Reduced Stability of Mitogen-activated Protein Kinase Kinase-2 mRNA and Phosphorylation of Poly(A)-binding Protein (PABP) in Cells Overexpressing PABP

Abstract

The poly(A)-binding protein (PABP) is an important regulator of mRNA translation and stability. The cellular level of PABP is controlled by regulating its mRNA translation by a feedback mechanism. The important aspect of this mechanism is that PABP binds to an adenosine-rich cis-element at the 5-untranslated region of its own mRNA and inhibits its translation. To assess the importance of controlling the PABP level, we studied the effect of PABP overexpression on the transcription profile using the microarray technique. In PABP-overexpressing cells, 19 mRNAs showed a reduction in cellular levels due to reduced mRNA stability, and one showed an increase due to increased mRNA stability. Among these mRNAs, the MKK-2 mRNA encodes the protein kinase activator of ERK1/2 kinase, which is involved in the phosphorylation of eukaryotic initiation factor (eIF) 4E. As a result, mRNA translation may be regulated by the cellular level of MKK-2. In this study, we show that the abundance of the MKK-2 polypeptide is reduced in PABP-overexpressing cells. In these cells, the levels of phosphorylated PABP, eIF4E, and ERK2 are also reduced. Treatment of HeLa cells with the MKK-2 inhibitor U0126 reduced PABP phosphorylation, suggesting that the phosphorylation of PABP is mediated by the MKK-2/ ERK signaling pathway. Thus, a novel signaling pathway involving MKK-2 and ERK1/2 may down-regulate the activity of PABP and eIF4E by controlling their phosphorylation and compensates for the effect of excess cellular PABP.Regulation of the rate of protein synthesis is important for the control of cellular growth and differentiation. Cells respond to changes in growth conditions by fine-tuning the rate of mRNA translation. Initiation of mRNA translation is the rate-limiting step and is often regulated by controlling the interaction between the 5Ј-cap of the mRNA and several initiation factors, including eukaryotic initiation factor (eIF) 2 4E, eIF4G, and eIF4A (1). A recent study has shown that the poly(A)-binding protein (PABP) also behaves like a genuine initiation factor because depletion of PABP from a cell-free extract prevents initiation of mRNA translation (2). The 3Ј-poly(A) tail-bound PABP interacts with eIF4G and circularizes the translating mRNA. This process is believed to enhance mRNA translation by promoting recirculation of terminating ribosomal subunits from the 3Ј-end of the mRNA for another round of initiation (3-6). Although this model is widely accepted, it has not been proven to occur in vivo. In addition, if PABP is essential for mRNA translation, it is possible that PABP participates in mRNA translation by a yet unknown mechanism, as the presence of 3Ј-poly(A) (and thus circularization of mRNA) is not essential for translation initiation of capped mRNAs (7,8).PABP binds to the 3Ј-poly(A) track of eukaryotic mRNA. It also interacts with polypeptides involved in regulating the translation and stability of mRNA (9 -12). Among the four highly conserved RNA-binding domains of PABP, the first two show specificity toward poly(A),...

Poly(A) Binding Protein C1 Is Essential for Efficient L1 Retrotransposition and Affects L1 RNP Formation

“…Using wild-type and mutant L1 constructs, we demonstrated that excessive overexpression of both PABPs caused hypersensitivity to puromycin. This is consistent with previous studies which showed that the consequences of unregulated overexpression of PABPN1 and PABPC1 included an increased cell doubling time, a reduced cloning efficiency, and a significantly changed expression level for at least 202 different genes (12,14,47,49).…”

Ataxin-2 Interacts with the DEAD/H-Box RNA Helicase DDX6 and Interferes with P-Bodies and Stress Granules

“…Alterations in the cellular level of PABP do not only affect synthesis of a variety of proteins, but the cellular protein expression profile as well (Ma et al, 2006). Overexpression of PABP leads to defects in cell division of the fission yeast Saccharomyces pombe (Tallada et al, 2002) and increases the translation termination efficiency in Saccharomyces cerevisiae (Cosson et al, 2002).…”

Systems perspectives on mRNA processing

“…Microarray profiling of cells aberrantly expressing RBPs has been employed to demarcate mRNA/RBP networks [25][26][27][28][29][30][31][32][33]. While this approach has been useful in identifying potential targets of tissue-specific RBPs [29,31], it has also revealed transcripts affected by proteins considered to be general processing factors.…”