The long-term goal of this project is to help determine the mechanisms that regulate cell adhesion and migration. Dynamic regulation of integrin affinity for ligand (activation) is important in a broad array of cellular functions including adhesion, migration, signal transduction, and the assembly of extracellular matrices. The major objective of the proposed research plan is to understand the molecular mechanism by which the ERK MAP kinase pathway modulates integrin function. RSK2 is a substrate of ERK and can mediate many of the biological functions of the Ras/ERK MAP kinase pathway including cell survival, proliferation and transcription. RSKs are large kinases consisting of two kinase domains. The regulation of RSK activity is complex and requires phosphorylation at several sites including auto-phosphorylation. We have recently shown that the protein PEA-15 binds to RSK2 and acts as a scaffold to enhance ERK activation of RSK2. Moreover, overexpression of PEA-15 can impair ERK activation of RSK2. Similarly overexpression of PEA-15 impairs ERK regulation of integrin activity and blocks cell migration. We therefore sought to determine if the effects of overexpression of PEA-15 were due to inhibition of ERK activation of RSK2. We show in the preliminary results that indeed overexpression of PEA-15 blocks RSK2 phosphorylation of filamin. Moreover dominant-active RSK2 blocks integrin activation and integrin-mediated fibronectin matrix assembly. Finally we show that the Ras/ERK affect on integrin activity is blocked by a dominant- negative RSK2 construct. Our Hypothesis is that RSK2 modulates integrin function downstream of ERK by regulating integrin 2 tail interaction with the actin cytoskeleton. The research plan has three specific aims: (1) Establish the molecular mechanism by which RSK2 modulates integrin activation. We provide preliminary data that active RSK2 blocks integrin ligand-binding. We will investigate the molecular mechanism of this action with particular focus on the RSK2 substrate filamin and the integrin activation protein talin. (2) Determine the functional consequences of RSK2 regulation of integrin activity. Integrins are involved in cell adhesion, migration, fibronectin matrix assembly and signal transduction. We will investigate the involvement of RSK2 in these processes. (3) Investigate the mechanism of PEA-15 regulation of RSK2 activity. We have reported that PEA-15 acts as a scaffold for the activation of RSK2 by ERK. We will further determine how PEA-15 modulates RSK2 activation and how this affects cell adhesion and migration. (4) To determine how RSK2 regulation of integrin activation affects glioma invasion. We previously reported that PEA-15 expression blocks glioma invasion. We will now explore how RSK2 contributes to changes in the adhesion and invasion of glioma.
These aims will be pursued in tissue culture cells using flow cytometry, confocal microscopy, immunofluorescence, molecular biology, and biochemistry. We will also employ mouse embryo fibroblasts and primary culture cells derived from mouse knock-out models. Significance: The proposed research will provide significant advances in our understanding of how the complex kinase RSK2 can play a central role in modulating cellular adhesion and migration. Changes in integrin activation are linked to diverse diseases including cancer metastasis. Many signal transduction pathways are known to modulate integrin function. However, the mechanisms by which these signals are transformed into changes in integrin conformation are not well understood. At the end of these studies we will better understand how the ERK MAP kinase signal transduction pathway modulates integrin activation through RSK2 and how this may contribute to disease states such as tumor metastasis. We will also have determined if RSK2 can serve as a potential new therapeutic target in the treatment of gliomas.
Control of cell adhesion is important in targeting immune cells to sites of inflammation, in platelet aggregation, and in cancer cell metastasis among other functions. We have found that the proteins PEA-15 and RSK2 may conspire to influence cell adhesion and migration. In particular we propose that PEA-15 regulates RSK2 activity while RSK2 in turn modulates the interaction of the cell cytoskeleton with adhesion proteins called integrins. At the conclusion of these studies the molecular mechanism of PEA-15 and RSK2 modulation of cell adhesion will be better understood. This will determine if these proteins are suitable targets for the development of new adhesion specific disease therapies. In particular we will test if RSK2 can serve as a potential new therapeutic target in glioma.
|Sulzmaier, Florian J; Ramos, Joe W (2013) RSK isoforms in cancer cell invasion and metastasis. Cancer Res 73:6099-105|
|Griffiths, Genevieve S; Grundl, Melanie; Leychenko, Anna et al. (2011) Bit-1 mediates integrin-dependent cell survival through activation of the NFkappaB pathway. J Biol Chem 286:14713-23|
|Liang, Zhibin; Sorribas, Analia; Sulzmaier, Florian J et al. (2011) Stictamides A-C, MMP12 inhibitors containing 4-amino-3-hydroxy-5-phenylpentanoic acid subunits. J Org Chem 76:3635-43|