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Differential regulation of p34cdc2 and p33cdk2 by transforming growth factor-beta 1 in murine mammary epithelial
M P Fautsch1, S T Eblen, R A Anders
1Thoracic Diseases Research Unit, Mayo Clinic, Rochester, Minnesota 55905, USA.
Abstract:
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.
Insights
Transforming growth factor-beta 1 (TGF-beta 1) inhibits cell cycle kinases. TGF-beta 1 reduces cdc2 protein synthesis and cdk2 kinase activity via distinct mechanisms in mammary epithelial cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cyclin-dependent kinases (cdks) regulate cell cycle progression.
- Transforming growth factor-beta 1 (TGF-beta 1) inhibits epithelial cell growth.
- CDKs are potential biomarkers for TGF-beta-induced growth inhibition.
Purpose of the Study:
- Investigate TGF-beta 1's effect on cdc2 and cdk2 in mouse mammary epithelial cells.
- Determine the mechanisms by which TGF-beta 1 affects cdk activity.
- Compare effects in normal (MME) and TGF-beta-resistant (BG18.2) cell lines.
Main Methods:
- Treatment of MME and BG18.2 cells with TGF-beta 1.
- Measurement of newly synthesized cdc2 protein levels.
- Assay of cdc2 and cdk2 kinase activity (histone H1 phosphorylation).
- Analysis of cdc2 mRNA and total protein levels.
Main Results:
- TGF-beta 1 decreased newly synthesized cdc2 protein within 6 hours.
- Histone H1 phosphorylation by cdc2 significantly decreased post-TGF-beta 1 addition.
- cdc2 mRNA and total protein levels were reduced only after 12 hours.
- TGF-beta 1 did not affect cdk2 protein synthesis but reduced its kinase activity.
- Distinct mechanisms were observed for cdc2 and cdk2 regulation by TGF-beta 1.
Conclusions:
- TGF-beta 1 impacts cdc2 kinase activity through reduced protein synthesis.
- TGF-beta 1 inhibits cdk2 kinase activity independently of protein synthesis.
- TGF-beta 1 utilizes distinct pathways to decrease the kinase activity of cdc2 and cdk2.