Phosphorylation of heterogeneous nuclear ribonucleoprotein K at an extracellular signal-regulated kinase phosphorylation site promotes neurofilament-medium protein expression and axon outgrowth in Xenopus

Highlights

• ERK1 phosphorylates a single serine residue (S257) on Xenopus hnRNPK.

• ERK site mutations at S257(A/D) fail to alter subcellular localization of hnRNPK.

• Phosphorylation of S257 is necessary for both NF-M translation and axon outgrowth.

Abstract

Post-transcriptional control of cytoskeletal genes fine-tunes the supply of structural materials to growing axons in response to extracellular cues. In Xenopus, heterogeneous nuclear ribonucleoprotein K (hnRNPK) plays a crucial role in the nuclear export and translation of multiple cytoskeletal-related mRNAs required for axon outgrowth, and as a substrate of multiple kinases, is thus a likely molecular target of cell signaling pathways regulating such outgrowth. To study the role of hnRNPK’s phosphorylation by extracellular signal-regulated kinase (ERK) in Xenopus axon outgrowth, we identified the only ERK1 phosphorylation site on Xenopus hnRNPK (S257; homologous with S284 of human hnRNPK) using an in vitro phosphorylation assay and tested its function in vivo by expressing phosphomimetic (S257D) and phosphodeficient (S257A) forms of hnRNPK in Xenopus embryos. Although neither form altered hnRNPK nuclear export, only the phosphomimetic form significantly rescued both neurofilament protein expression and axon outgrowth from hnRNPK knockdown. This finding represents a previously unidentified function of phosphorylation at this phylogenetically conserved site and implicates hnRNPK as an intracellular molecular target of ERK-mediated signaling in axon outgrowth.

Introduction

Post-transcriptional control of cytoskeletal gene expression is crucial for the development and maintenance of healthy axons [23]. By enabling neurons to respond more effectively to extracellular cues, it augments fine control over transcription in supplying structural proteins needed to build and maintain the axon. Such control is largely mediated through the actions of RNA-binding proteins, which coordinate cell signaling with the intracellular trafficking, stability, and translation of mRNAs [17]. In neurons of the frog Xenopus laevis, the RNA-binding protein hnRNPK regulates nuclear export and translation of multiple cytoskeletal-related transcripts, including those of Type IV neurofilament (NF) proteins, as well as others associated with microtubules and microfilaments (e.g., tau, ARP2) [13]. Knockdown of hnRNPK by antisense morpholino oligonucleotide (MO) collectively suppresses translation but not transcription of these mRNAs, leading to failure of embryonic and regenerative axon outgrowth [13], [14].

hnRNPK’s functions are regulated by multiple kinases [2], including two mitogen-activated protein kinases, c-Jun N-terminal kinase (JNK) [7] and extracellular signal-regulated kinase (ERK) [8]. In developing Xenopus neurons, JNK phosphorylation of Xenopus hnRNPK (XhnRNPK) is required for both axonogenesis and translation of hnRNPK’s targeted cytoskeletal transcripts [10]. Although, like JNK activity, ERK activity also promotes neurite and axon outgrowth [9], [26], its effects on hnRNPK in this context have yet to be examined. In human cell lines, ERK directly stimulates hnRNPK’s nuclear export [8], raising the hypothesis that it does the same for XhnRNPK. However, other possibilities exist, since ERK exerts effects on cells that are mediated by hnRNPK but not directly attributable to nuclear export [5], [11]. Moreover, the studies in mammalian cell lines made no distinction between two ERK phosphorylation sites of hnRNPK. Whereas one site lies within a well-characterized nucleocytoplasmic shuttling (KNS) domain, the other lies within a protein–protein interaction domain (KI) having multiple functions [2]. Also, XhnRNPK lacks a functioning KNS domain [15]. Collectively, these observations suggest that ERK phosphorylation of XhnRNPK could operate through a different mechanism, or alternatively play no role whatsoever, in axon development. Here, we provide evidence that a phylogenetically conserved, proline-directed serine residue within the KI domain of XhnRNPK (S257) is the only site phosphorylated by ERK and that phosphorylation at this site indeed promotes axon outgrowth. However, it does so not by stimulating nuclear export of XhnRNPK mRNP complexes, but rather by promoting translation of XhnRNPK-targeted mRNAs of axonal cytoskeletal proteins.