RSK1 drives p27^Kip1phosphorylation at T198 to promote RhoA inhibition and increase cell motility

Abstract

p90 ribosomal S6 kinase (RSK1) is an effector of both Ras/MEK/MAPK and PI3K/PDK1 pathways. We present evidence that RSK1 drives p27 phosphorylation at T198 to increase RhoA-p27 binding and cell motility. RSK1 activation and p27pT198 both increase in early G 1. As for many kinase-substrate pairs, cellular RSK1 coprecipitates with p27. siRNA to RSK1 and RSK1 inhibition both rapidly reduce cellular p27pT198. RSK1 overexpression increases p27pT198, p27-cyclin D1-Cdk4 complexes, and p27 stability. Moreover, RSK1 transfectants show mislocalization of p27 to cytoplasm, increased motility, and reduced RhoA-GTP, phospho-cofilin, and actin stress fibers, all of which were reversed by shRNA to p27. Phosphorylation by RSK1 increased p27pT198 binding to RhoA in vitro, whereas p27T157A/ T198A bound poorly to RhoA compared with WTp27 in cells. Coprecipitation of cellular p27-RhoA was increased in cells with constitutive PI3K activation and increased in early G 1. Thus T198 phosphorylation not only stabilizes p27 and mislocalizes p27 to the cytoplasm but also promotes RhoA-p27 interaction and RhoA pathway inhibition. These data link p27 phosphorylation at T198 and cell motility. As for other PI3K effectors, RSK1 phosphorylates p27 at T198. Because RSK1 is also activated by MAPK, the increased cell motility and metastatic potential of cancer cells with PI3K and/or MAPK pathway activation may result in part from RSK1 activation, leading to accumulation of p27T198 in the cytoplasm, p27:RhoA binding, inhibition of RhoA/Rock pathway activation, and loss of actomyosin stability. p27 phosphorylation ͉ PI3K ͉ actin cytoskeleton

p27 as Jekyll and Hyde: Regulation of cell cycle and cell motility

“…RSK1 overexpression reduced RhoA-GTP and led to loss of actin stress fibers, both of which were reverted by p27 knockdown. 37 Moreover, these effects on cytoskeletal dynamics correlated with enhanced cell migration in both transwell and wound-healing assays. This work also brought to light a previously undefined role for the p27pT198 isoform; in both in vitro assays and in cells with transfected RSK1 or PI3K pathway activation due to PTEN loss, RSK1-mediated T198 phosphorylation led to enhanced RhoA-p27 binding.…”

“…[36][37][38] While SGK1 contributes to T157 phosphorylation and cytoplasmic mislocalization of p27, 36 RSK1 appears to phosphorylate p27 predominantly at T198. 37 The mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell growth, cell proliferation and cell motility. mTOR can partner with raptor (mTORC1) or rictor (mTORC2) to regulate both protein synthesis and the cell cycle.…”

“…This work also brought to light a previously undefined role for the p27pT198 isoform; in both in vitro assays and in cells with transfected RSK1 or PI3K pathway activation due to PTEN loss, RSK1-mediated T198 phosphorylation led to enhanced RhoA-p27 binding. 37 This represents an important link between signal transduction cascades that converge on p27 and enhanced cell motility, and may be particularly germane to cancer metastasis.…”

“…These include: (1) S10, which mediates p27 export [44][45][46] ; (2) Y74, Y88, Y89, which make p27 a poor inhibitor of cyclin E-Cdk2 and facilitate cyclin E-Cdk2-mediated T187 phosphorylation and SCF Skp2 -dependent p27 proteolysis 23,47 ; (3) T157, whose phosphorylation impairs nuclear import [32][33][34] and increases cyclin D1-Cdk4 assembly 21 ; (4) T187, that targets p27 for SCF Skp2 -dependent proteolysis 48,49 ; and (5) T198, that regulates p27 stability 50,51 and increases p27-dependent motility. 37 Changes in p27 phosphorylation can lead to loss of p27 protein stability and alterations in its function and/or localization, all of which may contribute to oncogenesis.…”

Progesterone Stimulates Proliferation and Promotes Cytoplasmic Localization of the Cell Cycle Inhibitor p27 in Steroid Receptor Positive Breast Cancers

“…In the nucleus, p27 acts as a potent inhibitor of proliferation in the normal breast and breast cancer [13,14]. In the cytosol of breast cancer cells, p27 promotes cytoskeletal remodeling and cell motility [15,16]. Decreased nuclear and increased cytoplasmic expression of p27 is frequently observed in primary breast cancers and associated with poor clinical outcome [17,18].…”