title: The pseudophosphatase MK-STYX induces neurite-like outgrowths in PC12 cells.
abstract: The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and implicate this pseudophosphatase as a regulator of neuronal differentiation.
authors.affiliation: Department of Biology, Integrated Science Center, College of William and Mary, Williamsburg, VA, United States of America.
authors.forename: Brittany M
authors.initials: BM
authors.lastname: Flowers
authors.affiliation: Department of Biology, Integrated Science Center, College of William and Mary, Williamsburg, VA, United States of America.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Rusnak
authors.affiliation: Department of Biology, Integrated Science Center, College of William and Mary, Williamsburg, VA, United States of America.
authors.forename: Kristen E
authors.initials: KE
authors.lastname: Wong
authors.affiliation: Department of Biology, Integrated Science Center, College of William and Mary, Williamsburg, VA, United States of America.
authors.forename: Dallas A
authors.initials: DA
authors.lastname: Banks
authors.affiliation: Department of Biology, Integrated Science Center, College of William and Mary, Williamsburg, VA, United States of America.
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Munyikwa
authors.affiliation: Department of Biology, Integrated Science Center, College of William and Mary, Williamsburg, VA, United States of America.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: McFarland
authors.affiliation: Department of Biology, Integrated Science Center, College of William and Mary, Williamsburg, VA, United States of America.
authors.forename: Shantá D
authors.initials: SD
authors.lastname: Hinton
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D009154:Mutation; D016501:Neurites / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; D020742:rhoA GTP-Binding Protein