Opposing Action of Casein Kinase 1 and Calcineurin in Nucleo-cytoplasmic Shuttling of Mammalian Translation Initiation Factor eIF6

Abstract

Eukaryotic initiation factor 6 (eIF6), a highly conserved protein from yeast to mammals, is essential for 60 S ribosome biogenesis and assembly. Both yeast and mammalian eIF6 are phosphorylated at Ser-174 and Ser-175 by the nuclear isoform of casein kinase 1 (CK1). The molecular basis of eIF6 phosphorylation, however, remains elusive. In the present work, we show that subcellular distribution of eIF6 in the nuclei and the cytoplasm of mammalian cells is mediated by dephosphorylation and phosphorylation, respectively. This nucleo-cytoplasmic shuttling is dependent on the phosphorylation status at Ser-174 and Ser-175 of eIF6. We demonstrate that Ca 2؉ -activated calcineurin phosphatase binds to and promotes nuclear localization of eIF6. Increase in intracellular concentration of Ca 2؉ leads to rapid translocation of eIF6 from the cytoplasm to the nucleus, an event that is blocked by specific calcineurin inhibitors cyclosporin A or FK520. Nuclear export of eIF6 is regulated by phosphorylation at Ser-174 and Ser-175 by the nuclear isoform of CK1. Mutation of eIF6 at the phosphorylatable Ser-174 and Ser-175 to alanine or treatment of cells with the CK1 inhibitor, D4476 inhibits nuclear export of eIF6 and results in nuclear accumulation of eIF6. Together, these results establish eIF6 as a substrate for calcineurin and suggest a novel paradigm for calcineurin function in 60 S ribosome biogenesis via regulating the nuclear accumulation of eIF6.Eukaryotic translation initiation factor 6 (eIF6) 3 is a highly conserved protein between yeast and mammals. Both the mammalian and yeast proteins are each 245 amino acid long and 72% identical in amino acid sequence (1, 2). Although eIF6 was originally isolated and characterized from the postribosomal supernatant of both wheat germ (3, 4) and mammalian cell extracts including anucleated rabbit reticulocyte lysates (1, 5, 6), most of our knowledge of the functional properties of eIF6 have been derived from molecular genetic studies examining the yeast eIF6 ortholog Tif6p in the yeast Saccharomyces cerevisiae (2, 7). These studies have provided compelling evidence that, at least in yeast cells, Tif6p, encoded by a single copy essential gene, does not function as a canonical translation initiation factor (2). Rather, Tif6p is essential for the biogenesis of 60 S ribosomal subunits in S. cerevisiae (2,7,8). Specifically, the lack of Tif6p prevents the processing of the 27SB pre-rRNA to form the mature 25 S and 5.8 S rRNAs, the constituents of the 60 S ribosomal particle (8). In agreement with the essential requirement of Tif6p in pre-ribosomal RNA processing, Tif6p is found to be a constituent of a multiprotein assembly complex associated with the pre-60 S ribosomal particles in the nucleolus, where biogenesis and maturation of 60 S ribosomal subunits take place (2, 9, 10). Indeed, in exponentially growing yeast cells, Tif6p is localized primarily in the nucleolus where most of the steps of 60 S ribosome biogenesis occur (11,12).In previous studies, we have observed that in both mamm...

Casein Kinase 1γ1 Inhibits the RIG-I/TLR Signaling Pathway through Phosphorylating p65 and Promoting Its Degradation

“…It has been reported that p65 can be phosphorylated at Ser 468 and Ser 536 and these modifications related to the activity of p65 (20)(21)(22), and it is known that CK1 family members regulate the biological process by phosphorylating their target molecules (10)(11)(12)(13)(14). We therefore examined whether CK1g1 phosphorylates p65.…”

“…The casein kinase 1 (CK1) is a family of Ser/Thr protein kinases that functions as regulator in signal transduction of eukaryotic cells (10). Studies have shown that CK1 family members were involved in many biological processes, such as the regulation of Wnt signaling (11), circadian rhythm (12), nucleus-cytoplasm shuttling of transcriptional factors (13), mitosis, and DNA repair (14). In this study, we demonstrated that CK1g1 phosphorylates NF-kB subunit p65 and promotes its ubiquitin-mediated degradation and therefore inhibits the RIG-I signaling pathway.…”

The Activity-Dependent Regulation of Protein Kinase Stability by the Localization to P-Bodies

“…Hrr25 belongs to the casein kinase 1 (CK1) family of protein kinases and is the yeast ortholog of the mammalian CK1d enzyme (DeMaggio et al 1992). The Hrr25/CK1d proteins have conserved roles in ribosome maturation, vesicle trafficking, DNA repair, and chromosome segregation during meiosis (Hoekstra et al 1991;Petronczki et al 2006;Schafer et al 2006;Ray et al 2008;Grozav et al 2009;Biswas et al 2011;Isoda et al 2011;Lord et al 2011). We report here that this critical regulator of cell proliferation is efficiently recruited to P-bodies in both yeast and mammalian cells under all conditions examined.…”

CK1δ and CK1ε are components of human 40S subunit precursors required for cytoplasmic 40S maturation

“…Notably, CK1 has also been linked to 60S biogenesis because it has been shown that mammalian eIF6, the ortholog of yeast Tif6, is phosphorylated by CK1 (Basu et al, 2001), which leads to changes in the subcellular distribution of eIF6 (Basu et al, 2001;Biswas et al, 2011). However, it remains unclear how this phosphorylation affects nuclear export or cytoplasmic maturation of 60S precursors.…”