Phosphorylation of KIBRA by the extracellular signal-regulated kinase (ERK)–ribosomal S6 kinase (RSK) cascade modulates cell proliferation and migration

Highlights

• ERK–RSK cascade phosphorylates KIBRA in vitro and in cells.

• KIBRA knockdown impairs cell proliferation and migration.

• ERK–RSK phosphorylation of KIBRA modulates cell proliferation and migration.

Abstract

In mammals, KIBRA is defined as a memory performance-associated protein. The physiological function and regulation of KIBRA in non-neuronal cells are much less understood. Recent studies have identified KIBRA as a novel regulator of the Hippo signaling pathway, which plays a critical role in tumorigenesis by inhibiting cell proliferation and promoting apoptosis. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora and cyclin-dependent kinase 1 during mitosis. In this current study, we show that KIBRA is also phosphorylated by the ERK (extracellular signal-regulated kinases)–RSK (p90 ribosomal S6 kinases) cascade. We demonstrated that ERK1/2 phosphorylate KIBRA at Ser⁵⁴⁸ in cells as well as in vitro. Moreover, we found that RSK1/2 specifically phosphorylates KIBRA at two highly conserved sites (Thr⁹²⁹ and Ser⁹⁴⁷) in vitro and in cells. RSK-mediated phosphorylation is required for KIBRA binding to RSK1, but not RSK2. Surprisingly, KIBRA knockdown impaired cell migration and proliferation in breast cancer cells. By using inducible-expression cell lines, we further show that phospho-regulation of KIBRA by ERK1/2 and RSK1/2 is required for proper cell proliferation and RSK-mediated phosphorylation also modulates KIBRA's migratory activity in MDA-MB-231 breast cancer cells. Our findings uncover unexpected results and a new mechanism through which KIBRA regulates cell migration and proliferation.

Introduction

KIBRA (expression enriched in kidney and brain), also called WW and C2 domain containing 1 (WWC1), contains two WW domains at the N-terminus and a C2 domain in the C-terminal region [1]. KIBRA was originally identified as a memory performance and cognition-associated protein [2]; this function was recently confirmed by genetic models in mice [3] and studies in humans [4], [5], [6], [7], [8]. Increasing evidence also links the KIBRA locus to Alzheimer's disease [9], [10], [11]. While the role of KIBRA in neurons is well defined, its physiological function in non-neuronal cells is relatively less understood. We and others recently demonstrated that KIBRA is a regulator of the Hippo pathway in both Drosophila [12], [13], [14] and mammalian cells [15], suggesting a function of KIBRA in epithelial cell growth. Previous studies also indicated that KIBRA is involved in other biological processes including cell polarity, mitosis and cell migration. For example, KIBRA positively regulates directional migration in podocytes in conjunction with the polarity protein PALS1-associated tight junction (PATJ) and synaptopodin [16]. This positive regulation in migration was further demonstrated in NRK cells [17]. However, KIBRA was shown to be a negative regulator in cell motility in immortalized breast epithelial cells [18]. Despite the established role of KIBRA in cell migration, it is unknown how KIBRA is regulated during this process.

The Ras-activated MAPK/ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway plays a central role in diverse cellular processes [19]. MEK1/2 kinases phosphorylate and activate the mitogen-activated protein kinases (MAPKs) ERK1/2. Once activated, ERK1/2 transduce the extracellular signals to intracellular proteins by phosphorylating their substrates, including the 90 kDa ribosomal S6 kinase (RSK) family. The human RSK kinase family comprises four highly related serine/threonine isoforms (RSK1–4) [20]. The RSK kinases are the major downstream effectors of the RAS/MAPK signaling pathway and modulate various biological responses, including cell proliferation, survival, growth, and cell motility/invasiveness [20], [21]. Interestingly, KIBRA was shown to be required for collagen-induced ERK signaling activity via interactions with discoidin domain receptor tyrosine kinase 1 [22], providing a potential connection between MEK–ERK signaling and KIBRA.

KIBRA is a phospho-protein and we previously reported that KIBRA is phosphorylated by the mitotic kinases Aurora and cyclin dependent kinase 1 (CDK1) during mitosis [23], [24]. During the process of identifying the kinases for KIBRA shift/phosphorylation, we noticed that MEK–ERK inhibitors can significantly enhance the mobility of KIBRA on SDS-PAGE gels. In this report, we further characterize the phosphorylation of KIBRA. We show that the MEK–ERK–RSK cascade is also able to phosphorylate KIBRA and, importantly, this phosphorylation is required for KIBRA-mediated cell migration and proliferation in MDA-MB-231 cells. Thus, our study reveals an unrecognized mechanism through which KIBRA regulates cell migration and proliferation in breast cancer cells.