| Summary: | The Rho family of smallpTP-binding proteins includes Rndl, Rnd2 and Rnd3/RhoE, which are characterised by their inability to bind to GDP and therefore exist primarily in a GTPbound form. RhoA stimulates stress fibre formation whereas Rndl and Rnd3/RhoE induce the disassembly of actin stress fibres. Both RhoA and RhoE interact with the serine/threonine kinase ROCK I, which is a downstream target for RhoA. This thesis examines the interaction between RhoE and ROCK I and its effect on the actin cytoskeleton. Co-expression ofRhoE with ROCK I in Swiss 3T3 fibroblasts inhibited ROCK I-induced stellate stress fibre assembly. A series of ROCK I deletion mutants were used to identify residues 1-420 ofROCK I as the minimum region required to bind to RhoE. ROCK I 1-420 was successfully purified as an active kinase from mammalian and insect cells, but was insoluble in E. coli. Cross-linking experiments as well as co-expression of GST-mycROCK I 1-420 and myc-ROCK I 1-420 in Cos 7 cells revealed that ROCK I 1-420 forms a dimer. Deletion of the N-terminal region (residues 1-76) of ROCK I prevented dimerisation and RhoE binding suggesting that N-terminus- mediated dimerisation of ROCK I was required for RhoE binding. In addition, ROCK I 375-727, which includes the central coiledcoil domain could form dimers, but was unable to bind to RhoE demonstrating that two regions ofROCK I mediate dimerisation. To identify ROCK I 1-420 autophosphorylation sites, recombinant ROCK I 1-420 produced in baculovirus-infected insect cells was subjected to mass spectrometry analysis and Edman degradation. Eight autophosphorylation sites were identified and the SerlThr sites were mutated to alanine. Of these, Thr-398 phosphorylation was required for ROCK I to form dimers, bind to and phosphorylate RhoE and induce stellate structures in Swiss 3T3 fibroblasts. The findings reported here show that the interaction between RhoE and ROCK I is important for actin cytoskeleton regulation and remodelling.
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