Abstract
Rho kinase (ROCK), a downstream effector of Rho GTPase, is a serine/threonine protein kinase that regulates many crucial cellular processes via control of cytoskeletal structures. The C-terminal PH-C1 tandem of ROCKs has been implicated to play an autoinhibitory role by sequestering the N-terminal kinase domain and reducing its kinase activity. The binding of lipids to the pleckstrin homology (PH) domain not only regulates the localization of the protein but also releases the kinase domain from the close conformation and thereby activates its kinase activity. However, the molecular mechanism governing the ROCK PH-C1 tandem-mediated lipid membrane interaction is not known. In this study, we demonstrate that ROCK is a new member of the split PH domain family of proteins. The ROCK split PH domain folds into a canonical PH domain structure. The insertion of the atypical C1 domain in the middle does not alter the structure of the PH domain. We further show that the C1 domain of ROCK lacks the diacylglycerol/phorbol ester binding pocket seen in other canonical C1 domains. Instead, the inserted C1 domain and the PH domain function cooperatively in binding to membrane bilayers via the unconventional positively charged surfaces on each domain. Finally, the analysis of all split PH domains with known structures indicates that split PH domains represent a unique class of tandem protein modules, each possessing distinct structural and functional features.
Highlights
By the insertion of one or more autonomously folded protein modules
It is not known whether the C1 domain of ROCK can bind to DAG/phorbol ester and thereby regulate its kinase activity, as the ROCK C1 domain is distinct from the C1 domains of myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) and citron Rho-interacting kinase (CRIK)
Structural analysis revealed that the C1 domain of ROCK adopts an atypical structure and that the domain cannot bind to DAG/phorbol ester
Summary
Protein Expression and Purification—The ROCK I PHN-C1PHC tandem (residues 1119 –1319), the ROCK II PHN-C1-PHC tandem (residues 1142–1342), the joined PHN-PHC (residues 1142–1227, 1312–1342), the C1 domain (residues 1228 –1311), and various mutants of the PHN-C1-PHC tandem were PCRamplified from the rat hippocampal cDNA library with specific primers. After dialysis in 30 mM MES buffer (pH 5.5, with 75 mM Na2SO4, 20 M ZnCl2, and 10 M -mecaptoethanol) overnight, the proteins were purified using size-exclusion chromatography (Hiload 26/60 Superdex 200, preparation grade). NMR Structure Determination—The NMR samples used contained ϳ1.0 mM of the joint PHN-PHC or C1 domain in 30 mM MES (pH 5.5, with 1 mM dithiothreitol, and 75 mM Na2SO4). The backbone and side-chain resonance assignments of the protein were obtained by standard heteronuclear correlation experiments [29]. Hydrogen bonding restraints were generated from the standard secondary structures of the protein based on the NOE patterns and backbone secondary chemical shifts. The calculations of the C1 domain structures were first performed in the absence of zinc ligation restraints. A total of 200 structures were calculated with the final set of restraints, and the 20 structures with the lowest NOE energies were selected. Defined liposomes were reconstituted from synthetic L-␣-phosphatidylcholine (PC) and L-␣-phosphatidylserine (PS) (Avanti Polar Lipids) with or without various PIPs (Echelon Biosciences; see Ref. 1 for details)
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