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.

MK-STYX Alters the Morphology of Primary Neurons, and Outgrowths in MK-STYX Overexpressing PC-12 Cells Develop a Neuronal Phenotype

“…NGF causes sustained activation of MAPK (Traverse et al, 1992 ; Keegan and Halegoua, 1993 ; Grewal et al, 1999 ; Daniele et al, 2010 ) and is well characterized as an inducer of neuronal differentiation of PC-12 cells (Traverse et al, 1992 ; Pang et al, 1995 ). Thus, we suspected that MK-STYX, which alone induces neurite-outgrowth in PC-12 cells (Flowers et al, 2014 ), might increase neurite differentiation. To analyze MK-STYX's effects on primary neurites (extensions originating from the cell body) in response to a neurotrophin, we stimulated cells co-expressing pEGFP and pMT2 or MK-STYX with NGF.…”

“…PC-12 cells were transfected with pEGFP and pMT2 or MK-STYX and observed for neurite outgrowth 3 days later. Cells were imaged and the extensions were measured from the cell body to the end as depicted and reported previously (Flowers et al, 2014 ). An extension ≥20 μm was considered to be a neurite-like outgrowth.…”

“…This cell line was derived from rat embryonic neural crest, which has a mixture of eosinophilic and neuroblastic cells that allow PC-12 cells to differentiate into neuron-like cells (Greene and Tischler, 1976 ). Thus, it was imperative to determine whether the neurite-like outgrowths originating from MK-STYX-expressing PC-12 cells that we observed previously (Flowers et al, 2014 ; Dahal and Hinton, 2017 ) formed connections and had pre-synaptic and post-synaptic properties. PC-12 cells release neurotransmitters, such as dopamine and acetylcholine (Schubert and Klier, 1977 ) and form typical synapses with neurons in primary culture (Schubert et al, 1977 ; Zhou et al, 2006 ).…”

“…It is also a master regulator of apoptotic potential of mitochondria by negatively regulating PTPM1 (PTP localized to the mitochondrion) (Niemi et al, 2011 , 2014 ). Recently, we reported that it induces neurite-like outgrowths in rat adrenal pheochromocytoma (PC-12) cells through the RhoA signaling pathway (Flowers et al, 2014 ). MK-STYX over-expression decreased RhoA activation, while RhoA activation increased when MK-STYX was down-regulated.…”

“…Cofilin dephosphorylation is required for the induction of neurites (Zhang et al, 2006 ). MK-STYX decreased cofilin phosphorylation in the absence of NGF (Flowers et al, 2014 ), but increased cofilin phosphorylation upon NGF stimulation (Flowers et al, 2014 ). In addition to inducing outgrowths, when PC-12 cells expressing MK-STYX were stimulated with NGF, longer neurites formed and more branching occurred (Flowers et al, 2014 ; Dahal and Hinton, 2017 ), pointing toward the intriguing possibility that these cells were developing a neuronal phenotype.…”