CHARACTERIZATION OF P38alpha AND NITRIC OXIDE SYNTHASE 3 BINDING

P38alpha is a member of the mitogen activated protein kinase (MAPK) family. This family is important for mediating signal transduction pathways through phosphorylation of downstream substrates. Previous work has described the ability of p38alpha to bind and phosphorylate nitric oxide synthase 3 (NOS3) at two sites, the autoinhibitory loop (AI) at S600 and the N-terminal MAPK binding site (NtMAP) at S114. P38alpha has two substrate binding domains, the common docking groove (CD-groove) and a hydrophobic pocket (DEF) that are important in binding and phosphorylating various substrates specific to each pocket. Utilizing biolayer interferometry, this work describes the importance of both pockets in binding NOS3. AI peptides preferentially bound an interfering DEF pocket variant of p38alpha suggesting the CD-groove is important for binding whereas NtMAP peptides preferentially bound an interfering CD-groove variant, demonstrating reliance on DEF pocket binding. Phosphorylation of p38alpha (WT and variants) at the activation loop demonstrated an overall better affinity to NOS3 peptides. Oxidation of p38alpha induces a disulfide bond in the CD-groove and we found an increase in affinity to the NtMAP, suggesting binding of the disulfide form is mediated through the DEF pocket. Overall, NOS3 phosphorylation at S114 and S600 is DEF pocket dependent. NOS3 and p38 binding in situ detected with proximity ligation assay revealed localization of phosphorylated S114 to the nucleus in HMEC-1 cells. All together these findings provide insight into the complex interactions of binding and activity leading to new questions about the impact in regulation of NOS3 and p38.