Caspase-3 cleaves hnRNP K in erythroid differentiation

Abstract

Post-transcriptional control of gene expression is crucial for the control of cellular differentiation. Erythroid precursor cells loose their organelles in a timely controlled manner during terminal maturation to functional erythrocytes. Extrusion of the nucleus precedes the release of young reticulocytes into the blood stream. The degradation of mitochondria is initiated by reticulocyte 15-lipoxygenase (r15-LOX) in mature reticulocytes. At that terminal stage the release of r15-LOX mRNA from its translational silenced state induces the synthesis of r15-LOX. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key regulator of r15-LOX mRNA translation. HnRNP K that binds to the differentiation control element (DICE) in the 3′ untranslated region (UTR) inhibits r15-LOX mRNA translation initiation. During erythroid cell maturation, activation of r15-LOX mRNA translation is mediated by post-translational modifications of hnRNP K and a decrease of the hnRNP K level. To further elucidate its function in the post-transcriptional control of gene expression, we investigated hnRNP K degradation employing an inducible erythroid cell system that recapitulates both nuclear extrusion and the timely controlled degradation of mitochondria, mediated by the activation of r15-LOX synthesis. Interestingly, we detected a specific N-terminal cleavage intermediate of hnRNP K lacking DICE-binding activity that appeared during erythroid differentiation and puromycin-induced apoptosis. Employing mass spectrometry and enzymatic analyses, we identified Caspase-3 as the enzyme that cleaves hnRNP K specifically. In vitro studies revealed that cleavage by Caspase-3 at amino acids (aa) D334-G335 removes the C-terminal hnRNP K homology (KH) domain 3 that confers binding of hnRNP K to the DICE. Our data suggest that the processing of hnRNP K by Caspase-3 provides a save-lock mechanism for its timely release from the r15-LOX mRNA silencing complex and activation of r15-LOX mRNA synthesis in erythroid cell differentiation.

Translation control of TAK1 mRNA by hnRNP K modulates LPS-induced macrophage activation

“…The reticulocyte 15-lipoxygenase (r15-LOX) mRNA 3 ′ UTR differentiation control element (DICE, 190 nt) that bears 5 ′ UC 3-4 3 ′ repeats and confers hnRNP K KH3 domain binding served as a specificity control (Ostareck et al 1997;Messias et al 2006;Naarmann-de Vries et al 2013 ) or BMDMs from healthy C57BL/6 mice (right panel) were treated for 6 h with 10 ng/mL or 80 ng/mL LPS, respectively. Equal amounts of cytoplasmic extracts were used for immunoprecipitation with an anti-hnRNP K or a control antibody.…”

“…His-hnRNP K, His-hnRNP K (Δ KH3) , and His-KH3 were expressed and purified as described in Naarmann-de Vries et al (2013).…”

Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids

“…Both proteins inhibit translation initiation (Ostareck et al, 1997(Ostareck et al, , 2001). Phosphorylation by c-Src and cleavage by caspase-3 lead to release of hnRNP K from the DICE as a prerequisite for the initiation of r15-LOX synthesis (Ostareck-Lederer et al, 2002;Messias et al, 2006;Adolph et al, 2007;Ostareck-Lederer and Ostareck, 2012;Naarmann-de Vries et al, 2013). In addition, hnRNP K and hnRNP E1 stimulate c-Myc protein synthesis by direct interaction with a C-rich sequence in the internal ribosome entry site of c-Myc mRNA (Evans et al, 2003).…”

Aberrant hnRNP K expression: All roads lead to cancer

“…For example, hnRNP K has been shown to positively stimulate MYC translation, 8,34,35 while it inhibits the translation of 15-LOX, a key regulator of erythroid differentiation. 36,37 Collectively, these duplicitous and opposing mechanisms of gene regulation present a complex situation, whereby hnRNP K may play a critical balancing act between its roles in directly and globally activating and repressing gene expression, and simultaneously controlling translation of mRNA transcripts. Thus, it is easy to envision scenarios where small changes in hnRNP K expression (either increased or decreased) could result in drastic cellular defects that impact oncogenic or tumor suppressor pathways and directly impact tumorigenesis.…”