||Cells sense changes in their environment via specific sensors, so-called receptors that are located on the outer surface, at the plasma membrane. Receptors are activated by binding of their cognate ligands, signal molecules such as growth factors, which leads to distinct cellular responses. Stimulated receptors pass on the message by triggering a wide range of signalling cascades involving successive activation of enzymes and generation of “second messengers” (signal transduction). Which cellular responses arise after stimulation of a receptor depends on the signal molecule (agonist), the type of receptor, and the combination of both. In this way, growth factors that bind to their specific receptor encourage cells to divide. Similarly, next to affecting cell growth, receptor agonists can promote alterations in cellular morphology and motility. Changes in cell morphology and polarity are fundamental for many physiological (and pathological) processes, and are brought about by the dynamic regulation and reorganisation of the cytoskeleton.
The cytoskeleton provides the strength and motility of a cell and consists of three types of networks: the actin cytoskeleton, the microtubule network and intermediate filaments. These networks are characterised by filaments, which arise through the joining of single subunits into large strands (polymerisation). A higher order structure of the actin cytoskeleton is accomplished by the organisation of actin filaments into networks by actin-crosslinking and -bundling proteins. Other actin-binding proteins (ABPs) function as scaffolds holding the actin network and associating proteins together. In addition, various ABPs stimulate or reduce polymerisation of actin, thereby affecting the length and amount of actin filaments. So, the organisation of the actin network is determined by the combined activities of ABPs.
The Rho family of small GTPases, in particular the members RhoA, Rac, and Cdc42, has a central role in the regulation of the actin cytoskeleton. Rho GTPases act as molecular switches that are inactive when complexed with GDP and active when bound to GTP. Receptors that induce remodelling of the actin cytoskeleton all converge on these Rho GTPases to elicit their effects. Activated Rho GTPases transmit the signal to proteins that directly affect the assembly and disassembly of the cytoskeleton, such as ABPs. For example, activation of RhoA leads to cell contraction by the actin-associated motor protein myosin, which exerts force on actin filaments.
Besides their effects on actin reorganisation, Rho GTPases are involved in many other cellular responses, such as gene transcription leading to changes in protein expression. Serum or its major constituent lysophosphatidic acid activate for instance the serum response factor (SRF), which controls many growth factor-regulated genes, through RhoA-dependent increases in actin polymerisation.
Deregulation of Rho GTPase activity can contribute to various aspects of tumourigenesis. Therefore, studying the Rho GTPase signal transduction pathway will increase our understanding of altered cell behaviour as occurs during the migration, invasion, and metastasis of cancer cells.
The p116Rip protein was first identified as a putative RhoA-interacting protein. It contains several protein interaction domains and is widely expressed throughout the body. Initial results suggested that p116Rip expression in neuronal cells inhibited RhoA-induced contraction of the actin cytoskeleton. This thesis focuses on the function of p116Rip with emphasis on its role in RhoA-induced remodelling of the actin cytoskeleton and induction of gene transcription.
Chapter 1 describes the major signal transduction pathways downstream of RhoA leading to gene transcription and remodelling of the actin cytoskeleton, especially in neuronal cells. Moreover, we characterise p116Rip: the conservation of p116Rip among diverse organisms, its chromosomal location, occurrence of splice variants, related proteins, domain structure, and potential protein-protein interaction and regulatory motifs. Furthermore, chapter 1 summarizes published data on the presence of p116Rip in various protein complexes and its regulation under physiological conditions.
In chapter 2 we show that p116Rip is an F-actin binding protein that co-localises to, binds and bundles filamentous actin through actin-binding domain(s) located at its N-terminus. In addition, we show that overexpression of p116Rip in NIH3T3 cells leads to disruption of RhoA-dependent stress fibres and Rac-mediated lamellipodia formation, as shown by the formation of dendrite-like extensions. These findings indicate that p116Rip can affect, either directly or indirectly, the integrity of the actin cytoskeleton.
In chapter 3 we show that p116Rip, through its C-terminal coiled-coil domain, interacts directly with the regulatory myosin-binding subunits (MBS) of myosin light chain (MLC) phosphatase, MBS85 and MBS130. MLC-phosphatase is a crucial component in the RhoA pathway. The function of p116Rip is investigated by blocking its expression using a recently developed technique, RNA interference (RNAi). This technique involves the production of small specific pieces of RNA that block expression of the corresponding protein. RNAi-induced knockdown of p116Rip inhibits cell spreading and neurite outgrowth in response to extracellular stimuli, demonstrating that p116Rip is essential for neurite outgrowth. RNAi-induced knockdown of p116Rip does not affect MLC phosphorylation; the association of MBS with the cytoskeleton appears to depend on p116Rip. We suggest a model in which p116Rip acts as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.
Chapter 4 reports on the inhibition of RhoA-induced activation of the transcription factor SRF by p116Rip. However, overexpression of p116Rip does not directly affect RhoA activation. In addition, we demonstrate that p116Rip can oligomerise through its C-terminal coiled-coil domain. Mutational analysis revealed that transcriptional inhibition by p116Rip does not depend on the p116Rip-MBS interaction (chapter 3) or on oligomerisation. Exactly how this inhibition occurs is as yet unclear, but we propose that p116Rip abrogates RhoA-induced SRF activation due to its ability to disassemble the F-actin cytoskeleton (chapter 2). We conclude that p116Rip counteracts RhoA signalling downstream of RhoA and Rho-kinase (ROCK).
In conclusion, we have shown that p116Rip is essential for neurite outgrowth and counteracts RhoA action. This occurs downstream of RhoA and ROCK, probably at multiple levels: 1) by its direct effect on actin remodelling, 2) by acting as a scaffold linking MBS to the cytoskeleton. Further studies in primary neurons, should reveal how p116Rip regulates neurite outgrowth. Since p116Rip counteracts the RhoA pathway, it will be interesting to investigate the possible role of p116Rip in RhoA-dependent tumour development.