ERK‐mediated Phosphorylation of the Egr Transcriptional Coregulator NAB2 at Multiple Sites

Abstract

The early growth response (Egr) family of transcription factors comprises five members, Egr1‐4 and Wilms Tumor 1 (WT1), which play roles in various cell behaviors including proliferation, apoptosis, and differentiation in a cell type‐ and stimulus‐specific manner. Roles for Egr1‐3 are particularly well‐documented in the nervous system. Egr1 and 3 contribute to learning and memory through regulation of genes that mediate synaptic plasticity and long‐term potentiation. Egr2 controls hindbrain development through regulation of Hox gene expression and drives peripheral nerve myelination through, at least in part, activation of the gene encoding myelin protein zero in Schwann cells. Egr1 and 2 also contribute to transcriptional network that drives neuronal differentiation of PC12 cells. However, the mechanisms that regulate Egr1‐3 activities in the nervous system are not fully understood. One mechanism of regulation involves protein‐protein interactions with co‐regulators NAB1 and 2 through a conserved R1 domain in Egr1‐3. Most reports demonstrate that NABs repress Egr1‐3 transactivation activity when bound, however, NABs can also enhance transactivation in a target gene‐specific manner. The mechanisms controlling the effect of NABs on Egr activities are not clear. Here, we provide evidence that NAB2 regulation may involve its phosphorylation at several sites by ERK. Using PC12 cells as a model system, our data demonstrate that nerve growth factor (NGF) induces a NAB2 mobility shift in SDS‐PAGE within 10 minutes of treatment. The mobility shift was reversed when lysates from NGF‐treated cells were treated with lambda phosphatase, suggesting the shift is due to phosphorylation. Inhibition of ERK signaling during NGF treatment blocked the mobility shift, while recombinant ERK phosphorylated recombinant NAB2 in an in vitro kinase assay, together indicating that direct phosphorylation of NAB2 by ERK may be responsible for the NAB2 mobility shift in response to NGF. Nine potential ERK phosphorylation sites were identified in NAB2, which were mutated individually to alanine within a NAB2 expression construct to evaluate phosphorylation at those sites based on their mobility in SDS‐PAGE. Of the nine mutants, three exhibited clear changes in NAB2 mobility, which were additive in double and triple mutants for those sites. Ongoing experiments are evaluating the effect of NAB2 phosphorylation on its localization and regulation of Egr1‐3. Support or Funding Information Bridgewater State University Undergraduate Research Program

ERK‐dependent induction of the immediate‐early gene Egr1 and the late gene Gpr50 contribute to two distinct phases of PACAP Gs‐GPCR signaling for neuritogenesis

“…Thus, the continued requirement for ERK activation in neuritogenesis is not solely the induction and then continued activation of Egr1 transcriptional activity by ERK-dependent cofactor phosphorylation, as has been suggested in other systems. 50 Rather, late gene induction during neuritogenesis likely requires the direct effects of ERK signaling for transcription of these late genes, rather than an indirect effect through sustaining Egr1-dependent transcriptional effects.…”