Bridging the Gap - Cytoskeleton Regulation by DMPK Kinases

In order to grow, divide, and replicate, cells must be able to change their shape. The actomyosin cytoskeleton allows cells to change their shape in response to environmental signals: migration, adhesion, cytokinesis, chemotaxis, and vesicular transport all rely on the coordinated remodeling of the cytoskeleton within the cell. Pathophysiological processes, including cancer cell metastasis and neurodegeneration, are associated with cytoskeletal dysfunction. ROCK and MRCK, protein kinases of the DMPK-family, integrate multiple membrane-based signals, including both lipids and proteins, to phosphorylate downstream effector proteins that modulate actomyosin contractility and permit the cell to change its morphology according to its needs. We previously determined the structure of a full-length ROCK for the first time. The structure revealed a 107 nm long tether linking the kinase domain to the membrane binding domains. The tether functions as a molecular ruler and the ability of ROCK to phosphorylate substrates in the cell depends on the correct length of its coiled-coil. We propose to extend this new model of kinase regulation to MRCK by characterizing its structure, conformation and mechanism of kinase regulation. The localization of ROCK and MRCK inside cells is of fundamental importance, but to date the membrane ligands to which they bind have not been described. We propose to take a quantitative, systematic, and combinatorial high-throughput approach to determine the membrane ligands of ROCK and MRCK. In parallel, we will determine the structure of the membrane binding domains of MRCK. Combining knowledge about the membrane ligands and the 3-dimensional arrangement of the regulatory domains is expected to provide significant insight into how the specific localization of these important kinases is achieved. Finally, we will study the localization of ROCK and MRCK inside cells directly. Supported by two expert groups in Germany, we will apply state-of-the-art high-resolution fluorescence microscopy approaches to address the localization, conformation, and proximity to cytoskeletal components of ROCK and MRCK in cells. By combining structural, biophysical, biochemical, and cell biological techniques to study their localization in cells and their structure and membrane binding properties in vitro, we expect to learn significantly more about how the cytoskeleton is regulated by these essential kinases.

Kinases are important enzymes that enable cells to respond to environmental cues by phosphorylating down stream effector targets that regulate essential pathways inside cells including proliferation, survival, movement and transport. Pathophysiological processes, including cancer cell metastasis and neurodegeneration are associated with kinase dysfunction. ROCK and MRCK, two kinases of the AGC family of protein kinases integrate multiple membrane-based signals, including both lipids and proteins, to phosphorylate downstream effectors that modulate actomyosin contractility and permit the cell to change its morphology according to its needs. We previously determined the structure of full-length ROCK and revealed that its kinase and regulatory domains are tethered by a conserved 107 nm long coiled-coil domain. The tether functions as a molecular ruler and the ability of ROCK to phosphorylate substrates in the cell depends on the correct length of it. Interestingly also the kinase domain of MRCK is tethered to its membrane binding domains by a coiled-coil domain which is conserved in length. In this study we combined structural, biophysical, biochemical, and cell biological techniques to study ROCK and MRCK localization in cells and to explore their structure and membrane binding properties in vitro. We were able to solve the crystal structure of the regulatory domains of MRCK (C1-PH-CNH domains) and getting first insights into how membrane ligands are detected by MRCK. Particularly remarkable is that this is first structure of an CNH domain ever and, as the structure includes all three regulatory domains of MRCK, we now understand better how MRCK detects different lipid signals by coincidence detection. Using rotary shadowing electron microscopy we determined the low-resolution full length structure of MRCK and found that, as in ROCK, particles are in an extended open conformation. In addition we used a quantitative and systematic high-throughput approach (LIMA) to determine the lipid ligands that are detected by ROCK and MRCK. Together with the Biooptics facility of the Max Perutz Labs, this highly useful technique was implemented at the Vienna Biocenter Campus and is available to new users. Combining knowledge about the membrane ligands and the 3-dimensional arrangement of the regulatory domains is expected to help to understand how the specific localization of these important kinases is achieved. Furthermore we made important contributions to answer questions about the regulation of another essential kinase of the AGC kinase family, namely Akt1. We were able to solve the near full length structure of Akt1 in its autoinhibitory conformation using a llama-derived nanobody. Furthermore we define features about the regulation of the kinase activity of this important enzyme and our findings change the view about where, when and how Akt is active. Our results are published in the journal PNAS entitled: "Structure of autoinhibited Akt1 reveals mechanism of PIP3-mediated activation".