Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation

Abstract

Multisite phosphorylation regulates many transcription factors, including the Serum Response Factor partner Elk-1. Phosphorylation of the transcriptional activation domain (TAD) of Elk-1 by the protein kinase ERK at multiple sites potentiates recruitment of the Mediator transcriptional coactivator complex and transcriptional activation, but the roles of individual phosphorylation events remained unclear. Using time-resolved nuclear magnetic resonance spectroscopy, we found that ERK2 phosphorylation proceeds at markedly different rates at eight TAD sites in vitro, which we classified as fast, intermediate and slow. Mutagenesis experiments showed that phosphorylation of fast and intermediate sites promoted Mediator interaction and transcriptional activation, whereas modification of slow sites counteracted both functions, thereby limiting Elk-1 output. Progressive Elk-1 phosphorylation thus ensures a self-limiting response to ERK activation, which occurs independently of antagonizing phosphatase activity. Results and DiscussionMultisite protein phosphorylation increases the complexity of functional signaling outputs that can be generated from single protein kinase inputs. It can set thresholds for activity, or transform graded signals into switch-like responses (1-4). Many transcription factors and their interacting regulatory proteins are subject to multisite phosphorylation, which allows distinct aspects of protein function, including protein turnover, nuclear import and export, and specific protein interactions, to be controlled independently (5). However, in general, the † The ternary complex factor (TCF) subfamily of Ets-domain transcription factors, consisting of Elk-1, SAP-1, and Net, provides an example of multisite phosphorylation in transcriptional activation. TCFs, together with their partner protein SRF, function in many biological processes by coupling SRF target genes to mitogen-activated protein kinase (MAP kinase) signaling (5). Mitogenic and stress stimuli induce phosphorylation of TCF Cterminal transcriptional activation domains (TADs) at multiple S/T-P (Ser-or Thr-Pro) phosphorylation sequences, of which eight are conserved across the family ( Fig. 1A; fig. S1) (6-11). Two MAP kinase-docking sites, the D-box and the Phe-Gln-Phe-Pro (FQFP) motif, control phosphorylation of these sites (12-15). Multisite phosphorylation triggers transcriptional activation by TCFs, facilitating their interaction with the Mediator transcriptional co-activator complex (16-19) but the kinetics with which the different sites are phosphorylated, and whether they serve distinct functions, remain unclear.To obtain atomic-resolution insights into phosphorylation of the Elk-1 TAD, we used nuclear magnetic resonance (NMR) spectroscopy (20) Fig. 2A). In the fast site mutant Elk-1F, phosphorylation rates of intermediate and slow sites increased, whereas those of the fast and slow sites increased in the intermediate-site mutant Elk-1I; in both cases the altered kinetics fit well with those predicted by the model (Fig 2B,...

Homeostatic Control of Hpo/MST Kinase Activity through Autophosphorylation-Dependent Recruitment of the STRIPAK PP2A Phosphatase Complex

“…This molecular design provides a most efficient way to couple a “signal-on” state to a “signal-off” state, and may serve to maintain a homeostatic level of Hippo signaling or prevent spurious/prolonged pathway activation that can be detrimental to an organism. Interestingly, multisite phosphorylation with opposing effects was also documented in a recent study of the Elk-1 transcriptional factor (Mylona et al, 2016), suggesting that this may be a common design principle in signal transduction.…”

SRF Co-factors Control the Balance between Cell Proliferation and Contractility

“…In a direct comparison, SAP-1 both activated transcription and restored proliferation more effectively than Elk-1, suggesting that it may restore a greater fraction of TCF-dependent gene expression. However, expression of mouse Elk-1 was also more effective than human Elk-1 in restoring proliferation (Mylona et al., 2016). Resolution of these issues will require direct comparative analysis of TCF recruitment with genomic targets.…”

Protein Phosphorylation Dynamics: Unexplored Because of Current Methodological Limitations

“…This is the reason for which only a few examples have been reported in the literature regarding the intradynamics of phosphorylation within polypeptide chains, [ 81 ] in which proteins inactivate the partner through phosphorylation, [ 86 ] or exert opposite effects. [ 87 ] More importantly, this time lapse monitoring of phospho addition will have to be coupled with the scrutiny of the activation process to establish correlation and causality. Contrary to the common “bottom‐up” mass spectrometry that is applied to proteolyzed proteins, “top‐down” MS starts with intact proteins in the first step without prior digestion.…”