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. Split pleckstrin homology (PH)3 domains are a unique subclass of PH domains in which the domain is split into two halves by the insertion of one or more autonomously folded protein modules. To date, only six proteins with diverse functions have been found to contain split PH domains in the eukaryotic genome as follows: the syntrophin family scaffold proteins; the second messenger metabolizing enzymes phospholipase C␥ (PLC␥); the Vps36 subunit of the yeast ESCRT-II sorting machinery; the neuronal GTPase PI 3-kinase enhancer (PIKE, also named AGAP and GGAP); the actin filament-based molecular motor myosin X; and the ROCK family serine/threonine protein kinases (supplemental Fig. 1A). Recent structural studies of ␣-syntrophin, PLC␥, Vps36, and PIKE showed that the split PH domains in these proteins all fold into a canonical PH domain conformation with or without the insertions of various protein domains (1-4). The functional significance of such domain organization has also been evaluated. It was demonstrated in ␣-syntrophin that the PDZ domain insertion functions synergistically with the split PH domain in binding to lipid membranes (1). Similarly, the nuclear localization sequence (NLS) insertion in the split PH domain of PIKE was found to coordinate with the split PH domain in the membrane attachment of the protein, and the PH N -NLS-PH C supramodule may function as a cytoplasmic/nuclear shuttling switch to regulate the subcellular localization of both PIKE-L and PIKE-A (4). Although the SH2-SH2-SH3 tandem insertion in the split PH domain of PLC␥ was implicated in the regu...

Mitogen-activated protein kinase kinase 6 (MKK6) is a member of the mitogen-activated protein kinase (MAPK) kinase (MAP2K) subfamily that specifically phosphorylates and activates the p38 MAPKs. Based on both biochemical and cellular assays, we found that MKK6 was extremely sensitive to oxidation: It was inactivated by oxidation and its kinase activity was fully restored upon treatment with a reducing agent. Detailed mechanistic studies showed that cysteines 109 and 196, two of the six cysteines in MKK6, formed an intramolecular disulfide bond upon oxidation that inactivated MKK6 by inhibiting its ATP binding. This mechanism is distinct from that seen in other redox-sensitive kinases. The two cysteines involved in intramolecular disulfide formation are conserved in all seven members of the MAP2K family. Consistently, we confirmed that other MAP2Ks were also sensitive to oxidation. Our work reveals that MKK6 and other MAP2Ks are a distinct class of cellular redox sensors.

The vast majority of PDZ domains are known to bind to a few C-terminal tail residues of target proteins with modest binding affinities and specificities. Such promiscuous PDZ/target interactions are not compatible with highly specific physiological functions of PDZ domain proteins and their targets. Here, we report an unexpected PDZ/target binding occurring between the scaffold protein inactivation no afterpotential D (INAD) and transient receptor potential (TRP) channel in Drosophila photoreceptors. The C-terminal 15 residues of TRP are required for the specific interaction with INAD PDZ3. The INAD PDZ3/TRP peptide complex structure reveals that only the extreme C-terminal Leu of TRP binds to the canonical αB/βB groove of INAD PDZ3. The rest of the TRP peptide, by forming a β hairpin structure, binds to a surface away from the αB/βB groove of PDZ3 and contributes to the majority of the binding energy. Thus, the INAD PDZ3/TRP channel interaction is exquisitely specific and represents a new mode of PDZ/target recognitions.