Shoc2-tranduced ERK1/2 motility signals — Novel insights from functional genomics

Highlights

• Depletion of the Shoc2 scaffold attenuates cell motility and adhesion.

• Shoc2-mediated ERK1/2 signals control expression of multiple proteins, including LGALS3BP.

• Ectopic expression of LGALS3BP rescues adhesion deficiency of the Shoc2-depleted cells.

Abstract

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway plays a central role in defining various cellular fates. Scaffold proteins modulating ERK1/2 activity control growth factor signals transduced by the pathway. Here, we analyzed signals transduced by Shoc2, a critical positive modulator of ERK1/2 activity. We found that loss of Shoc2 results in impaired cell motility and delays cell attachment. As ERKs control cellular fates by stimulating transcriptional response, we hypothesized that the mechanisms underlying changes in cell adhesion could be revealed by assessing the changes in transcription of Shoc2-depleted cells. Using quantitative RNA-seq analysis, we identified 853 differentially expressed transcripts. Characterization of the differentially expressed genes showed that Shoc2 regulates the pathway at several levels, including expression of genes controlling cell motility, adhesion, crosstalk with the transforming growth factor beta (TGFβ) pathway, and expression of transcription factors. To understand the mechanisms underlying delayed attachment of cells depleted of Shoc2, changes in expression of the protein of extracellular matrix (lectin galactoside-binding soluble 3-binding protein; LGALS3BP) were functionally analyzed. We demonstrated that delayed adhesion of the Shoc2-depleted cells is a result of attenuated expression and secretion of LGALS3BP. Together our results suggest that Shoc2 regulates cell motility by modulating ERK1/2 signals to cell adhesion.

Introduction

The mechanisms leading to activation of RAF, MEK and ERK kinases in the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway have been studied extensively [1], [2]. However, what determines the activity of the pathway in the context of a specific set of downstream targets and how ERK signals result in distinct biological outcomes are not yet clear. Several studies suggest that divergent cell fates induced by the ERK pathway are the result of a tight spatio-temporal control of ERK1/2 targeting, sequestration and activation of the kinases and phosphatases, as well as modulation of the strength and duration of the ERK signaling [3], [4]. Scaffold proteins have been proposed to fulfill some of these requirements. Scaffolds have been implicated in controlling the spatial organization of signaling enzymes, insulation of active modules and prevention of a spurious cross-talk of signaling networks [5], [6]. Yet, detailed mechanisms allowing scaffolds to elicit specific cellular responses at the molecular level remain to be elucidated.

Scaffold proteins of the ERK1/2 pathway represent a diverse group of proteins [2]. A well-studied scaffold kinase suppressor of Ras 1 (KSR1) [7], [8], [9] is the multifunctional protein that binds to and accelerates activity of MEK and RAF kinases thereby stimulating expression of genes that drive cell proliferation and differentiation [10], [11]. Other ERK1/2 scaffolds, mitogen-activated protein 1 (MP1) and p14 (also called the LAMTOR2/3 complex) are believed to regulate cytoskeletal dynamics [12], [13], [14], [15], and the MP1/p14 complex is involved in remodeling of focal adhesion and actin structures during cell spreading [16].

Ras–RAF-1–ERK1/2 signaling is accelerated by the scaffolding protein Shoc2 [17], [18]. This evolutionarily well-conserved protein is essential for normal development [19], [20], [21]. Loss of Shoc2 in mammalian cultured cells and Caenorhabditis elegans leads to a dramatic decrease in ERK1/2 activity [17], [22], [23]. As a scaffold protein, Shoc2 provides a molecular platform for multi-protein assemblies that modulate ERK1/2 activity [24], [25]. In addition to its signaling partners Ras and RAF-1, Shoc2 tethers the catalytic subunit of protein phosphatase 1c (PP1c) as well as proteins of the ubiquitin machinery HUWE1 and PSMC5 [23], [26], [27]. The ability of this non-catalytic scaffold to mediate ERK1/2 signaling is controlled through allosteric ubiquitination [24]. Alterations in the mechanisms controlling ubiquitination of the scaffold affect Shoc2-mediated ERK1/2 signals and cell motility [27].

Activation of the ERK1/2 pathway in response to epidermal growth factor (EGF) stimulation of the EGF receptor falls into three major regulatory loops: immediate, delayed, and late (secondary) [28], [29], [30]. The immediate regulatory loop induces phosphorylation of transcription factors such as FOS, Jun and EGR1 and does not require new protein synthesis for their transcription [30]. Expression of the genes of the immediate response induces transcription of delayed genes, such as the RNA-binding protein ZFP36 or dual specific phosphatases, which dephosphorylate ERK1/2 kinases that terminate the activity of the immediate loop [30]. Late (secondary) transcriptional response leads to expression of genes such as actin-binding proteins or genes encoding proteins that are involved in cell metabolism and biogenesis of membranes and appear to define cellular outcomes [31].

In the current study, we aimed to determine the specific ERK1/2 response elicited through the Shoc2 scaffolding module. Results of this study provide evidence that Shoc2-mediated ERK1/2 activity contributes to maintenance of the ERK1/2 feedback loop that regulates expression of genes of the TGFβ pathway. We also found that Shoc2–ERK1/2 signals control cell motility and adhesion, in part, through mechanisms that monitor expression of the protein of extracellular matrix — lectin galactoside-binding soluble 3-binding protein or LGALS3BP (also called Mac-2 binding protein) [32]. Deficient expression and secretion of this heavily glycosylated protein led to attenuated attachment of Shoc2-depleted cells. These results indicate that Shoc2 transduces signals to unique cellular responses and identifies novel molecular targets of the Shoc2–ERK1/2 signaling axis.