Proteomic Investigation of Novel Nuclear JAK2 Pathways

Myeloproliferative neoplasms represent different cancer entities, which all originate from clonal deregulation of hematopoietic stem cells (HSCs). Most disease entities share the same activating mutation of the tyrosine kinase JAK2. Constitutively activated JAK2-V617F mutated HSCs are thought to promote disease progression by stimulating uncontrolled gene expression through the activation of signal transducers and activators of transcription (STAT). Whereas this canonical JAK2/STAT pathway has been thoroughly investigated, research within the nuclear compartment revealed previously unknown functions of JAK2. The kinase was shown to be involved with other transcription factors like NF1-C2 or RUSH. Moreover, through phosphorylation of histone H3 and subsequent release of HP1a, JAK2 is also responsible for epigenetic regulation. Discovery of these pathways gives rise to the question: which other pathways are influenced by nuclear JAK2? Striving to uncover further mechanisms of nuclear JAK2, I initially utilized mass spectrometric pYome and interactome analyses which led me to the recognition of proteins involved in mRNA splicing as thus far unknown targets: hnRNPA1-Y341&Y347, hnRNPA2B1-Y336&Y347, Raver1-Y303 and SF3b155-Y84. Introducing a mutated hnRNPA1 sequence and applying a CRISPR-Cas9 based approach to knock-out the endogenous one, ensured that phosphorylation of the mentioned tyrosine-residues was inhibited. Through a subsequent RNA-sequencing analysis, the differences in phosphorylated vs. non-phosphorylatable hnRNPA1 transcriptomes were examined. With this, I was able to show that nuclear JAK2 mediated phosphorylation of hnRNPA1-Y341&Y347 aids in activating the NFkB pathway by fostering expression of the easily degradable IkBb-1 isoform. In conclusion, I showed that inhibition of hnRNPA1 phosphorylation led to reduced expression of NFkB target genes