Ribosomal RACK1:Protein Kinase C βII Phosphorylates Eukaryotic Initiation Factor 4G1 at S1093 To Modulate Cap-Dependent and -Independent Translation Initiation

Abstract

Eukaryotic ribosomes contain the high-affinity protein kinase C βII (PKCβII) scaffold, receptor for activated C kinase (RACK1), but its role in protein synthesis control remains unclear. We found that RACK1:PKCβII phosphorylates eukaryotic initiation factor 4G1 (eIF4G1) at S1093 and eIF3a at S1364. We showed that reversible eIF4G(S1093) phosphorylation is involved in a global protein synthesis surge upon PKC-Raf-extracellular signal-regulated kinase 1/2 (ERK1/2) activation and in induction of phorbol ester-responsive transcripts, such as cyclooxygenase 2 (Cox-2) and cyclin-dependent kinase inhibitor (p21), or in 5' 7-methylguanosine (mG) cap-independent enterovirus translation. Comparison of mRNA and protein levels revealed that eIF4G1 or RACK1 depletion blocked phorbol ester-induced Cox-2 or p21 expression mostly at the translational level, whereas PKCβ inhibition reduced them both at the translational and transcript levels. Our findings reveal a physiological role for ribosomal RACK1 in providing the molecular scaffold for PKCβII and its role in coordinating the translational response to PKC-Raf-ERK1/2 activation.

Ribosomal protein RACK1 facilitates efficient translation of poliovirus and other viral IRESs

“…We thus employed RACK1 knockout cells generated by CRISPR-Cas9 mediated genome editing (45) and transduced them with lentiviruses to express RACK1-FLAG, which was incorporated into translating ribosomes (figure 1). In contrast to others who have reported that RACK1 depletion reduces cap-dependent translation (30, 56), we do not observe major changes in cap-dependent translation in RACK1 KO cells ((45), figure 4A and unpublished data). It is possible that the contrasting observations are due to cell line specific differences.…”

“…It is possible that the contrasting observations are due to cell line specific differences. While HAP1 cells are derived from the chronic myelogenous leukemia (CML) cell line KBM-7, all studies that found that RACK1 influenced cap-dependent and -independent translation were performed in HEK293, HEK293T, and HeLa cells (30, 31, 56). Since RACK1 stimulates global translation in a PKCβII-dependent manner (30, 43), potentially higher PKCβII expression levels found in HEK293 and HeLa cells or other cell line-specific differences might explain the opposing effect on translation ((57), https://www.proteinatlas.org/ENSG00000166501-PRKCB/cell).…”

“…Directly and/or indirectly, RACK1 also influences translation of cellular mRNAs. The Saccharomyces cerevisiae RACK1 homolog, Asc1, facilitates efficient translation of mRNAs containing a short open reading frame (42), while in mammalian cells, RACK1 appears to stimulate cell proliferation in a PKCβII-dependent manner (30, 43, 44).…”

Ribosomal RACK1:Protein Kinase C βII Modulates Intramolecular Interactions between Unstructured Regions of Eukaryotic Initiation Factor 4G (eIF4G) That Control eIF4E and eIF3 Binding

“…Cell lines, DNA transfections, and eIF4G expression plasmids. HEK293 cells were grown, transfected, synchronized, and treated with kinase inhibitors and/or activators as described earlier (4). Construction of Myc/Flag-tagged eIF4G expression plasmids has been described previously (4); the eIF4G fragments used in this study were generated by PCR with the corresponding primers (Table 1), and mutations were introduced by overlapping PCR as described earlier (4).…”

“…phorbol-13-acetate (TPA)-responsive template-specific translation in an eIF4G-, PKC␤-, and RACK1-dependent manner (4). We showed anomalous TPA-inducible eIF3 binding to nonphospho (S1093A) or phosphomimic (S1093E) variants of eIF4G, indicating that reversible S1093 phosphorylation regulates eIF4G's assembly with eIF3 and 40S ribosomal subunits (4).…”