Proximity Proteomics Identifies a Role of MAP2K4 (MKK4) in JAK2 V617F Signaling

JAK inhibitors reduce symptomatic burden of myeloproliferative neoplasms (MPN), including attenuation of disease-driving JAK-STAT activation, but their curative efficacy is limited. To identify non-canonical activities of JAK that contribute to disease pathology, we used proximity dependent biotin labeling followed by mass spectrometry of JAK2 V617F and JAK2 enabled by enhanced biotin ligase TurboID. Using proximity labeling-based proteomics, supported by our newly developed proximity proteomics data bioinformatics analysis pipeline, we performed a systematic analysis of spatially restricted JAK2 V617F-specific proteomes using cellular models of FDC-P1 and BA/F3 myeloid cell lines engineered to express TurboID-JAK2 V617F or TurboID-JAK2 WT at the near physiological levels. We identified robust proximal interaction signature of JAK2 V617F with MAP2K4 (MKK4, SEK1, JNKK1). MAP2K4 is an established tumor suppressor and is downregulated in blood cancers. Moreover, it was recently shown that negative regulation of MAPK signaling mediated by DUSP1 phosphatase enforces JAK2 V617F clonal advantage, affecting ERK, p38, and JNK activation (Kesarwani et al, Leukemia, 2024). Using proximity proteomics data, we explored MAP2K4 in the context of DUSP1 and JAK2 V617F signaling and assessed how JAK2 V617F-associated dysregulation of MAPK signaling affects apoptosis and survival. Immunoblotting analyses on our cellular models showed decreased activation of MAP2K4 in JAK2 V617F-expressing myeloid cells, consistent with increased inhibitory phosphorylation of upstream MAP2K4 activator MAP3K5. Proximity proteomics data indicated significant proximal interaction of JAK2 WT with MAP3K5 inhibitory 14-3-3 proteins. There was also significant proximal interaction between JAK2 V617F and DAB2IP and TRADD, which are known to form a complex that promotes 14-3-3 degradation and MAP3K5 activation in response to TNFR activation. RIPK1, which is also part of this complex and is involved in apoptosis, was measured as a significant proximal interactor of JAK2 WT. Consistently, decreased levels of pro-apoptotic RIPK1 S166 phosphorylation in JAK2 V617F cells was detected. Bioinformatic analysis of significant proximal interactors detected as common to both JAK2 WT and JAK2 V617F enriched for 'Negative regulation of MAPK cascade“ pathway with 13 component proteins including LYN, ITCH, PTPN6, and NF2. Proximal interactors significantly enriched by JAK2 WT identified 5 proteins in ”MAPKinase Signaling“ pathway including MAP3K1, MAP4K5, RIPK1, and IKBKB. Proximal interactors significantly enriched by JAK2 V617F identified 9 proteins in ”Positive regulation of MAPK cascade“ including DAB2IP, MAP4K1, FGFR1, and TRAF7. Together, this supports the constitutive role of JAK2 in MAPK regulation that is dysregulated by mutation. To further inform mechanisms by which JAK2 V617F affects MAPK, in alignment with Kesarwani et al (2024), we intersected JAK2 proximal interactors with known DUSP1 interactors. Of 135 known DUSP1 interactors (BioGRID), 35 were enriched as significant JAK2 proximal interactors including MAP2K4, MAP2K1, MAPK1, CHUK, IKBKB, ATF2, STAT1. Interestingly, of these proteins, only MAP2K4 was significantly more enriched by JAK2 V617F than by JAK2 WT. These findings suggest a role of MAP2K4 in mediating JAK2 V617F-associated signaling, providing a link between MAPK and NF-kB, JNK, and STAT1 inflammatory activation affected by V617F mutation. Together, these data suggest a previously unexplored JAK2 V617F inhibitory function that modulates signal propagation from MAPK to inflammatory pathways. This work challenges current perception of JAK2 signaling to focus on inhibitory signalosomes in search for more effective therapeutic targets to treat MPNs.