines, p21 and p27 were localized primarily in the cytoplasm. Decreases in nuclear Cdk2 concentrations correlated with increased binding of Cdk2 to cytoplasmic p21 and p27.


CONCLUSIONS
Cooperative growth arrest induced by treatment with TGF-beta + rapamycin causes inhibition of nuclear Cdk2 activity through multiple mechanisms, including Cdk2 relocalization to the cytoplasm, increased p27 and p21 binding to Cdk2, and increased phosphorylation of nuclear Cdk2 on its inhibitory site, Tyr15.
Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA.
Kimberly A
KA
Brown
Richard L
RL
Roberts
Carlos L
CL
Arteaga
Brian K
BK
Law
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D042846:CDC2-CDC28 Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003593:Cytoplasm / Q000201:enzymology*; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000166:cytology; D042361:Mammary Glands, Human / Q000166:cytology; D051379:Mice; D010766:Phosphorylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D016160:Retinoblastoma Protein; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D020123:Sirolimus