c-Jun N-terminal kinase phosphorylation of heterogeneous nuclear ribonucleoprotein K regulates vertebrate axon outgrowth via a posttranscriptional mechanism.
c-Jun N-terminal kinase (JNK) mediates cell signaling essential for axon outgrowth, but the associated substrates and underlying mechanisms are poorly understood. We identified in Xenopus laevis embryos a novel posttranscriptional mechanism whereby JNK regulates axonogenesis by phosphorylating a specific site on heterogeneous nuclear ribonucleoprotein K (hnRNP K). Both JNK inhibition and hnRNP K knockdown inhibited axon outgrowth and translation of hnRNP K-regulated cytoskeletal RNAs (tau and neurofilament medium), effects that were alleviated by expressing phosphomimetic, but not phosphodeficient, forms of hnRNP K. Immunohistochemical and biochemical analyses indicated that JNK phosphorylation of hnRNP K occurred within the cytoplasm and was necessary for the translational initiation of hnRNP K-targeted RNAs but not for hnRNP K intracellular localization or RNA binding. Thus, in addition to its known roles in transcription and cytoskeletal organization, JNK acts posttranscriptionally through hnRNP K to regulate translation of proteins crucial for axonogenesis.
PubMed ID: 24027268
Article link: J Neurosci.
Genes referenced: dio3 gapdh gnl3 hnrnpc hnrnpk igf2bp3 jun mapk1 mapk8 mapt nefm prph sst
Antibodies referenced: Nuclear Lamins 2/3 Ab1
Morpholinos referenced: hnrnpk MO1
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|Figure 2. Serine 189, the JNK target site on Xenopus hnRNP K, is phosphorylated in neurons in vivo. A, Multiple sequence alignment of the region of hnRNP K surrounding X. laevis hnRNP K S189 (indicated by *) from five species shows that the prospective JNK site is highly conserved. Black and gray shading indicates 100 and 80% conservation among the different species examined, respectively. The black line indicates the region of Xenopus hnRNP K used to generate the phospho-specific antibody (pS189). B, C, Immunohistochemistry (IHC) on transverse sections of spinal cord and somites of stage 37/38 embryos. B, Sections of embryos unilaterally injected with hnRNP K MO at the two-cell stage. Overlays indicate locations of spinal cord (SC) and somites (Som). Unilateral MO suppression of endogenous hnRNP K caused a similar reduction of immunostaining (p = 0.9, two-sided t test, 5 sections per embryo, n = 3 embryos per condition) for both total hnRNP K (55 ± 1% injected/uninjected, p = 0.03, one-sided t test as before; B2) and pS189 (56 ± 5% injected/uninjected, p = 0.01, one-sided t test as before; B4) on the injected side, indicating that pS189 is specific for hnRNP K. Phase contrast images (B1, B3) demonstrated that loss of staining in spinal cord and somites was attributable to loss of expression on the injected side and not to loss of cells. C, Sections of uninjected embryos treated with alkaline phosphatase before immunostaining for total hnRNP K (C2) or pS189 (C4), demonstrating specificity of pS189 for phosphorylated hnRNP K. C1, C3, SYBR Green staining of nuclei demonstrated that loss of staining was not attributable to cell loss. D, E, Representative images of immunostaining (magenta) of stage 37/38 hindbrain sections (D; confocal microscopy) and neurons from dissociated neural tube/myotome cultures (E; conventional microscopy) with the pS189 antibody indicated hnRNP K was phosphorylated at S189 in vivo during nervous system development. Whereas total hnRNP K shuttles to the cytoplasm but localized predominantly to the nucleus during axon outgrowth (D1, D2, SYBR Green; E1–E3, DAPI, blue), phospho-hnRNP K (pS189) was primarily cytoplasmic and perinuclear (D3, D4, E4–E6). Arrows indicate regions measured for intensity profiles (E3, E6). Scale bars, 20 μm.|
|Figure 3. Phosphorylation at serine 189 on Xenopus hnRNP K is reduced by pharmacological inhibition of JNK. A–D, Representative neurons from control or SP600125-treated cultures stained for total hnRNP K and peripherin (XIF3; A, C) or pS189 and N-β-tubulin (B, D). E–H, Background intensity measurements for control (E, F) or SP600125-treated (G, H) cultures were taken from cultures stained without primary antibodies (Ab) to total hnRNP K (E, G) or pS189 (F, H). JNK inhibition caused a significant decrease in staining for pS189 but not total hnRNP K (I). Scale bars, 20 μm. ns, Nonsignificant, p = 0.5; *p = 0.02, two-sided t test, 30 neurons/culture, n = 5 cultures.|
|Figure 8. Phosphorylation of the JNK site on EGFP–hnRNP K regulates handoff of its RNA targets to the translational machinery. Polysome profiling followed by Western blot analysis of cytosolic extracts from embryos unilaterally coinjected with hnRNP K MO and either EGFP–hnRNP K (A), S189A (B), or S189D mRNA (C). The black line indicates RNA absorbance at 260 nm (A260) across fractions, and premonosomal (Pre), monosomal (Mono), and polysomal (Poly) fractions are indicated (top). Western blots were probed with anti-GFP to visualize the EGFP fusions and anti-S6 to demonstrate proper polysomal separation (bottom). A, EGFP–hnRNP K was most abundant in the premonosomal fractions (94.6%) but was also present to a lesser extent in the monosomal (3.9%) and polysomal fractions (1.5%). B, The loss of phosphorylation at the JNK site prevented S189A from moving beyond the premonosomal fraction (99.7%) into heavier, translating fractions (0.3%). C, S189D, like EGFP–hnRNP K (A), also appeared across all fractions (premonosome, 95.4%; monosome, 3.5%; polysome, 1.1%). D, Quantitation by real-time qRT-PCR of the target (NF-M and tau) and nontarget (peripherin) RNAs of hnRNP K among non-premonosomal fractions (monosome + polysome) with real-time qRT-PCR. Relative amounts (corrected against uninjected WT; see Results) of NF-M (*p = 0.04, one-way ANOVA with Tukey's post hoc test; 3 replicates, 25 embryos per sample) and tau (*p = 0.01, as before) mRNAs in these fractions for embryos unilaterally coinjected with hnRNP K MO and S189A RNA were significantly reduced compared with those coinjected with EGFP–hnRNP K or S189D RNA, whereas relative amounts of peripherin mRNA were not significantly different among groups (p = 0.2, one-way ANOVA as above). Error bars indicate pooled SDs. ns, Nonsignificant.|