c-Src-Mediated Phosphorylation of hnRNP K Drives Translational Activation of Specifically Silenced mRNAs

Abstract

hnRNP K and hnRNP E1/E2 regulate human papilloma virus type 16 (HPV-16) L2 capsid protein mRNA and reticulocyte 15-lipoxygenase (LOX) mRNA expression in the course of cellular differentiation. The expression of the virus capsid protein L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium by repression of L2 mRNA translation in the deeper layers (13). The underlying inhibitory mechanism employs hnRNPs K and E1/E2 interacting with a specific cis-acting element in the 3Ј end of L2 mRNA (5). LOX is a key enzyme in erythroid cell differentiation. It can attack phospholipids of the mitochondrial membranes and participates in their breakdown in late reticulocytes (20,24). LOX expression is temporally restricted by translational silencing of LOX mRNA in erythroid precursor cells in the bone marrow and in early reticulocytes (12). The differentiation control element (DICE) in the 3Ј untranslated region (UTR) of LOX mRNA binds the KH domain proteins hnRNP K and hnRNP E1 to form translationally silenced mRNPs in immature erythroid cells (15,16). The hnRNP K/E1-DICE complex blocks 80S ribosome assembly by inhibition of 60S ribosomal subunit joining (15). This erythroid silencing mechanism can be recapitulated in vitro and in HeLa cells transfected with DICE-regulated reporter mRNAs and hnRNP K alone or together with hnRNP E1 (16). Like LOX mRNA silencing, the HPV-16 L2 mRNA mechanism has been shown to operate in HeLa cells as well (5).The C-terminal part of hnRNP K contains proline-rich domains, which enable hnRNP K to interact with the SH3 domains of members of the Src kinase family (3) such as c-Src itself (23,25,27), Fyn, and Lyn (27). c-Src and Lck, a further member of the Src kinase family, have been shown to be able to phosphorylate hnRNP K in vitro and to affect its binding to RNA (19). The functional significance of hnRNP K tyrosine phosphorylation by members of the Src family of kinases is as yet unknown.Here we show that hnRNP K specifically binds and activates c-Src. c-Src mediates tyrosine phosphorylation of hnRNP K and inhibition of its DICE binding activity. Moreover, we demonstrate that c-Src kinase specifically regulates hnRNP K function as a translational silencer in vivo. Our results identify a specific role of c-Src in posttranscriptional regulation via hnRNP K, and suggest a mechanism for how the differentiation-dependent translation of cellular and viral RNA could be activated in maturing cells. MATERIALS AND METHODSPlasmids. For luciferase (LUC) indicator constructs, the LUC cDNA from pGEM-LUC (Promega) was inserted into pSG5 (16). LUC-DICE and LUC-NR were made by insertion of the DICE or nonregulatory (NR) sequences of the LOX mRNA 3Ј UTR into the EcoRV site of pSG5 before insertion of the LUC open reading frame (18). pSG5-His-hnRNP K was generated using pSG5-hnRNP K (16) by insertion of an oligonucleotide coding for 10 histidine residues between the SmaI and XhoI sites, N-terminal to hnRNP K. The tyrosine-tophenylalanine mutants pSG5-His-...

Nuclear shift of hnRNP K protein in neoplasms and other states of enhanced cell proliferation

“…K protein is tyrosine phosphorylated by Src-family of kinases (Ostrowski et al, 2000a;Ostareck-Lederer et al, 2002). Serum treatment increases tyrosine phosphorylation of many proteins involved in signal transduction and gene expression (Wang, 1994).…”

“…K protein is modified in response to changes in extracellular environment including cytokines, growth factors and oxidative stress (Ostrowski et al, 1991;2000a). Changes in K protein phosphorylation regulate its interaction with nucleic acids and protein partners (Schullery et al, 1999;Ostrowski et al, 2000a;Ostareck-Lederer et al, 2002). These properties suggest that K protein bridges signal transduction pathways to nucleic aciddirected processes.…”

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors

“…33 However, it is likely that multiple signalling events are required for ITAF activation and it has been shown recently that the cmyc-IRES-ITAF, hnRNPK, interacts with c-Src kinase, leading to c-Src activation and tyrosine phosphorylation of hnRNPK in vivo and in vitro. 72 This raises the possibility that regulation of hnRNPK through changes in phosphorylation could affect c-myc IRES function.…”

Control of mRNA translation and stability in haematopoietic cells: The function of hnRNPs K and E1/E2

“…C-Src is activated by its substrate hnRNP K. Tyrosine phosphorylation of hnRNP K by c-Src inhibits the binding of hnRNP K to the DICE and leads to translational activation of silenced DICE bearing mRNAs. In contrast to hnRNP K, hnRNP E1 is not an activator or substrate of c-Src (Ostareck-Lederer et al, 2002). We have not been able to identify a modification of hnRNP E1 that interferes with its DICE binding activity.…”