Disease relevance of RHOK

• Peptide maps of rhodopsin phosphorylated by protein kinase C or rhodopsin kinase were compared using limited Staphylococcus aureus V8 protease digestion or complete tryptic digestion [1].
• The Ca2+ dependence of the kinetics and light sensitivity of light-activated phosphodiesterase was studied with a pH assay in toad and bovine rod disk membranes (RDM), and in a reconstituted system containing GTP-binding protein, phosphodiesterase and rhodopsin kinase [2].
• Another Rho-kinase inhibitor, HA-1077 or Fasudil, is currently used in the treatment of cerebral vasospasm; the related nanomolar inhibitor H-1152P improves on its selectivity and potency [3].
• Although recent investigations have suggested that a Rho-kinase-mediated Ca2+ sensitization of vascular smooth muscle contraction plays a critical role in the pathogenesis of cerebral and coronary vasospasm, the upstream of this signal transduction has not been elucidated [4].

High impact information on RHOK

• The latter is mediated by a unique protein kinase, rhodopsin kinase, which phosphorylates only the light-bleached form of rhodopsin [5].
• Rod outer segments (ROSs) of vertebrate photoreceptor cells have been reported to contain several enzyme systems including a dark, Ca2+-stimulated ATPase, a rhodopsin kinase, a phosphodiesterase and a GTPase, all of which are light-stimulated [6].
• I have indeed found that light induces profound changes in the interation of rhodopsin kinase, GTPase (reported here), and other proteins with the photoreceptor membrane [7].
• The catalytic domain of Pkn2 contains 27% and 25% identity to rat Ca2+/calmodulin-dependent protein kinase and Bos taurus rhodopsin kinase, respectively [8].
• A three- to fourfold increase in GRK activity was also observed in a population of T cell blasts (> 97% CD3+) exposed to PHA for 48-72 h [9].

Biological context of RHOK

• Limited proteolysis of RK with endoproteinase Asp-N removes the phosphorylation sites [10].
• This finding indicates that isoprenylation of RK requires Cys558 [11].
• Recoverin is an EF-hand Ca(2+)-binding protein that is suggested to control the activity of the G-protein-coupled receptor kinase GRK-1 or rhodopsin kinase in a Ca(2+)-dependent manner [12].
• Recoverin is a Ca(2+)-binding protein that may play a role in vertebrate photoreceptor light adaptation by imparting Ca2+ sensitivity to rhodopsin kinase [13].
• One of the Ca2+ binding sites of recoverin exclusively controls interaction with rhodopsin kinase [14].

Anatomical context of RHOK

• We relate this effect to the high cholesterol content of DRMs because manipulating the cholesterol content of rod outer segment membranes by methyl-beta-cyclodextrin yielded a similar shift of the Ca2+-dependent dose-response curve of rhodopsin kinase inhibition [15].
• Furthermore, the Ca2+ sensitivity of rhodopsin kinase inhibition in DRMs was shifted to lower free Ca2+ concentration in comparison with the initial rod outer segment membranes (IC50=0.76 microm in DRMs and 1.91 microm in rod outer segments) [15].
• Transient expression of RK in COS-7 cells revealed the presence of two immunoreactive protein species [11].
• One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells [16].
• Indirect immunofluorescence of the intact ciliary epithelium with monoclonal antibodies (MAbs) against rhodopsin, rhodopsin kinase, and visual arrestin revealed labeling preferentially restricted to the NPE cells [17].

Associations of RHOK with chemical compounds

• Rhodopsin kinase (RK), interphotoreceptor retinoid-binding protein, and tubulin interact with Rv in the presence of Ca2+ [18].
• Moreover, a serine substitution for Cys558 resulted in a single RK species whose migration on sodium dodecyl sulfate-polyacrylamide gels was identical to that of RK from cells treated with mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and, thus, of isoprenoid biosynthesis [11].
• Rhodopsin phosphorylation in the reconstituted system consisting of urea-washed photoreceptor membranes, rhodopsin kinase and recoverin is regulated by Ca2+: the process takes place at low [Ca2+] but is suppressed at high [Ca2+] [19].
• Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function [20].
• We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2Delta1, respectively) suppressed stress fiber formation, but fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency) [21].

Physical interactions of RHOK

• A competition binding assay in which transducin competes with rhodopsin kinase for binding sites on rhodopsin was used to assess the interaction of purified transducin subunits with rhodopsin [22].

Enzymatic interactions of RHOK

• In this work we test this idea by determining whether two constitutively active opsin mutants are phosphorylated by rhodopsin kinase [23].
• The analog-reconstituted rhodopsin activated transducin and was phosphorylated by rhodopsin kinase on illumination [24].

Regulatory relationships of RHOK

• The available data suggest that the functions of recoverin myristoylation in the living rod are to induce a sharp Ca2+ dependence of rhodopsin kinase inhibition and to bring this dependence into the rod's physiological Ca2+ concentration range [13].
• Calcium-bound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation [25].
• These results suggest that MBS is recruited with moesin to the plasma membrane and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of moesin downstream of Rho [26].
• TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol [27].

Other interactions of RHOK

• The importance of the Rv/RK interaction was further characterized [18].
• The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase [28].
• Recoverin binds exclusively to an amphipathic peptide at the N terminus of rhodopsin kinase, inhibiting rhodopsin phosphorylation without affecting catalytic activity of the kinase [29].
• GRK5 possesses neither a CAAX motif specifying protein prenylation like rhodopsin kinase nor similarity to the G protein beta gamma-subunit binding domain of beta-adrenergic receptor kinases [30].
• We found that opsin mutants where Lys-296 is replaced either by Glu (K296E) or by Gly (K296G) are not substrates of rhodopsin kinase in the absence of chromophore [23].

Analytical, diagnostic and therapeutic context of RHOK

• Furthermore, Western blot analysis of whole lysates from the pars plicata region of the human ciliary epithelium with MAbs demonstrated immunochemical cross-reactivity with proteins of molecular mass similar to rhodopsin (36 kDa), rhodopsin kinase (64 to 66 kDa), and arrestin (48-52 kDa) from the human retina [17].
• By RT-PCR and DNA sequencing we identified the expression of rhodopsin and components linked to its deactivation, including rhodopsin kinase, recoverin, and visual arrestin [17].
• Purification of rhodopsin kinase by recoverin affinity chromatography [31].
• Rhodopsin kinase is a single polypeptide chain with Mr = 67,000-70,000 as determined by gel filtration and SDS-PAGE [32].
• In vivo interaction of GRK and tubulin was suggested by the following: (i) co-purification of betaARK with tubulin from brain tissue; (ii) co-immunoprecipitation of betaARK and tubulin from COS-1 cells; and (iii) co-localization of betaARK and GRK5 with microtubule structures in COS-1 cells [33].

References