The cyclin D3-CDK4-p27kip1 holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFbeta, and phosphorylations of its subunits demonstrated by two-dimensional gel electrophoresis.
The cAMP-dependent mitogenic stimulation elicited by thyroid-stimulating hormone (TSH) in primary cultures of canine thyroid epithelial cells is unique as it upregulates the cyclin-dependent kinase (CDK) inhibitor p27kip1 but not D-type cyclins. TSH and cAMP promote the assembly of required cyclin D3-CDK4 complexes and their nuclear import. Here, the nuclear translocation of these complexes strictly correlated in individual cells with the enhanced presence of nuclear p27. p27, like cyclin D3, supported the TSH-stimulated pRb-kinase activity of the CDK4 complex and, as demonstrated using the high-resolution power of the two-dimensional (2D) gel electrophoresis, the phosphorylation of CDK4, presumably by the nuclear CDK-activating kinase. In the presence of TSH, transforming growth factor beta (TGFbeta) did not affect the assembly of cyclin D3-CDK4, but it strongly inhibited the pRb-kinase activity associated with both cyclin D3 and p27, not only by preventing the nuclear import of cyclin D3-CDK4 and its binding to p27, but also by inhibiting CDK4 phosphorylation within residual p27-bound cyclin D3-CDK4 complexes. No alterations of the relative abundance of multiple (un)phosphorylated forms of cyclin D3 and p27 demonstrated by 2D-gel electrophoresis were associated with these processes. This study suggests a crucial positive role of p27 in the TSH-stimulated nuclear import, phosphorylation, and catalytic activity of cyclin D3-bound CDK4. Moreover, it demonstrates a technique to directly assess the in vivo phosphorylation of endogenous CDK4, which might appear as a last regulated step targeted by the antagonistic cell cycle effects of TSH and TGFbeta.
Institute of Interdisciplinary Research, Faculté de Médecine, Université Libre de Bruxelles, Campus Erasme, B-1070 Brussels, Belgium.
Katia
K
Coulonval
Laurence
L
Bockstaele
Sabine
S
Paternot
Jacques E
JE
Dumont
Pierre P
PP
Roger
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000235:genetics; D000818:Animals; D020134:Catalytic Domain / Q000187:drug effects / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D056743:Cyclin D3; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000187:drug effects / Q000378:metabolism*; D016213:Cyclins / Q000187:drug effects / Q000378:metabolism*; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D020035:Holoenzymes / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D013972:Thyrotropin / Q000378:metabolism / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins / Q000187:drug effects / Q000378:metabolism*
D018797:Cell Cycle Proteins; D056743:Cyclin D3; D016213:Cyclins; D020035:Holoenzymes; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013972:Thyrotropin; D017868:Cyclic AMP-Dependent Protein Kinases; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases