IDENTIFICATION OF NEW ROCK SUBSTRATES USING A QUANTITATIVE PROTEOMICS APPROACH

Abstract

The identification of protein phosphorylation sites and establishing their biological function are important challenges. Small G-proteins of the Rho and Ras families use multiple effector protein kinases to initiate signalling pathways that control various aspects of the cell cycle, cell differentiation and cytoskeletal changes. The ROCK kinases were the first class of effectors to be described that act downstream of RhoA. These protein kinases can regulate the actin cytoskeleton by promoting myosin II assembly and contractility. Here, I have used two proteomic mass spectrometry (MS)-based methods to attempt to identify new substrates of ROCK (other than MLC2), based on their sensitivity to the ROCK-specific inhibitor Y-27632. Firstly I attempted to identify changes in total phospho-peptide levels (after chemical enrichment) in cell treated with Y-27632. In the second approach I have used Hela cell line stably expressing GFP-tagged 14-3-3 protein. This abundant adaptor protein interacts with moderate affinity (0.1- 5uM) with ~300 cellular protein when they are phosphorylated on two or more specific serine/threonine residues. This method combined with Stable isotope labelling with amino acids in cell culture (SILAC) identified 9 potential ROCK targets [LMO7, TEF5, EFTUD2, TAF4, TOP1, PUF60, MRIP, NAT10, and snRNPQ1] with 2-15 fold decrease in 14-3-3 binding after Y-27632 treatment. Some of these proteins, such as LMO7 and snRNPQ1, represent previously identified ROCK substrates. Several potential new ROCK substrates were also identified including MRIP, and TAF4. In order to validate this MS data. I analyzed ROCK phosphorylation of MRIP in some detail to confirm that it interacts directly with 14-3-3 in an ROCK-dependent manner both in vivo and in vitro. These results confirmed 14-3-3 affinity analyses has the potential to identify other biologically important phosphorylation events in vivo, and provide a platform to elucidate signalling pathways downstream of kinase activation.