The Pseudophosphatase MK-STYX Induces Neuronal Differentiation in PC12 Cells

MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/ tyrosine binding protein] is a pseudophosphatase of the MAPK phosphatase family. Though structurally related to the MAPK dual specificity phosphatases, MK-STYX lacks both the critical nucleophilic cysteine and adjacent histidine residues in the active site signature motif (HCX5R) required for catalysis. Thus, MK-STYX is catalytically inactive. Despite its lack of catalytic activity, MK-STYX maintains its ability to bind phosphorylated proteins but not dephosphorylate them. This thesis focuses on the role of MK-STYX in neuronal differentiation signal transduction cascades. The rat pheochromocytoma (PC12) cell line was used as a model system to study neuronal differentiation. Prior studies have shown that stimulation by neurotrophin nerve growth factor initiates sustained activation of a Ras-dependent MAPK phosphorylation cascade. Specifically, it is the sustained activation of extracellular signal-regulated kinase (ERK) 1/2 that leads to neuronal differentiation in PC12 cells. The results presented here confirm that MK-STYX causes neuronal differentiation in PC12 cells, suggesting a role in modulation of the MAPK pathway. Initially, MK-STYX modulation of the small G- protein Ras was investigated, because activation of Ras is known to lead to activation of the MAPK signal transduction cascade. This thesis shows that MK-STYX causes a very transient decrease in the activation of Ras. To further investigate the role of MK-STYX in the MAPK cascade, the kinase activity of MEK was inhibited.Without MEK activation of ERK 1/2, PC12 cells should not be able to differentiate. However, despite the presence of an inhibitor, MK-STYX continued to induce neuronal differentiation, suggesting MK- STYX acts independently of the MAPK pathway. This finding led to investigation of the small G-protein, RhoA. RhoA is involved in actin cytoskeleton remodeling. Prior studies have shown that activation of RhoA inhibits the initiation of neuronal outgrowths, whereas inactivation of RhoA promotes it. These studies provide evidence that MK- STYX decreases activation RhoA leading to the induction of neurite outgrowth. In summary, this thesis demonstrates that MK-STYX can induce PC12 neuronal differentiation through inactivation of RhoA and independently of the MAPK pathway. This strongly supports a model in which the pseudophosphatase MK-STYX has a critical role as a regulator in PC12 neuronal differentiation