ZFP-36 proteins in monocyte and macrophage inflammatory signaling and differentiation. Model-ling their post-translational modifications, intracellular locations, interactome, structural hetero-geneity and halflife

Abstract

ZFP36 and ZFP36L1 (L1) are RNA-binding nucleo-cytoplasmic phospho-proteins with zinc-fingers, modulating the decay of AU-rich mRNAs such as those of some inflammatory cytokines. Their individual roles or redundancy in macrophages are unknown. Both are post-translationally modified with multi-site phosphorylation by many kinases and by other PTMs. The impact of all PTMs on their roles, location, mRNA binding, folding, modification code, half-life and interactions are understudied. We aimed to study them in THP1 and HeLa cell lines by subcellular fractionation, immunoprecipitation, immunoblotting, far-WB, dye binding, transfection, radiolabeling and 1D/2D gels, also using enzyme inhibitors and TLR ligands for cell treatments. We studied rZFP36 mutants by kinase assays and ZFP36 by informatic, interactome and MS analysis. As novel PTM, we considered the isomerization in proline-directed phosphosites. By densitometry of their isoforms in gels, results suggested with statistical significance that the hyperphosphorylated forms of L1 and ZFP36 were cytoplasmic but insoluble, interacting with the cytoskeleton. Thus, both distribute in at least 6 locations: cytosol, cytoskeleton, mRNAs, stress-granules, P-bodies and nucleus. Besides,both cellular and rZFP36 isomerize. ZFP36 becomes a model for multi-site phosphorylation and isomerization. L1 levels were different in monocytic and macrophage states, suggesting a role in adifferentiation switch but not in inflammation. ZFP36 was affected by inflammatory signaling but not by macrophage adherence or multinucleation or ribotoxic stress. We visualize a complex rheostatic regulation in which ZFP36 is controlled by interactions with ions, proteins, mRNAs and proteasomes, behaving as a polyanion with electrostatic interactions and disordered regions that can isomerize. More studies are needed to understand their molecular heterogeneity and if they will become drug targets, to fine-tune their many activities without side effects.