RESUMO
Cyclin-dependent kinases (cdks) are a family of proteins whose function plays a critical role in cell cycle traverse. Transforming growth factor-beta 1 (TGF-beta 1) is a potent growth inhibitor of epithelial cells. Since cdks have been suggested as possible biochemical markers for TGF-beta growth inhibition, we investigated the effect of TGF-beta 1 on cdc2 and cdk2 in a normal mouse mammary epithelial cell line (MME) and a TGF-beta-resistant MME cell line (BG18.2). TGF-beta 1 decreases newly synthesized cdc2 protein levels within 6 h after addition. Coincident with this decrease in newly synthesized cdc2 protein was a marked reduction in its ability to phosphorylate histone H1. This decrease in kinase activity is not due to a change in steady-state levels of cdc2 protein, since mRNA and total protein levels of cdc2 are not reduced until 12 h after TGF-beta 1 addition. This suggests that the kinase activity of cdc2 is dependent on newly synthesized cdc2 protein. Moreover, the protein synthesis of another cyclin-dependent kinase, cdk2, is not effected by TGF-beta 1 addition, but its kinase activity is substantially reduced. Thus, it appears that TGF-beta decreases the kinase activity of both cdc2 and cdk2 by distinct mechanisms.
Assuntos
Proteína Quinase CDC2/farmacologia , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos , Northern Blotting , Linhagem Celular , Quinase 2 Dependente de Ciclina , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/enzimologia , Feminino , Humanos , Immunoblotting , Cinética , Glândulas Mamárias Animais , Camundongos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Fosforilação , Protamina Quinase/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Fatores de TempoRESUMO
The cell cycle inhibitor mimosine was used to examine the activation of the p34cdc2 protein kinase in S phase of the cell cycle. Addition of mimosine to cycling epithelial cells halted cell cycle traverse in S phase, coincident with an inhibition of p34cdc2 histone H1 kinase activity. Mimosine treatment did not alter p34cdc2 synthesis or turnover; however, overall phosphorylation of p34cdc2 was decreased to near undetectable levels. Although activity of p34cdc2 was inhibited, the ability of the protein to form high molecular weight complexes, a phenomenon associated with kinase activation in vivo, was not affected. These results indicate that p34cdc2 complex formation can occur in the absence of phosphorylation and that phosphorylation of p34cdc2 is then required to activate these preformed complexes.
Assuntos
Proteína Quinase CDC2/metabolismo , Fator Promotor de Maturação/metabolismo , Protamina Quinase/metabolismo , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/biossíntese , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Células Cultivadas , Ativação Enzimática , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/enzimologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Mimosina/farmacologia , Vison , Peso Molecular , Fosforilação , Protamina Quinase/antagonistas & inibidores , Protamina Quinase/biossíntese , Fase S/efeitos dos fármacos , Fase S/fisiologiaRESUMO
Cycling epithelial cells were shown to reversibly arrest in late G1 phase following treatment with transforming growth factor beta 1. Associated with this G1-S phase arrest was a decrease in the synthesis and histone H1 kinase activity of p34cdc2. Transforming growth factor beta 1 did not reduce p34cdc2 levels by modulating the turnover of newly synthesized p34cdc2. The decrease in p34cdc2 synthesis preceded any detectable effect on DNA synthesis. Moreover, the action of transforming growth factor beta 1 was regulated in a cell cycle-specific manner; epithelial cells were sensitive to transforming growth factor beta 1 only during the G1 phase. The results suggest that p34cdc2 might be a useful biochemical marker for investigating the mechanism(s) of transforming growth factor beta 1 signaling.