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Phosphatase regulation of gene expression during development of the palate
Wayde M Weston1, Angela B Freeman, Christian Haberecht
1Pulmonary/Diabetes Therapeutic Unit, UP4310, SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Road, PO Box 5089, Collegeville, PA 19426, USA.
Abstract:
In mammalian cells, including those of the embryonic palate, the level of phosphorylation of cellular proteins at any given time reflects the activities of protein kinases and protein phosphatases. Both protein phosphatase-1 (PP-1) and PP-2A inhibit cAMP-mediated increases in transcription by dephosphorylating CREB at ser-133. Western blot analysis indicated that protein phosphatase 1 (PP-1) was expressed constitutively in palatal tissue during its development. Expression of PP-2A was regulated developmentally with maximal expression on gestational day (gd) 14. Densitometric scanning revealed a 30% increase in expression from gd 13 to gd 14. Virtually all phosphatase activity in the tissue extracts could be inhibited by 5 microM okadaic acid, demonstrating that PP-1 and PP-2A account for all detectable ser/thr protein phosphatase activity present in the developing palate. Moreover, no significant differences in PP-1 and PP-2A activities were observed during the period of palate development. Treatment of primary cultures of murine embryonic palate mesenchymal (MEPM) cells with forskolin (20 microM) to elevate intracellular cAMP levels, resulted in a time-dependent increase in CREB ser-133 phosphorylation and a corresponding time dependent decrease in PP-1 and PP-2A levels. Moreover, treatment of MEPM cells with okadaic acid resulted in a dramatic increase in basal CREB ser-133 phosphorylation. This suggests that PP-1 activity may contribute to transcriptional regulation of CREB and that PP-1 and PP-2A are regulated differentially by cAMP. Treatment of MEPM cells with TGF beta 1 (1 ng/ml) under conditions of TGF beta-induced CREB phosphorylation resulted in no effect on the expression of either PP-1 or PP-2A proteins and no significant alterations in total basal protein phosphatase activity. These results demonstrate that transcriptional regulation of CREB in embryonic palatal issue is dependent on the coordinate activity of specific kinases and phosphatases.
Insights
Protein phosphatases, specifically PP-1 and PP-2A, regulate CREB phosphorylation in developing mammalian palate. Their activity is crucial for transcriptional regulation, with differential responses to cAMP signaling.
Area of Science:
- Cellular Biology
- Molecular Biology
- Developmental Biology
Background:
- Protein phosphorylation dynamics, governed by kinases and phosphatases, dictate cellular protein activity.
- Protein Phosphatase-1 (PP-1) and Protein Phosphatase-2A (PP-2A) dephosphorylate CREB at Ser-133, inhibiting cAMP-mediated transcription.
Purpose of the Study:
- To investigate the roles and regulation of PP-1 and PP-2A in the developing mammalian palate.
- To elucidate the involvement of these phosphatases in the transcriptional regulation of CREB.
Main Methods:
- Western blot analysis to assess PP-1 and PP-2A expression in developing palatal tissue.
- Enzyme activity assays using okadaic acid to quantify phosphatase activity.
- Treatment of murine embryonic palate mesenchymal (MEPM) cells with forskolin and TGF-β1 to study signaling pathways.
Main Results:
- PP-1 was constitutively expressed, while PP-2A expression peaked at gestational day 14 in palatal tissue.
- Forskolin treatment increased CREB phosphorylation and decreased PP-1/PP-2A levels in MEPM cells, indicating differential regulation by cAMP.
- Okadaic acid treatment significantly increased basal CREB phosphorylation, highlighting the role of phosphatases.
Conclusions:
- PP-1 and PP-2A are the primary serine/threonine phosphatases in the developing palate.
- Transcriptional regulation of CREB in the embryonic palate relies on the coordinated action of kinases and phosphatases.
- PP-1 and PP-2A exhibit differential regulation by cAMP signaling pathways.