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Phosphorylation of Recombinant Tristetraprolin In vitro

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Abstract

Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some proinflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of proinflammatory cytokines. TTP gene expression is induced by various factors including insulin, cinnamon, and green tea extracts. Previous studies have shown that TTP is highly phosphorylated in vivo and multiple phosphorylation sites are identified in human TTP. This study evaluated the potential protein kinases that could phosphorylate recombinant TTP in vitro. Motif scanning suggested that TTP was a potential substrate for various kinases. SDS-PAGE showed that in vitro phosphorylation of TTP with p42 and p38 MAP kinases resulted in visible electrophoretic mobility shift of TTP to higher molecular masses. Autoradiography showed that TTP was phosphorylated in vitro by GSK3b, PKA, PKB, PKC, but not Cdc2, in addition to p42, p38, and JNK. These results demonstrate that TTP is a substrate for a number of protein kinases in vitro.

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Abbreviations

TTP:

Tristetraprolin

hTTP:

Human TTP

mTTP:

Mouse TTP

ARE:

AU-rich element

Cdc2:

Cyclin-dependent kinase 2

CIAP:

Calf intestine alkaline phosphatase

GSK3b:

Glycogen synthase kinase 3B

JNK:

c-Jun-N-terminal kinase

LPS:

Lipopolysaccharide

MAPK:

Mitogen-activated protein kinase

MBP:

Maltose binding protein

MK2:

MAP kinase-activated protein kinase 2

PK:

Protein kinase

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TNF:

Tumor necrosis factor alpha

ZFP:

Zinc finger protein

ZFP36L:

ZFP36-like

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Acknowledgements

This work was supported in part by USDA-ARS Human Nutrition Research Program and NIH-NIEHS Intramural Research Program. A preliminary report of this study was presented at the Experimental Biology 2006 in San Francisco, California, on April 1–5, 2006. We greatly appreciate Dr. Perry J. Blackshear (NIH/NIEHS) for his generous support. We also thank Dr. Joseph F. Urban Jr. (USDA/ARS) for his helpful comments on the manuscript.

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Author notes

  1. Rui Lin

    Present address: National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA

Authors and Affiliations

  1. Diet, Genomics and Immunology Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, US Department of Agriculture, Building 307C, BARC-East, 10300 Baltimore Avenue, Beltsville, MD, 20705, USA

    Heping Cao

  2. Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA

    Heping Cao & Rui Lin

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  1. Heping Cao
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  2. Rui Lin
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Correspondence to Heping Cao.

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Cao, H., Lin, R. Phosphorylation of Recombinant Tristetraprolin In vitro. Protein J 27, 163–169 (2008). https://doi.org/10.1007/s10930-007-9119-7

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