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Growth arrest induced by transforming growth factor beta 1 is accompanied by protein phosphatase activation in human
P A Gruppuso1, R Mikumo, D L Brautigan
1Department of Pediatrics, Brown University, Providence, Rhode Island 02912.
Abstract:
Protein phosphorylation and dephosphorylation are involved in regulation of cell growth. We tested the hypothesis that the growth inhibitory effect of transforming growth factor beta 1 (TGF-beta 1) involves activation of protein phosphatases. Exposure of human keratinocytes in culture to 400 pM TGF-beta 1 for 48 h led to 80% inhibition of DNA synthesis as measured by nuclear labeling. Incubation of cultured keratinocytes with 400 pM TGF-beta 1 rapidly activated (within 30 min) protein serine/threonine phosphatase, measured using phosphorylase as a substrate. Based on several criteria, including neutralization of activity with specific antibodies and inhibitor-2, TGF-beta 1-activated phosphorylase phosphatase was identified as protein phosphatase 1. TGF-beta 1 did not have rapid effects on protein serine/threonine phosphatase activity (type 2A) measured with histone phosphorylated by protein kinase C or on protein tyrosine phosphatase activity. However, protein tyrosine phosphatase was activated at 48 h, coincident with growth arrest. Differentiation, induced by the combination of TGF-beta 1 plus calcium or by serum, was not accompanied by further serine/threonine or tyrosine phosphatase activation. We conclude that induction of growth arrest in keratinocytes by TGF-beta 1 involves acute activation of protein phosphatase 1, while activation of protein tyrosine phosphatase may represent an additional mechanism for maintaining cells in a growth-arrested state.
Insights
Transforming growth factor beta 1 (TGF-beta 1) inhibits keratinocyte DNA synthesis by activating protein phosphatase 1. Protein tyrosine phosphatase activation may sustain this growth arrest.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell growth regulation is intricately linked to protein phosphorylation and dephosphorylation.
- Transforming growth factor beta 1 (TGF-beta 1) is a key regulator of cell proliferation and differentiation.
Purpose of the Study:
- To investigate the role of protein phosphatases in the growth inhibitory effects of TGF-beta 1 on human keratinocytes.
- To identify specific protein phosphatase types activated by TGF-beta 1 during growth arrest.
Main Methods:
- Human keratinocytes were treated with TGF-beta 1, and DNA synthesis was measured via nuclear labeling.
- Protein phosphatase activities (serine/threonine and tyrosine) were assayed using specific substrates.
- Immunological methods (antibodies and inhibitor-2) were used to identify the activated phosphatases.
Main Results:
- TGF-beta 1 (400 pM, 48 h) inhibited keratinocyte DNA synthesis by 80%.
- Rapid activation (30 min) of protein serine/threonine phosphatase, identified as protein phosphatase 1, was observed.
- Protein tyrosine phosphatase activation occurred at 48 h, coinciding with growth arrest.
Conclusions:
- TGF-beta 1 induces keratinocyte growth arrest through the acute activation of protein phosphatase 1.
- Subsequent activation of protein tyrosine phosphatase may contribute to maintaining the growth-arrested state.