The nuclear import mechanism of SRF co-activator MKL1
Year: 2015
Type: dissertation
Abstract: An essential transcription factor Serum response factor (SRF) and its co-activators, Myocardin related transcription factors (MRTFs) regulate the expression of many target genes required for normal growth and actin cytoskeleton regulation. MKL1 (also known as MRTF-A and MAL) is one family member of MRTFs and mediates the signals from the cytoplasm to the nuclear SRF in response to changes in actin dynamics. Although it is well established that actin regulates nucleo-cytoplasmic shuttling of MKL1, the molecular mechanism of this regulation has not been characterized. Therefore the aim of this thesis was to reveal the mechanisms of MKL1 nuclear import. RNA interference (RNAi) screen identified two proteins as putative proteins mediating MKL1 nuclear localization: Importin-β (Ipoβ), which is the main import receptor in cells, and mRNA export factor Ddx19. The main purpose of this study was to confirm the hits from the RNAi screen and assess their specificity in regulating MKL1 localization. This work revealed that both Ipoβ and Ddx19 are specific and necessary factors for MKL1 nuclear import and thus required for SRF activation. We show that Ipoβ together with its adaptor protein Importin-α (Ipoα), binds to a bipartite nuclear localization signal (NLS) of MKL1, which is located in the actin-binding RPEL repeat and composed of two basic elements. Furthermore, the biochemical assays demonstrate that actin competes with Ipoα/Ipoβ heterodimer for access to the MKL1 NLS, thus explaining the inhibitory effect that actin binding has on MKL1 nuclear localization. By using advanced microscopy techniques, we show that Ddx19 adds an additional regulatory step for MKL1 nuclear import by modulating the conformation of MKL1, which affects its interaction with Ipoβ for efficient nuclear import. The ATPase cycle of Ddx19, which is crucial for its role in mRNA export, is not required in MKL1 nuclear import. In contrast, the RNA-binding activity of Ddx19 seems to be required. My work thus proposes a novel role for Ddx19, a well-known mRNA export factor and regulator of translation, in nuclear import of MKL1. In addition to MKL1, the conserved actin-binding RPEL repeat is also present in the Phosphatase and actin-regulating proteins (Phactrs). Our work demonstrates that the RPEL repeat of Phactr4 does not determine its localization in cells, but instead facilitates the competitive binding of monomeric actin and Protein phosphatase 1 (PPI) to Phactr4. This mechanism is required to control the phosphorylation status of cofilin, one of the downstream targets of PPI. Upon decrease in the cellular G-actin levels, Phactr4 activates cofilin through its binding to PPI, which leads to increase in the cellular levels of monomeric actin. Therefore our data pointed to an important role for Phactr4 in a feedback system, where actin monomers can locally regulate their own abundance. Thus this work highlights the role of RPEL repeat as a universal actin-binding site, which regulates actin homeostasis in cells.
Source: Työväentutkimus Vuosikirja
Author: Eeva Kaisa Rajakylä
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2149319184