Abstract
Tristetraprolin (TTP) is a potential transcription factor that contains three PPPPG repeats and two putative CCCH zinc fingers. TTP is encoded by the early response gene Zfp-36, which is highly expressed in response to growth factors and in several hematopoietic cell lines. In the present studies, we investigated the possibility that TTP is phosphorylated in intact cells. In NIH/3T3 cells that were made to overexpress TTP constitutively, we found that the protein was phosphorylated on serine residues, and that this phosphorylation was rapidly (within 10 min) stimulated by several mitogens. In cell-free assays, recombinant mouse TTP was a substrate for the mitogen-activated protein (MAP) kinase. By a combination of protease digestion experiments and site-directed mutagenesis strategies, we found that serine 220 was phosphorylated by p42 MAP kinase in vitro. Expression of mutant TTP in fibroblasts confirmed that serine 220 was one of the major, mitogen-stimulated phosphorylation sites on the protein in intact cells. These results suggest that TTP may be phosphorylated by MAP kinases in vivo and that this phosphorylation may regulate its function.
Highlights
~ Associate of the Howard Hughes Medical Institute
2 TTP has been localized to the cell nucleus in mouse fibroblasts [2] and may well be a transcription factor, neither TTP nor any of the related zinc finger proteins has been shown to bind to specific DNA target sequences
The mRNA is constitutively highly expressed in several hematopoietic cells, with very high expression in macrophages and neutrophils, and somewhat lower expression in Band T lymphocytes." Recently, targeted disruption of Zfp-36 in mouse embryonic stem cells has been used to create mice that do not express TTP. 4 These mice are nornral at birth, but soon become runted and cachectic, develop patchy alopecia and dermatitis, and often die within the first few months after birth
Summary
Vol 270, No 22, Issue of June 2, pp. 13341-13347, 1995 Printed in U.S.A. Phosphorylation of Tristetraprolin, a Potential Zinc Finger Transcription Factor, by Mitogen Stimulation in Intact Cells and by Mitogen-activated Protein Kinase in Vitro*. Expression of mutant TTP in fibroblasts confirmed that serine 220 was one of the major, mitogen-stimulated phosphorylation sites on the protein in intact cells. These results suggest that TTP may be phosphorylated by MAP kinases in vivo and that this phosphorylation may regulate its function. Furthernrore, we show that serine 220 is one of the residues that is phosphorylated by p42 MAPK in vitro and that this site is phosphorylated in intact cells These experiments suggest that, in addition to controlling the availability of TIP protein by regulating gene transcription, certain mitogens may control its function by regulating its reversible phosphorylation
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