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P40 AUF1 and mRNA Regulation

Holly Porter, Gary Brewer's Lab, UMDNJ

The regulation of mRNA decay and stability is critical in gene expression.  In the 3’UTR of some labile mRNAs there is an A + U rich element (ARE) that affects mRNA stability.  In rapidly degraded mRNAs this cis-acting element is bound by AUF1 binding protein (1).  Phosphorylation of p40AUF1 causes changes to the 3D structure of the STAR (Signal Transduction and Regulation) complex, ARE binding affinity, and the structure of the p40AUF1-ARE complex (2).  For p40AUF1, Ser87 is phosphorylated by Protein Kinase A and Ser83 is phosphorylated by Glycogen Synthase Kinase-3b in vitro (2).  Under normal conditions, steady state levels of many cytokines are low, but upon monocyte adherence, the mRNAs of some cytokines become more stable and their translation is upregulated.  After deadherence, these monocytes show destabilization of the cytokines mRNAs, and reactivation of ARE-binding complexes (3).  It is difficult to study monocytes because of their nonproliferative status.  THP1 cells are used because these cells are cancerous and can be used as promonocytes.  THP1 cells are transfected with similar approaches to that of monocytes, and upon adhesion undergo a somewhat similar pattern of phosphorylation events (3). When THP1 cells are treated with phorbol esters their cytokine mRNAs are stabilized and their p40AUF1 proteins are unphosphorylated at Ser83 and Ser87 (2).  Objectives are to confirm that GSK-3b  phosphorylates Ser83 and that PKA phosphorylates Ser87 in vivo.  One or both kinases in THP1 cells will be either knocked out with the use of siRNA or inactivated with the use of inhibitors

To see which forms of AUF1 (unphosphorylated, Ser-83-P, Ser 87-P, bisphosphorylated) are present by Western blot using their corresponding antibodies.  Methods used include kinase assays, titrations and specificity assays to optimize conditions, and cell culturing.  Current results showed that both kinase reactions worked in vitro.  The ntibody (crude serum) raised to Ser-87-P peptide is very specific for Ser-87-P p40AUF1.  This antibody can be used to detect the presence of Ser-87-P p40AUF1.  The antibody (crude serum) raised to unphosphorylated peptide not only recognizes unphosphorylated p40AUF1, but also Ser-83-P p40AUF1, and Ser-87-P p40AUF1. 

Antibody (crude serum) raised to Ser-83-P peptide not only recognizes the Ser-83-P p40AUF1, but also Ser-87-P p40AUF1, and the unphosphorylated p40AUF1. This suggests that neither of these antibodies can be used to differentiate between AUF1 forms.  A different method must be used to detect the presence of Ser-83-P p40AUF1, unphosphorylated p40AUF1 and bisphosphorylated p40AUF1.  In the future, an isoelectric focusing gel will be used to detect the presence of the unphosphorylated, bisphosphorylated, and either Ser-83-P or Ser-87-P form of p40AUF1.  One future goal is to discover the phosphatases acting to remove the phosphates from AUF1 during cytokine adherence. 

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