PP2A regulates upstream members of the c-jun N-terminal kinase mitogen-activated protein kinase signaling pathway

We have previously demonstrated that inhibition of the serine-threonine phosphatase PP2A resulted in increased c-jun N-terminal kinase (JNK) activity, and that the regulatory subunit, A/α of PP2A, was physically associated with the JNK. Because there exists additional examples of phosphatases serving as negative regulators of multiple members of mitogen-activated protein kinase (MAPK) pathways in Drosophila and yeast, we hypothesized that PP2A may serve a homologous function in mammalian cells affording the regulation of additional upstream kinases in the JNK pathway. In human monocytes, activation of JNK by LPS proceeds through the MAPK kinase kinase MEKK-1 and, subsequently, the MAPK kinases MKK4 and/or MKK7. Using the human monocyte cell line THP-1, we show that pharmacological manipulation of the activity of PP2A seemed to regulate not only JNK but also the upstream kinases MKK4 and MEKK-1. Using coimmunoprecipitation, overexpression of tagged recombinant JNK, and bacterial two-hybrid strategies, evidence for physical interactions between the structural subunit, PP2A-A/α and MEKK-1, MKK4, and JNK was observed. These studies suggest that the target of regulation by PP2A includes upstream kinases in the JNK MAPK pathway. Furthermore, PP2A-A/α seems to serve as a structural protein to foster protein-protein interactions affording specificity of the regulation among members of this MAP kinase pathway.