Abstract
Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.
Highlights
Phosphorylation of 4E binding protein 1 (4E-BP1) plays a critical role in controlling its ability to inhibit protein translation
Identification of PPM1G as the Phosphatase for 4E-BP1—In our studies to examine the function of mammalian target of rapamycin (mTOR) in regulating protein translation, we became interested in determining which protein phosphatases may be involved in dephosphorylating 4E-BP1, the key downstream effector of mTOR
Increased levels of 4E-BP1 phosphorylation and expression of eIF4E have been found to be associated with higher tumor grade and decreased survival in breast cancer [23]
Summary
Phosphorylation of 4E-BP1 plays a critical role in controlling its ability to inhibit protein translation. Results: PPM1G is identified as a protein phosphatase that dephosphorylates 4E-BP1 in vitro and in cells. Our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1. Numerous studies have demonstrated that mammalian target of rapamycin (mTOR) plays a critical role in controlling the translation initiation step in protein synthesis [5]. We report the identification of PPM1G as the phosphatase that directly dephosphorylates multiple phosphorylation sites in 4E-BP1 both in vitro and in cells. We examine the role of PPM1G in regulating cap-dependent protein translation by controlling the binding of 4E-BP1 with the cap structure
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